arsenic is a ubiquitous metalloid that 
 is introduced into the environment through both geological and anthropogenic 
 sources , such as mining activities , utilization of arsenic - based pesticides 
 or herbicides . arsenic in soils and sediments enters food chain 
 from edible parts of vegetables or cereals that have accumulated high 
 levels of arsenic and poses a serious health risk to humans . inorganic 
 arsenic has been classified as class one carcinogen by the international 
 agency for research on cancer . long - term 
 exposure has resulted in a global epidemic of arsenic - related diseases , 
 with many people having developed skin lesions , kidney , lung , and 
 bladder cancers . there is an urgent need to efficiently 
 remove arsenic from contaminated water and soils . in situ bioremediation 
 of arsenic by microorganisms has been widely 
 hailed because of their potential advantages in facilitating cost - effective 
 and environmental friendly technologies . bacteria containing 
 the arsenic(iii ) s - adenosylmethionine methyltransferase ( arsm ) gene were able to sequentially methylate toxic inorganic arsenic to less toxic pentavalent methylated arsenicals such as methylarsenate 
 ( mas(v ) ) , dimethylarsenate ( dmas(v ) ) and trimethylarsine oxide ( tmas(v)o ) . 
 whether biomethylation of arsenic was considered as a detoxification 
 process is still controversial . the intermediate 
 metabolites methylarsonous acid ( mas(iii ) ) and dimethylarsinous acid 
 ( dmas(iii ) ) are generated during this process . these trivalent methylated 
 arsenical intermediates are highly toxic and possibly carcinogenic , 
 but they can be easily oxidized to much less toxic mas(v ) and dmas(v ) , 
 so they are not readily detected in environmental settings . biovolatilization 
 of the final product , gaseous trimethylarsine ( tmas(iii ) ) , could remove 
 arsenicals from polluted sites , while 
 converting the extremely toxic inorganic species of arsenic to virtually 
 innocuous counterparts , thereby adding to the potential toolbox for 
 bioremediation of soil arsenic . bioremediation of arsenic from contaminated soils using genetically 
 engineered ( ge ) bacteria might be a better option as the amount of 
 volatile arsenic generated by indigenous microorganisms is limited . successfully overexpressed 
 an arsm gene in sphingomonas desiccabilis and bacillus idriensis and improved arsenic volatilization 
 from contaminated soil . pseudomonas putida kt2440 , 
 an efficient colonizer of the root system of various plants , is a 
 ubiquitous saprophytic bacterium endowed with a remarkable adaptability 
 to diverse environments , including soils heavily contaminated with 
 multiple metals . this soil microbe has 
 been studied extensively as an model organism for biodegradation of 
 aromatic compounds and it has been highlighted as an optimal chassis 
 for implantation of synthetic genetic circuits.p . putida kt2440 has two systems for arsenic 
 tolerance based on arsenate reduction to arsenite and ensuing export 
 of arsenite to the external medium , a feature that affords endurance 
 to very high concentrations of the corresponding anions . however , p. putida does not 
 have an arsm gene and can not methylate arsenic . we 
 previously reported the construction of ge p. putida expressing the chlamydomonas reinhardtii arsm gene 
 ( crarsm ) . recently arsm genes from a variety of microorganisms have been identified 
 and shown to exhibit quite variable capacity for arsenic methylation . the construction of new ge bacteria with higher arsenic methylation 
 become possible by selecting increasingly active products of arsm genes . one problem is that plasmids carrying exogenous 
 genes ( arsm genes ) are often lost during practical 
 application . we can solve this dilemma by inserting the cloned genes 
 into the chromosome rather than expressing from a plasmid . this also 
 increases the stability and safety of the strain , and reduces the 
 likelihood of horizontal gene transfer . in this study we also 
 chose p. putida kt2440 as 
 the host strain for arsenic removal . the rhodopseudomonas 
 palustris arsm ( rparsm ) gene was selected 
 because of its high rate of methylation . the rparsm gene was inserted into the chromosome of p. putida kt2440 using a synthetic transposon vector that allows random generation 
 of fusions between a promoterless arsm gene and external 
 transcription initiation signals , thereby allowing selection for different 
 degrees of arsenic methlyation and resistance . the selected genetically 
 engineered construct exhibited a high level of arsenic methylation 
 and volatilization compared to wild type . in the environmental arsenic - contaminated 
 soil assay , ge p. putida exhibited a high capacity 
 for arsenic volatilization , which is 9-fold higher than wild type 
 cells . further experiments for rhizosphere bioremediation by plants 
 and ge p. putida will be conducted . to monitor colonization 
 of ge p. putida in soil , the rparsm gene was fused with the gene for green fluorescent protein ( gfp ) , and the fusion gene was stably integrated into the 
 chromosome of p. putida kt2440 . the ge p. 
 putida exhibits a high level of fluorescence when exposed 
 to arsenic . these fluorescent bacteria can visualized in situ in the 
 environment and in the rhizosphere . the pseudomonas putida kt2440 strains , e. coli cc118pir , e. coli hb101 ( prk600 ) and pbam1 
 plasmid used in this study have been described before.e . coli strain dh5 ( promega , 
 madison , wi ) was used for plasmids construction , replication . strain 
 aw3110(de3 ) ( arsrbc ) , which is hypersensitive to arsenic(iii ) , was used for complementation 
 studies . selection of p. putida cells was made onto m9 minimal medium plates with citrate ( 2 antibiotics , 
 when needed , were added at the following final concentration : ampicillin 
 ( ap ) 100 g / ml for e. coli and 500 g / ml 
 for p. putida , kanamycin ( km ) 50 g / ml and 
 chloramphenicol ( cm ) 30 g / ml for both species . 
 to construct a vector for rparsm transfer , the rparsm gene was retrieved from plasmid pet28 . bacterial 
 growth was monitored by measuring the optical density at 600 nm ( od600 ) . all reagents were obtained from commercial sources . for expressing rparsm of r. palustris in p. putida kt2440 , 
 plasmid pbam1-rparsm , in which rparsm was promoterless and has the sequence of a c - terminal six - histidine 
 tag , was constructed . rparsm under control of t7 promoter and with a c - terminal six - histidine was cloned from 
 plasmid pet28a - rparsm . the promoterless arsm with a c - terminal his tag 
 was removed by double digestion with saci and xbai and ligated to pbam1 which was digested with the same 
 restriction enzymes . a gfp reporter plasmid was constructed 
 to form pbam1-rparsm - gfp , in which 
 the gfp was fused to the c - terminal of rparsm to replace of six - histidine tag . rparsm was cloned 
 from pet28a - rparsm using primers : 5- gcgagctcaaggagatataccatggc 3 ( saci site underlined ) and 5- gctctagaatgatgcccggaggagcg 3 ( xbai site 
 underlined ) . the gfp gene was obtained from 
 pgreen vector , which was digested by xbai . pbam1 
 was digested with the same restriction enzymes , generating plasmid 
 pbam1-rparsm - gfp . plasmid pbam1 
 and its derivatives were delivered from e. 
 coli cc118pir ( pbam1 or pbam1-rparsm - gfp ) donor cells into p. putida kt2440 by tripartite 
 mating with the assistance of the helper strain e. coli hb101 ( prk600 ) . the conjugation mixture 
 was incubated at 30 c for 6 to 8 h by tripartite mating on membrane 
 filters ( 0.45 m , millipore ) on lb agar plates . the filter was 
 transferred to 5 ml of 10 mm mgso4 and vortexed to suspend 
 the cells . afterward , appropriate dilutions were plated onto selective 
 medium as indicated for counter - selecting against the donor cells . p. putida kt2440 containing different 
 expression vectors were cultured overnight in lb medium at 30 c . cells were harvested and identified by sodium dodecyl sulfate polyacrylamide 
 gel electrophoresis ( sds - page ) . proteins separated 
 by sds page were transferred to a nitrocellulose membrane ( protran , 
 schleicher & schuell ) . western blot analysis was performed using 
 a western lighting ultra chemiluminescence substrate kit ( perkinelmer ) 
 using an antimouse igg to the six histidine tag . the arsenic resistance 
 phenotype of ge p. putida kt2440 cells was determined 
 in bacteria . cells of wild type bacteria ( bearing pbam1 ) , and bacteria 
 bearing pbam1-rparsm , which were used for inorganic 
 arsenic sensitivity assays , were grown overnight in m9 medium at 30 c , supplemented with containing 
 25 g / ml kanamycin and 0.2% glucose . overnight cultures were 
 adjusted to 1.0 od600 , diluted in a 10-fold series with 
 double distilled water , and spotted ( 2 l ) onto m9 solid medium 
 supplemented with the indicated concentrations of arsenite and antibiotics . growth was scored after incubation at 30 c 
 for 24 h. the growth rate of the two strains in liquid medium was 
 also monitored . after growing in lb liquid medium to log phase ( od600 0.60.8 ) , the growth of the cultures 
 was monitored over 24 h and the od600 was plotted as a 
 function of growth time . to investigate 
 the effects of arsenic methylation and volatilization by wild type 
 and ge p. putida ( expressing rparsm ) in the m9 medium , arsenic methylation by bacteria was performed 
 in capped 20 ml vials with a total volume of 4 ml and sodium arsenite 
 was added to each vial in a final concentration of 25 m . the supernatant were filtered and stored at 4 c until analysis . 
 to trap volatile arsenicals , 2 cm nitrocellulose membrane filters 
 were put in vial caps and impregnated with 0.15 ml of 6% h2o2 . the filters were digested 
 with 0.2 ml of 70% hno3 at 70 c for 20 min . arsenic speciation 
 analysis was conducted within 24 h by hplc - icp - ms ( series 2000 , perkinelmer ) 
 with anion exchange column . soil was sampled from farmland near a mining company 
 at zhuzhou , 
 hunan province , china . soil ph was 5.82 , organic matter was 3% and total arsenic was 20.0 
 mg / kg . the experimental device was described as reported . briefly each erlenmeyer flask ( 250 ml ) contained 
 unsterilized soil ( 40 g ) with water holding capacity at 30% , ( unflooded ) . the wild type or ge p. putida kt2440 were cultured 
 in the lb medium at exponential phase , and this culture medium ( 2 
 ml ) was inoculated into flasks containing soil . the control ( without 
 strain inoculation ) was supplemented with an equal volume of lb medium . trapping tubes for volatile arsenic were prepared by filling the silica 
 gel beads impregnated with 10% agno3 into glass tubes and 
 connected to the flask . the headspace was refreshed by pumping filtered 
 air with pumps at intervals ( 24 h ) for 10 min each time . in order 
 to increase bioavailable arsenic in the soil to investigate the effect 
 of arsenic bioavailability on arsenic volatilization , arsenite were 
 spiked into subsamples to a final concentration of 30 mg / kg . all flasks were shaken in 
 the dark at 150 rpm at 30 c for 6 days . all trapping tubes were 
 taken off , and samples were prepared as described and stored at 4 c until analysis . arsenic 
 speciation in the lb medium was determined by hplc - icp - ms . briefly , either a reverse - phase c18 column ( jupiter 
 300 ) was eluted isocratically with a mobile phase consisting of 3 
 mm malonic acid , 5 mm tetrabutylammonium hydroxide , and 5% methanol , 
 ph 5.6 , with a flow rate of 1.0 ml / min or an anion - exchange column 
 ( prp x100 , hamilton ) was eluted with a step gradient of mobile phase 
 a ( 20 mm ammonium bicarbonate , ph 8.5 ) and mobile phase b ( 20 mm ammonium 
 bicarbonate , ph 7.0 ) at a flow rate of 1.5 ml / min . indium ( 10 g / l ) 
 was added online postcolumn as the internal standard . retention times 
 for arsenic species were determined using a mixture of standards comprised 
 of 1 m each of as(iii ) , as(v ) , dmas(v ) , mas(v ) , and mas(iii ) . after 6 days inoculation of bacteria 
 in the soil , soil samples ( 0.5 g ) were washed with sterilized water 
 ( 5 ml ) . subsamples of supernatant ( 100 l ) were inoculated in 
 lb plates supplemented with kanamycin ( 50 g / ml ) to inhibit 
 growth of indigenous bacteria and stored at 30 c for 48 h. photographs 
 were taken in visible and uv light . many orthologous arsm encoding putative orthologs of as(iii ) methyltransferases 
 have been reported , including rparsm ( rhodopseudomonas palustris 
 cga009 ) , has3mt ( homo 
 sapiens ) nsarsm ( nostoc 
 sp . pcc7120 ) , crarsm ( chlamydomonas reinhardtii ) , and cmarsm ( cyanidioschyzon sp . 5508 ) . to choose a suitable arsm gene 
 for high arsenic methylation and volatilization in p. putida kt2440 , a comparison was made for all the arsms arsenic methylation activity . rparsm , crarsm and nsarsm were shown 
 to confer a high arsenic(iii ) resistance when expressed in an arsenic - sensitive 
 strain of e. coli aw3110 . cmarsm and has3mt showed a relatively low arsenic(iii ) resistance 
 ( supporting information ( si ) , figure s1a , 
 b ) . further arsenic speciation analysis showed that each arsm examined could convert as(iii ) to dmas(v ) or tmas(v)o after 12 h 
 of incubation ( figure 1a ) . tmas(iii ) and some 
 me2ash were the volatile products , both were easily oxidized 
 to tmas(v)o and dmas(v ) , respectively ( figure 1b ) . among these arsms , the intracellular arsenic 
 species of e. coli aw3110 expressing various arsms was quantified ( figure 1c ) . crarsm also 
 showed a high methylation acitivity with dmas(v ) ( 50% ) and tmas(v)o 
 ( 43% ) . cmarsm shows a least activity due to the incubation temperature 
 is not the optimal one ( 6070 c ) . to remove arsenic from soil , rparsm was 
 the best for arsenic volatilization by genetically engineered p. putida kt2440 . to obtain optimum expression of foreign rparsm gene in the p. putida kt2440 strain , fortunately the triplet codons for the rparsm are on their relative abundance in the p. 
 putida genome . formation 
 of methylated arsenicals was measured in cells of e. coli aw3110 ( de3 ) ( arsrbc ) , bearing 
 vector plasmid pet28a(+ ) as a control ; rparsm ( rhodopseudomonas palustris cga009 ) ; nsarsm ( nostoc sp . 5508 ) ; crarsm ( chlamydomonas reinhardtii ) ; has3mt ( ( homo sapiens ) . a , speciation of arsenic in the culture medium by different arsm genes ; b , volatilization of arsenic by e. coli aw3110 expressing different arsm genes . cells were 
 incubated for 12 h in lb medium with 25 m as(iii ) . cells were grown for 12 h. soluble 
 arsenic species were determined by hplc - icp - ms using an anion - exchange 
 column . the chromosomal engineering rparsm to p. putida kt2440 is a prerequisite 
 to realize the full potential of this species in the arsenic detoxification 
 and bioremediation . rparsm was cloned into the transposon 
 vector pbam1 ( si , figure s2 ) and transferred 
 from e. coli cc118pir to p. putida kt2440 by tripartite conjugation . to examine whether exconjugants 
 had undergone authentic transposition events or resulted from the 
 cointegration of pbam1-rparsm into the host genome , 
 100 colonies were randomly selected , and their sensitivity to the 
 plasmid marker ( ap ) was examined . all 100 km clones were sensitive to ampicillin ( 500 g / ml ) , thereby indicating 
 that the insertion of the minitransposon carried by pbam1 occurred 
 as expected . several colonies were resistant to as(iii ) , indicating 
 that the promoterless rparsm had been inserted into 
 chromosomal genes in the correct orientation and in the proper reading 
 frame . it was easier to screen the positive transformants for rparsm fused with gfp , where fluorescent 
 colonies could be chosen for the further analysis . putida kt2440 is a ubiquitous saprophytic bacterium endowed with a remarkable 
 adaptability to diverse environments including tolerance to different 
 heavy metals and metalloids . two copies of an arsrbch operons exist in the chromosome of p. putida kt2440 allowing it to grow in the presence of 2 mm 
 as(iii ) . the putative functions 
 of these genes products are proposed to be a phosphatase , a monooxygenase , 
 and a phosphinothricin n - acetyltransferase respectively , but their 
 relationship to arsenic resistance is unclear . neither ars operon has an arsm gene , so wild type p. 
 putida is unable to methylate as(iii ) . when exposed to 7.5 
 mm as(iii ) , cells expressing rparsm grew dramatically 
 better than those bearing the empty vector ( figure 2a ) . in addition , cells bearing vector plasmid pbam1 were sensitive 
 to 10 mm as(iii ) . in contrast , cells expressing rparsm could grow in as(iii ) concentration as high as 10 mm ( figure 2a ) , demonstrating that the gene products confer 
 tolerance to as(iii ) . furthermore , the growth curve in liquid m9 medium 
 were also consistent with the arsenic resistance results observed 
 on solid medium ( si , figure s3 ) . genetically 
 engineered p. putida kt2440 exhibits much more resistance 
 to as(iii ) , which can be used for in situ remediation of arsenic - polluted 
 soil . it also shows a high capacity for arsenic volatilization both 
 in culture and in environmentally similuated arsenic - contaminated 
 soil . therefore , practical application of ge p. putida for arsenic biovolatilization form arsenic contaminated soils is 
 becoming possible . growth of 
 cells in lb medium plates with 2 , 7.5 , and 10 mm as(iii ) . soluble ( solid symbols ) and 
 volatile ( open symbols ) arsenic species formed by e. coli strain bl21 ( de3 ) with vector plasmid pet28a ( circles ) or pet28a - rparsm ( inverted triangles ) in solution ( solid symbols ) 
 were determined following growth in lb medium in the presence of 25 
 m as(iii ) for 16 h. total arsenic in the culture medium and 
 cells ( black ) and volatilized arsenic ( white ) were determined by icp - ms , 
 as described above . cells were grown 
 for 24 h. soluble arsenic species were determined by hplc - icp - ms using 
 an anion - exchange column . the amount of volatilized arsenic was proportional to the loss 
 of arsenic from the medium ( figure 2b ) . ge p. putida biomethylated as(iii ) primarily to dmas(v ) , with 
 some tmas(v)o , after 12 h of incubation ( si , figure s4a and b ) . wild type p. putida cells exhibited 
 resistance to as(iii ) due to its two ars operons , 
 no methylated or volatilized arsenicals were detectable . the amount 
 of volatile tmas(iii ) increased with time ( figure 2b and si , figure s4b ) . after 48 
 h , me2ash ( 21  1.0% ) and tmas(iii ) ( 10  1.2% ) 
 were the volatile arsenic species . the arsenic species detected in 
 the medium were 62  2.2% dmas(v ) , 25  1.4% mas(v ) and 
 10  1.1% tmas(v)o of total arsenic ( figure 2c ) . due to the existence of two copies of arsc in the chromosome of p. putida kt2440 , we have shown that the initial product 
 of arsenic methylation is mas(iii ) , and it is likely that the second 
 product is dmas(iii ) , but these are rapidly 
 oxidized to the pentavalent species in soil , so only mas(v ) and dmas(v ) can be detected . to representative of 
 soil conditions , low phosphate medium and m9 medium were also used 
 to measure the ge p. putida kt2440 methylation activity . as(v ) was totally converted to methylated arsenic by ge p. 
 putida similar to the rate of methylation of as(iii ) in both 
 lb medium and a low phosphate medium ( data not shown ) . in m9 medium , the arsenic methylation and volatilization were not efficient due 
 to the high phosphate concentration in this medium , which inhibited 
 the uptake of arsenate by cells . these results demonstrated that heterologous 
 expression of rparsm in the chromosomal of p. putida conferred the ability to methylate and volatilize 
 arsenic . it efficiently decreased the levels of inorganic arsenic 
 and converted it to volatile arsenic species . like 
 as(iii ) , the protonated , uncharged forms of the methylated as species 
 mas(v ) and in contrast , mas(v ) and dmas(v ) 
 are not complexed by thiols , which is very mobile and more efficiently 
 translocated from roots to the shoot in rice . dmas(v ) can be removed from soil by plants by uptake through nodulin 
 26-like intrinsic ( nip ) aquaporin channels and accumulated in plant 
 tissues . the establishment of p. putida on plant 
 roots is a rapid and dynamic process . many plant species accumulate methylated arsenicals such as mas(v ) , 
 dmas(v ) , and tmas(v)o if they are available in soil . thus , using plant - rhizosphere bacterium symbiosis to clean up the 
 arsenic by harvesting plants is a promising and potentially practical 
 method for bioremediation . ge p. putida with rparsm has the prospect for efficiently removing arsenic 
 from contaminated soil by a combination of rhizosphere biovolatilization 
 and phytoextraction by host plants . the increased resistance to arsenic 
 by the engineered rhizobacteria p. putida at higher 
 concentrations is particularly important , as it could provide a competitive 
 advantage to the ge p. putida in a contaminated soil 
 environment . this could be crucial factor for sustaining the growth 
 of the engineered strain in the presence of the native bacterial population . 
 as a rhizosphere bacterium , p. putida can colonize 
 and persist in the root system of a number of plants . this is a promising 
 concept for rhizosphere bioremediation and phytoextraction . to elucidate the mechanism of arsenic methylation 
 by the ge p. putida , the protein expression level 
 of rparsm ( 29.7 kda ) in ge p. putida was determined . compared to rparsm expressed in e. coli ( bl21 ) behind 
 the t7 promoter , expression of rparsm was low in ge p. putida . it was expected that expression from chromosome 
 would be less than from a multicopy plasmid . there was no difference 
 from wild type with or without as(iii ) induction ( si , figure s5a ) . rparsm expression was probed by western blotting 
 with antibody agains the six histidine tag . rparsm expressed in bl21 
 ( de3 ) under control of the t7 promoter was used as positive 
 control . a band at 30 kda corresponding to the predicted size of rparsm 
 was detected in ge p. putida ( si , figure s5b ) . although chromosomal expression of rparsm in p. putida kt2440 was lower than 
 on a plasmid under the t7 promoter in e. coli . , high level of expression might constitute a selective disadvantage 
 to the organism , particularly when competing with indigenous organisms . microbes that carry multiple copies of foreign 
 genes or express those genes at a high level often compete poorly 
 with indigenous bacteria in their natural environment . plasmids are 
 often not maintained in genetically engineered bacteria in the absence 
 of selective pressure and are lost during attempts at practical application . 
 in genetically modified microorganisms , it is frequently preferable 
 in some circumstances to insert the cloned genes into the chromosome , 
 where they are maintained at a low , natural copy number and thus , 
 at least theoretically , they should be as stable as chromosomal genes . microorganisms bearing chromosomal inserts with 
 the genes of interest have advantages to compete with indigenous organisms 
 than their counterparts with plasmids . in our previous study , an arsm gene from the eukaryotic unicellular alga chlamydomonas 
 reinhardtii was inserted into the p. putida chromosome under control of the kanamycin promoter . compared to that construct , our present one in which the arsm gene from the bacterium r. palustris is chromosomally inserted showed a considerably higher capacity 
 for arsenic methylation and volatilization . to monitor growth of p. putida under environmental 
 conditions , we constructed plasmid pbam1-rparsm - gfp , in which green fluorescent protein was fused to the c - termus of 
 rparsm in place of the six histidine tag , allowing direct fluorescent 
 detection of hybrid protein production rather than just mrna synthesis . the exconjugant clones were screened on m9 agar plates for green fluorescent 
 colonies by illuminating the plate with uv light , and fluorescent 
 colonies were screened for methylation activity ( figure 3 ) . use of a gfp tag allows for facile selection 
 of recombinants with high methylation activity . only the colonies 
 with high fluorescence were picked up for further experiments , making 
 this time - consuming screening much easier . in addition , p. 
 putida , as a root - colonizing bacterium , are very relevant 
 in phytoremediation . it is important to understand the effect of p. putida as inoculator and whether they cause changes in 
 the microbial community structure in the rhizosphere and soil . using 
 this strategy , it conferred the ge p. putida have 
 fluorescence and the process of these labeled strains colonizing all 
 surfaces of plant root zones , such as roots hairs and lateral roots 
 can be easily monitored , including the effect of inoculation on soil 
 microbial populations . ( a ) , growth of the cells was 
 examined 
 by epifluorescence microscopy under white light . ( b ) , growth of the 
 cells was examined by epifluorescence microscopy under uv light . ( c ) , 
 higher magnification images from ( b ) . to examine the ability of ge p. putida kt2440 to volatilize arsenic in environmental soil samples , cells were inoculated in unsterilized soil , and volatilization of arsenic 
 was quantified . in comparison with soil not inoculated with bacteria , 
 wild type of p. putida kt2440 did not show increased 
 arsenic volatilization ( figure 4 ) , in contrast , 
 ge p. putida exhibited a high capacity for arsenic 
 volatilization from arsenic - contaminated soil . the amount of volatile 
 arsenic was about 9-fold of that of wild type cells , although volatilization 
 from soil was less than from lb medium , which may reflect lower arsenic 
 bioavailability in soil . arsenite was amended into the soil to increase 
 its bioaccessibility . in the control ( soil + as(iii ) ) and wild type 
 ( soil + wt + as(iii ) ) , there are no significant difference in arsenic 
 volatilization compared with unamended soil . however , addition of 
 as(iii ) significantly increased arsenic volatilization by ge p. putida kt2440 . the amount of volatilized arsenic was 
 49-fold that of unamended soil with the ge cells , indicating that 
 arsenic bioaccessibility is a major limiting step in arsenic removal 
 via arsenic volatilization . since bioavailability of as in contaminated 
 soils is an important factor determining the remediation efficiency , 
 future studies should also be directed to manipulating as bioavailability , 
 such as the amendment of soil organic matter and soil water management . under uv light it could be seen that there are many more fluorescent 
 cells with ge p. putida inoculation ( si , figure s6b ) than with wild type inoculation 
 ( si , figure s6a ) , even though growth of 
 both looked similar under visible light ( si , figure s6a , b ) . this result indicates that ge p. putida kt2440 grow better in arsenic - contaminated soil as arsenic is methylated 
 and volatilized . amount of volatilized arsenic by wild type and ge p. putida kt2440 in arsenic contaminated soil ( 20 mg / kg ) 
 and contaminated 
 soil amended with as(iii ) ( 10 mg / kg ) . the different letters above bars indicate 
 significant differences ( p < 0.005 ) . in future experiments , arsenic removal from contaminated 
 soil by 
 ge p. putida kt2440 expressing either rparm or rparsm - gfp in combination with plants will be 
 examined . the rhizosphere is the most active portion in soil , which 
 has important ecological implications for soil function , including 
 biogeochemical cycles . furthermore , a wider range of soil types , soil 
 properties and soil with different arsenic contamination levels will 
 be further utilized to evaluate the efficiency of ge p. putida in arsenic removal . the relative toxicity of an arsenical depends 
 primarily on the species , whether it is inorganic or organic , its 
 oxidation state and solubility . inorganic trivalent arsenite has high 
 toxicity and high mobility , but mineral - bound arsenicals such as sulfide 
 forms are highly insoluble , so the efficiency for ge p. putida to removal these arsenic compounds need to be further evaluated . a better understanding of these processes is critical for maintaining 
 the safety of our food supply . a genetically engineered 
 microbe that exhibits fluorescence when it methylates arsenic will 
 be especially useful for monitoring this critical biogeochemical process 
 at the plant root - soil interface . our results suggest the potential 
 feasibility of using symbosis between ge p. putida chromosomal expressing rparsm and plant as an efficient strategy 
 for arsenic remediation from contaminated soil . although more toxic 
 mas(iii ) and dmas(iii ) are generated during this methylation pathway , 
 both are rapidly methylated to the volatile end product tmas(v)o . in addition , they are chemically unstable and are quickly oxidized 
 to the nontoxic pentavalent forms . this process is promising for harnessing 
 the root system of plants to increase yields of staple food crops . of concern for any new bioremediation scheme is whether it is better 
 and safer than the methods that it replaces . large - scale interventions 
 on arsenic - contaminated soil based on the ge strain described in this 
 paper must be preceded by a thorough evidence - based risk assessment 
 study to ensure safety to the surrounding animal , plant , and human 
 landscape . in this context , the question of the toxicity and fate 
 of tmas(iii ) is key to diagnose the applicability of such an strategy . bartolomeo gasio ( 18631944 ) demonstrated that fungi grown 
 on arsenic produced a gas that could kill rats . however , recently cullen has called this an  urban myth  
 and the has showed that tmas(iii ) is entirely nontoxic . inhalation by animals or humans in the target 
 site would be expected to result in rapid elimination in urine , resulting 
 in a negligible hazard . if produced by soil bacteria during the day , 
 the half - life of atmospheric tmas(iii ) can be measured in minutes 
 or 8 h under daytime conditions . however , 
 at night when there is no photodegradation , tmas(iii ) is stable for 
 days , providing sufficient time for the gas to be diluted into the 
  infinite sink  of the atmosphere . these hypothetical 
 scenarios should be endowed with solid evidence and quantitative parameters 
 before large - scale applications can be contemplated in earnest . while 
 such studies are beyond the scope of this paper , we advocate our approach , 
 that is , engineering soil microbes for arsenic methylation and volatilization 
 as a valuable and worth considering avenue for tackling the phenomenal 
 challenge of this type of environmental pollution .
even 
 though arsenic is one of the most widespread environmental 
 carcinogens , methods of remediation are still limited . in this report 
 we demonstrate that a strain of pseudomonas putida kt2440 endowed with chromosomal expression of the arsm gene encoding the as(iii ) s - adenosylmethionine ( sam ) methyltransfase 
 from rhodopseudomonas palustris to remove arsenic 
 from contaminated soil . 
 we genetically engineered the p. putida kt2440 with stable expression of an arsm - gfp fusion 
 gene ( ge p. putida ) , which was inserted into the 
 bacterial chromosome . 
 ge p. putida showed high arsenic 
 methylation and volatilization activity . 
 when exposed to 25 m 
 arsenite or arsenate overnight , most inorganic arsenic was methylated 
 to the less toxic methylated arsenicals methylarsenate ( mas(v ) ) , dimethylarsenate 
 ( dmas(v ) ) and trimethylarsine oxide ( tmas(v)o ) . 
 of total added arsenic , 
 the species were about 62  2.2% dmas(v ) , 25  1.4% mas(v ) 
 and 10  1.2% tmas(v)o . 
 volatilized arsenicals were trapped , 
 and the predominant species were dimethylarsine ( me2ash ) 
 ( 21  1.0% ) and trimethylarsine ( tmas(iii ) ) ( 10  1.2% ) . 
 at later times , more dmas(v ) and volatile species were produced . 
 volatilization 
 of me2ash and tmas(iii ) from contaminated soil is thus 
 possible with this genetically engineered bacterium and could be instrumental 
 as an agent for reducing the inorganic arsenic content of soil and 
 agricultural products .