Biotransformation and Volatilization of Arsenic by Three ......Biotransformation and Volatilization...

Biotransformation and Volatilization of Arsenic by ThreePhotosynthetic Cyanobacteria1

Xi-Xiang Yin2, Jian Chen2, Jie Qin, Guo-Xin Sun, Barry P. Rosen, and Yong-Guan Zhu*

Key Lab of Urban Environment and Health, Institute of Urban Environment (X.-X.Y., Y.-G.Z.), and State KeyLab of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences (X.-X.Y., J.C., G.-X.S.,Y.-G.Z.), Chinese Academy of Sciences, Xiamen 361021, China; and Department of Cellular Biology andPharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida 33199(J.C., J.Q., B.P.R.)

Arsenic (As) is a pervasive and ubiquitous environmental toxin that has created worldwide human health problems. However,there are few studies about how organisms detoxify As. Cyanobacteria are capable of both photolithotrophic growth in thelight and heterotrophic growth in the dark and are ubiquitous in soils, aquatic systems, and wetlands. In this study, weinvestigated As biotransformation in three cyanobacterial species (Microcystis sp. PCC7806, Nostoc sp. PCC7120, andSynechocystis sp. PCC6803). Each accumulated large amounts of As, up to 0.39 g kg21 dry weight, 0.45 g kg21 dry weight,and 0.38 g kg21 dry weight when treated with 100 mM sodium arsenite for 14 d, respectively. Inorganic arsenate and arsenitewere the predominant species, with arsenate making up .80% of total As; methylated arsenicals were detected followingexposure to higher As concentrations. When treated with arsenate for 6 weeks, cells of each cyanobacterium produced volatilearsenicals. The genes encoding the As(III) S-adenosylmethionine methyltransferase (ArsM) were cloned from these threecyanobacteria. When expressed in an As-hypersensitive strain of Escherichia coli, each conferred resistance to arsenite. Two ofthe ArsM homologs (SsArsM from Synechocystis sp. PCC6803 and NsArsM from Nostoc sp. PCC7120) were purified and wereshown to methylate arsenite in vitro with trimethylarsine as the end product. Given that ArsM homologs are widespread incyanobacteria, we propose that they play an important role in As biogeochemistry.

Arsenic (As) is the most ubiquitous toxic environ-mental substance (Smedley and Kinniburgh, 2002).Anthropogenic activities, such as mining, burning offossil fuels, and pesticide application, also introduceAs into the environment (Bissen and Frimmel, 2003a,2003b). Major sources of human As exposure are drink-ing water and the food supply, leading to health prob-lems such as cancer, cerebrovascular disease, diabetesmellitus, and kidney disease (Meliker et al., 2007).Arsenic is first on the Centers for Disease Control’sAgency for Toxic Substances and Disease RegistryComprehensive Environmental Response, Compen-sation, and Liability Act Priority (Superfund) List ofHazardous Chemicals (http://www.atsdr.cdc.gov/cercla/07list.html) because of its ubiquity, health ef-fects, and potential for human exposure. The WorldHealth Organization has called As contamination of

drinking water in Bangladesh the worst environ-mental catastrophe in human history, and its control isbeyond the capability of many countries (Johnstonet al., 2010).

Arsenic biotransformation by microbes may play acritical role in the fate and toxicity of environmentalAs, which is present primarily as arsenate [As(V)] andarsenite [As(III)]. The metabolism of As(V) has beenstudied in many organisms, including mammals,plants, and microorganisms (Bhattacharjee and Rosen,2007). As(III) biotransformation exists in parallel path-ways, including oxidation to As(V) or methylation toproduce organic arsenicals. Oxidation of As(III) to As(V) is an energy-producing reaction that is also be-lieved to be a detoxification process since As(V) is lesstoxic than As(III) (Páez-Espino et al., 2009). In bothprokaryotic and eukaryotic microbes, As(III) methyl-ation is clearly a detoxification mechanism (Qin et al.,2006, 2009). The products include less toxic As species,such as methylarsenate [MAs(V)], dimethylarsenate[DMAs(V) or cacodylate], trimethylarsine oxide[TMAsO(V)], and the final product of the methyl-ation pathway, volatile trimethylarsine [TMAs(III)].Arsenic methylation is proposed to play an importantrole in As cycling among terrestrial, aquatic, and atmo-spheric surroundings (Bhattacharjee and Rosen, 2007;Rensing and Rosen, 2008).

Cyanobacteria are widespread in aquatic environ-ments and soils and are one of the main representatives

1 This work was supported in part by the Natural Science Foun-dation of China (grant no. 21077100), the Ministry of Science andTechnology of China (grant no. 2009DFB90120), and the U.S. Na-tional Institutes of Health (grant no. R37 GM55425 to B.P.R.).

2 These authors contributed equally to the article.* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Yong-Guan Zhu ([email protected]).

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in the microbiota of such sites (Sinha and Hader, 1996;Ladha and Reddy, 2003; Song et al., 2005). Cyanobac-teria are responsible for as much as 50% of earthly pho-tosynthesis and are major contributors to the globaloxygen cycle. Metals can exert a potential negativeinfluence on cyanobacterial photosynthesis, such ascopper in the atmospheric aerosols (Paytan et al.,2009). Moreover, due to their ability to adapt rapidlyto environmental changes and rapid growth rates, cya-nobacteria are often key players in toxic algal bloom-ing in various aquatic environments (Bianchi et al.,2000; Dokulil and Teubner, 2000). Nevertheless, thereis a dearth of studies on the role of freshwater and soilalgae in As biogeochemistry, not to mention the bio-chemical and molecular mechanisms.

Microcystis sp. PCC7806, Nostoc sp. PCC7120, andSynechocystis sp. PCC6803 are typical freshwater cya-nobacteria. All of them are dominant species in theblue algal eruption. However, little is known about Asmetabolism in these prokaryotic blue-green algae. Inthis study, we characterized the patterns and molecu-lar mechanisms of As biotransformation in these threespecies. By rapidly methylating and volatilizing As,these widespread cyanobacteria may be major con-tributors to the global As cycle.

RESULTS

Arsenic Speciation in Cyanobacteria

After 2 weeks of exposure to the indicated concen-trations of As(III), the resulting As species were ana-lyzed byHPLC-inductively coupled plasma (ICP)-massspectrometry (MS; Fig. 1). Only As(III) and As(V) weredetected after exposure to the lower As(III) concentra-tions (10 mM), and As(V) was the predominant intra-cellular species, accounting for 81% to 84% of the total.

When exposed to the higher As(III) concentrations(100 mM), both inorganic arsenicals were found as thedominant species. In addition, about 2% to 5% of thetotal As was in the form of DMAs(V), demonstratingthat this organism is capable of Asmethylation (Fig. 1).Meanwhile, the As species of the control [free As(III)]were determined.

Volatile As from Cyanobacteria

When the three cyanobacteria were treated witheither 0.1 or 0.4 mM As(V) for 6 weeks, 11.0 and 22.1 ng(Microcystis), 16.9 and 37.8 ng (Nostoc), and 16.4 and35.3 ng (Synechocystis), respectively, of volatile arsines,presumably TMAs(III), were trapped. It was notedthat the amount of volatile As increased along withincreasing external As concentrations, accounting for,0.1% of total As in the medium. At an As(V) con-centration of 0.4 mM, production of gaseous As in-creased sharply. In comparison, the amount of As inthe control chemotrap (including the glass tubes, glasswools, and silica gel) was determined (Fig. 2). Thisdemonstrates that the cells have the ability to methylateinorganic As to TMAs(III), the volatile product of themethylation pathway. It is also noted that the produc-tion of volatile As was up to the maximum generatedby Nostoc sp. PCC7120 among three cyanobacteria.

Cloning and Transcriptional Analysis of the arsM Genes

Using the ArsM sequences from the photosyntheticprokaryote Rhodopseudomonas palustris and the eukary-otic photosynthetic alga Cyanidioschyzon, we identifieda homolog in each of the three cyanobacteria (Fig. 3A).Each gene was 969 bp in length and was designated asNsarsM (GenBank accession number HQ891147),MsarsM (GenBank accession number HQ891148), andSsarsM (GenBank accession number HM776638), re-spectively. Each was cloned from total genomic DNAfrom the respective organisms by PCR. Each gene en-codes an ArsM homolog of 323 residues, with a pre-dicted molecular mass of 35.26, 35.26, and 35.27 kD.The three ArsM are closely related to RpArsM (283residues, 29.66 kD; 23.53%, 26.18%, and 25.29%, respec-tively) and CsArsM (400 residues, 43.73 kD; 22.22%,20.84%, and 20.05%, respectively). The sequence sim-ilarities were higher in the putative S-adenosyl Metbinding sites.

To investigate whether exposure to different As con-centrations affected the expression level of cyanobacte-rial arsM genes, semiquantitative reverse transcription(RT)-PCR analyses were carried out in the presence andabsence of 0.1 mM sodium As(III) (Fig. 3B). Total RNAwas extracted from three cyanobacteria. The house-keeping gene rnpB was included in all RT-PCR anal-yses to ensure that equivalent amounts of total RNAwere used in all reactions. In the absence of added As(III), none of the arsM genes were expressed. In con-trast, each was expressed in the presence of 0.1 mM As(III), suggesting that transcription of cyanobacteria

Figure 1. Arsenic speciation in cyanobacteria after exposure to theindicated concentrations of As(III) for 2 weeks, and the products wereanalyzed as described in “Materials and Methods”: black bars, As(III);white bars, As(V); gray bars, DMAs(V). All data are the mean 6 SE(n = 3). DW, Dry weight.

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arsM genes is regulated by external As(III). This isconsistent with the observation that in other bacteriaarsM genes are controlled by ArsR As(III) transcrip-tional repressors (Qin et al., 2006), though the regula-tion of arsM expression in cyanobacteria needs furtherinvestigation.

ArsM Enzymes Detoxify As(III)

To examine whether ArsM enhances As resistance,the arsM genes were cloned into an Escherichia colivector pET28a and expressed in E. coli strain AW3110,which has no orthologous arsM gene and is hypersen-sitive to As(III) resulting from the deletion of chromo-somal arsRBC operon (Carlin et al., 1995). When grownon As(III)-free Luria-Bertani (LB) medium, no differ-ence could be observed between cells bearing emptyvector and expressing ArsM. However, when exposedto 100 mM As(III), cells expressing ArsM grew dramat-ically better than those bearing empty vector (Fig. 4A).In addition, cells bearing vector plasmid pET28a weresensitive to 50 mM As(III). In contrast, cells expressingthe arsM genes could grow in As(III) concentration ashigh as 100 mM (Fig. 4B), demonstrating that the geneproducts confer tolerance to As(III).

As(III) Methylation and Volatilization of ArsM

Two different strains of E. coli, BL21(DE3), an As-tolerant strain with a chromosomal ars operon, andAW3110(DE3), an As-hypersensitive strain in whichthe ars operon was deleted, expressing cyanobacterialarsM biomethylated As(III) primarily to methylatedAs [DMAs(V) and some amount of TMAsO(V)] after12 h of incubation by analyzing the soluble As(III)transformation products in the reaction solution(Fig. 5, A and B). It should be noted that expressionof arsM in E. coli strain BL21 resulted in more volatil-ization than expression in AW3110. In addition, thevolatile product TMAs(III) was trapped by oxidation

Figure 3. Identification of ArsM homolog in cyanobacteria. A, Multiplealignment of ArsM homologs. Ss, Synechocystis sp. PCC6803; Ns,Nostoc sp. PCC7120; Ms, Microcystis sp. PCC7806; Rp, R. palustrisCGA009; Cs,Cyanidioschyzon sp. 5508; Cy, Cyanobium sp. PCC7001.B, Semiquantitative RT-PCR analyses of ArsM expression levels inNostoc,Microcystis, and Synechocystis after exposure to 100 mM As(III)concentrations. Total RNAwas extracted from the three cyanobacteria.rnpB was used as a control.

Figure 2. Volatile As levels (ng) exposed to the indicated concentra-tions of As(V) during a 6-week period. Data are the mean 6 SE (n = 3).

Arsenic Biotransformation by Cyanobacteria

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to TMAsO(V) when expressing arsM genes in the twocell strains (Fig. 5, C and D). Consistent with directtrapping of the gas, more TMAsO(V) was generated byoxidation when arsM was expressed in strain BL21than in AW3110. In particular, NsArsM showed thehighest methylation ability regardless of in whichstrain the gene was expressed. In addition, the productof the methylation reaction was quantified in cells of E.coli AW3110 expressing arsM (Fig. 5E). DMAs(V) wasthe predominant As species in the medium, accountingfor 43% (SsarsM), 90% (MsarsM), and 93% (NsarsM) oftotal As. Moreover, traces of TMAO(V) (, 1%) weredetected with expression of NsarsM. These resultsdemonstrate that heterologous expression of the cya-nobacteria arsM genes in E. coli conferred the ability tomethylate As.

Purification of ArsM and in Vitro Methylation

To elucidate the mechanism of As methylation bythe algal enzymes, NsArsM and SsArsMwere purifiedfrom E. coli BL21. Of the three, NsArsM exhibited the

highest As(III) methylation activity in vitro. PurifiedNsArsMmethylated As(III) to a variety of species (Fig.6B). Little As(III) remained, and the primary productwas DMAs(V), with lesser amounts of MAs(V). Puri-fied SsArsM produced some MAs(III) (Fig. 6B). SinceMAs(III) is the precursor of DMAs(V), this indicatesthat the reaction rate of SsArsM is slower thanNsArsM.TMAs(III) gas could be detected on H2O2-impregnatedfilters by two purified proteins, which oxidize it toTMAsO(V) (Fig. 6C). Less than 2% of the total As wasconverted to TMAs(III). These results demonstratethat the two ArsMs exhibit difference in methylationactivity in vitro, as was observed in vivo results. Ad-ditionally, with both NsArsM and SsArsM, the majorproduct was DMAs(V), indicating that addition of athird methyl group is most likely the rate-limiting stepin the overall reaction.

Methylation was determined with two different pro-tein concentrations or two different incubation timesusing purified NsArsM (Fig. 7). It is noted that moreDMAs(V) was produced at the higher concentration ofNsArsM or the longer reaction time. In addition, smallquantities of TMAsO(V)were detected after 12 h (Fig. 7,B and C).

DISCUSSION

Our results clearly demonstrate that three As-tolerantcyanobacteria not only grow at high concentrations ofthe toxic metalloid but also have the ability to accu-mulate large amounts of As (Fig. 1). These are possiblyrelated to the organisms’ adaption to their environment.Both prokaryotes, such as R. palustris, and eukaryoticalgae, such as Cyanidioschyzon, are widely distributedin aquatic environments and soils (Qin et al., 2006,2009), where soluble inorganic As is ubiquitous. As aconsequence, these photosynthetic microorganismshave evolved mechanisms to detoxify environmentalAs. Here, we showed that As(III) is efficiently detoxi-fied by methylation by these three cyanobacteria. Somealgae, such as Cyanidioschyzon, Chlorella vulgaris, andChlamydomonas reinhardtii have also been shown toaccumulate or be resistant to high levels of As (Kaiseet al., 1999; Suhendrayatna et al., 1999; Qin et al., 2009).Given the high abundance and ubiquity of cyanobacte-ria, our results imply that cyanobacteria may contributesubstantially the biogeochemistry of As, particularlyunder the situation of toxic algal blooming in lakes andponds also heavily contaminated with As, such asLake Yangzonghai in the southwest of China.

In microorganisms and plants, As(III) is detoxifiedby oxidation to relatively less toxic As(V) or formationof organoarsenicals, including methylated arsenicals,arsenosugars, and arsenolipids, and volatile As species(Bentley and Chasteen, 2002; Murray et al., 2003). Ourresults indicate that several of these parallel pathwaysof As(III) detoxification exist in the three cyanobacteria.First, when exposed to As(III), the cells accumulateprimarily As(V) by a process of As(III) oxidation. We

Figure 4. Cyanobacterial arsM genes enhance As resistance in E. colistrain AW3110 (DarsRBC). A, Growth of cells in LB medium without orwith 100 mM As(III). Control, Empty vector. The initial optical intensity(OD600) of cells is 1.5. B, Expression of algal arsM confers tolerance tosodium As(III), bearing vector plasmid pET28a(+) or pET28arsM withthe indicated As(III) concentrations. Vector plasmid pET28a, 50 mM(white triangles), 100 mM (black triangles); pET28a-SsarsM, 50 mM(white circles), 100 mM (black circles); pET28a-NsarsM, 50 mM (whitediamonds), 100 mM (black diamonds); pET28a-MsarsM, 50 mM (whitesquares), 100 mM (black squares). Data are the mean 6 SE (n = 3).

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speculated that this As(V) may be located in the vac-uole. This is similar to the response of the red algaCyanidioschyzon merolae, which oxidizes As(III) ex-tracellularly, takes up the resulting As(V), and thenrereduces it to As(III) for efflux or methylation (Qinet al., 2009). Second, when exposed to high concentra-tions of As up to 0.1 mM, the relatively nontoxic meth-ylated species DMAs(V) was formed as the majorspecies by ArsM methylation, a parallel pathway thatwas first identified in R. palustris and Cyanidioschyzon(Qin et al., 2006, 2009).

Biological production of volatile As compounds isconsidered to be an important part in the global Asbiogeochemical cycling (Michalke and Hensel, 2004).Bacteria, fungi, and alga can convert inorganic As intovolatile compounds for As detoxification (Qin et al.,2006, 2009; Urı́k et al., 2007). In this study, the amount ofvolatile arsenicals depended on the initial As concen-trations in the medium (Fig. 2). This may explain whygaseous TMAs(III), the final detoxification product ofthe ArsM reaction, is produced only when these cya-nobacteria are exposed to high concentrations of As.

Figure 5. In vivo formation of methylated arsenicals. A, Speciation of As in the culture medium by cyanobacterial arsM genesexpressed in E. coli BL21. B, Speciation of As in the culture medium of AW3110 expressing cyanobacterial arsM genes. C,Volatilization of As from cultures of E. coli BL21 expressing cyanobacterial arsM genes. D, Volatilization of As from cells of E. coliAW3110 expressing cyanobacterial arsM genes. Curve 1, Standard; curve 2, Microcystis arsM; curve 3, Nostoc arsM; curve 4,Synechocystis arsM. Cultures were incubated for 12 h in LB medium with 25 mM As(III). E, Speciation of As in the culturemedium. Cultures were grown for 12 h. Soluble As species were determined by HPLC-ICP-MS using an anion-exchange column.cps, Counts per second.





To examine whether the ArsM pathway exists inthese cyanobacteria, genes encoding three ArsM ho-mologs were cloned and expressed in E. coli, wherethey each conferred As(III) resistance and methylatedAs(III) to DMAs(V). Two, NsArsM and SsArsM, werepurified and shown to catalyze methylation of As(III)

to a variety of species in vitro, primarily DMAs(V) andlesser amounts of MAs(III), MAs(V), and TMAs(III)gas, demonstrating that they are genuine ArsM ortho-logs. To date, only two microbial ArsM homologs,RpArsM from the photosynthetic bacterium R. palust-ris and CmArsM from the eukaryotic alga C. merolae,have been characterized. For these organisms, meth-

Figure 6. Biomethylation of As(III) by purified ArsM enzymes. A,Purification of ArsM was tested by SDS-PAGE. B, Soluble As specieswere determined by HPLC-ICP-MS analysis using an anion-exchangecolumn after 12 h of incubation with purified enzyme, as described in“Materials and Methods.” Curve 1, Standard arsenicals; curve 2,starting material; curve 3, 5 mM SsArsM after 12 h at 37�C; curve 4, 5mM NsArsM after 12 h at 37�C. C, Volatile As species trapped on H2O2-impregnated filters and determined by HPLC-ICP-MS analysis using aC18 reverse phase column, as described in “Materials and Methods.”Curve 1, LBmedium after 12 h at 37�Cwithout any ArsM; curve 2, 5 mMSsArsM after 12 h at 37�C; curve 3, 5 mM NsArsM after 12 h at 37�C.

Figure 7. Identification of methylated As by purified NsArsM. A,Soluble As species were analyzed after 4 h of incubation with purifiedNsArsM. Curve 1, Starting material; curve 2, 1 mM NsArsM after 4 h at37�C; curve 3, 2 mM NsArsM after 4 h at 37�C. B, As species weredetermined after different incubation time with purified NsArsM. Curve1, Starting material; curve 2, 5 mM NsArsM after 4 h at 37�C; curve 3,5 mMNsArsM after 12 h at 37�C. C, The products of methylation by 5mMNsArsM after 4 or 12 h were examined. cps, Counts per second.

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ylation of As(III) by ArsM was demonstrated to be adetoxification mechanism, and we have proposed thatmethylation and volatilization by photosynthetic mi-crobes are significant contributors to global biogeo-chemical cycling of As (Mukhopadhyay et al., 2002).Moreover, the fact that both in vivo and in vitro resultsshowed that the ArsM fromNostoc exhibits the highestmethylation capacity implies that there is opportunityin improving methylation in cyanobacteria by geneticengineering.In conclusion, based on our data, we speculate that

the abilities of three cyanobacteria to accumulate andmethylate As could be useful for phytoremediationand phytofiltration of As-contaminated water or soil.Advantages of using these environmentally friendlycyanobacteria include rapid growth plus high levels ofaccumulation and biotransformation of As to less toxicforms. In addition, given the high abundance of cya-nobacteria in freshwater environment and soils, thesephotosynthetic prokaryotic microbes may play a sig-nificant role in the biogeochemical cycling of As inaquatic systems.

MATERIALS AND METHODS

Organism and Cultivation

Microcystis sp. PCC7806, Nostoc sp. PCC7120, and Synechocystis sp. PCC6803

were obtained from the Institute ofHydrobiology, Chinese Academyof Sciences,

Wuhan, China, and were grown in BG-11 medium (Rippka et al., 1979). Ex-

periments were carried out in a controlled-environment growth chamber under

the following conditions: 16-h light period d21 with a light intensity of approxi-

mately 280 mmol m22 s21, 25�C:20�C day:night temperatures, and 60% relativehumidity.

Arsenic Speciation in Cyanobacteria

Arsenic was supplied as NaAsO2 at the indicated concentrations (10 and

100 mM) to investigate As transformation in cyanobacteria. Three replicates

were used in each set. After exposure for up to 14 d, the cultures were

harvested and rinsed with deionized water and ice-cold phosphate buffer

[1 mM K2HPO4, 0.5 mM Ca(NO3)2, and 5 mM MES, pH 6.0]. Methylation of

As(III) was assayed both in vivo in cultures of cyanobacteria. Approximately

0.02 g (dry weight) of algal samples were put in 50-mL centrifuge tubes

and treated with 10 mL of 1% HNO3 overnight. The samples were heated in a

microwave accelerated reaction system (CEM Microwave Technology), and

As speciation in extracts was determined by HPLC-ICP-MS (7500a; Agilent

Technologies; Zhu et al., 2008). Chromatographic columns were obtained from

Hamilton and consisted of a precolumn (11.2 mm; 12–20 mm) and a PRP-X100

10-mm anion-exchange column (2503 4.1 mm). The mobile phase consisted of10 mM diammonium hydrogen phosphate [(NH4)2HPO4] and 10 mM ammo-

nium nitrate [NH4NO3] adjusted to pH 6.2 using nitric acid.

TMAs(III) Chemotrapping

Volatile TMAs(III) released from cyanobacteria was trapped as reported

(Mestrot et al., 2009). The preparation of the trap tube was as follows: silica gel

(0.5-mm diameter) was immersed in 5% HNO3 overnight, washed with

Millipore water, impregnated with 10% AgNO3 solution (w/v) overnight, and

dried in an oven at 70�C. Subsequently, the silica gel was loaded into a 3-mLburette and held in by a small quantity of quartz wool at each end. The tube

was covered with aluminum foil to avoid photodecomposition of silver

nitrate. Avessel was designed for volatile arsine trap consisting of a triangular

flask (250 mL) and a glass joint with inlet and outlet of gas. The trap tube was

connected to the one of the outlet, and a super silent adjustable air pump

(ACO-9601; 2-W power) was linked to the inlet to supply oxygen for the

growth of cyanobacterium. The trapping period lasted for 6 weeks with

100 mL cyanobacterium culture. Then, 5 mL of 1% (v/v) hot boiling nitric acid

was used to elute the collected As in traps. Total As was analyzed by ICP-MS.

Cloning of the arsM Gene and Expression inEscherichia coli

DNA were extracted from the cultures of each cyanobacterium using a

Genomic-Tip 20/G and buffer set following the company’s protocol (Qiagen).

Plasmid DNA was isolated with a QIA Prep Spin Miniprep kit (Qiagen). For

construction of plasmids for arsM gene expression in E. coli, three 0.96-kb

fragments containing the ATG start codon and the TGA stop codon were

amplified from genomic DNA using three pairs of primers: forward primer

5#-CGCCATATGACTTATCTGGAAGCTAC-3# (NdeI site underlined) and re-verse primer 5#-CCGCTCGAGTTAACAGCAA CCGCCCCCAT-3#(XhoI siteunderlined) for MsarsM; forward primer 5#-CGCCATATGACCTATTTAGA-AACAGC-3# and reverse primer 5#-CCGCTCGAGTTAACAGCAACCACC-ACCG-3# for NsarsM; and forward primer 5#-GGGAATTCCATATGACC-TATCTGCAAACCACCG-3# (NdeI site underlined) and reverse primer5#-CCGCTCGAGTCAACAACAG CCGCCACCGT (XhoI site underlined) forSsarsM. PCR (94�C for 5 min, 94�C for 30 s, 60�C for 30 s, and 72�C for 45 s,30 cycles) were performed with LA Taq DNA polymerase (Takara Bio). The

PCR product was cloned into PMD19Tsimple vector (Takara Bio) to create the

plasmid PMD19T-arsM. After digestion with NdeI and XhoI, the 0.96-kb

fragments were inserted into pET28a(+) to create plasmids of pET28a-arsM, in

which the arsM genes were controlled by the T7 promoter. As a consequence,

the N-terminal His-tagged ArsMs were established composed of 327 amino

acid residues. All of the sequences were verified by DNA sequencing.

Biomethylation of As(III) was measured in E. coli strains AW3110(DE3) and

BL21(DE3) expressing the cyanobacterial genes cultured in LB medium with

25 mM As(III) with 0.3 mM isopropylthio-b-galactoside at 37�C for 12 h. Thesoluble As(III) transformation products generated were analyzed by HPLC-

ICP-MS. TMAs(III) was trapped with three 2-cm nitrocellulose membrane

filters (Schleicher and Schuell) in the cap. The filters were impregnated with

0.20 mL of 6% H202 to oxidize TMAs(III) to TMAsO(V). After the reaction, the

filters were digested with 0.2 mL of 70% HNO3 at 70�C for 20 min, which wasdiluted 20-fold and analyzed by HPLC-ICP-MS (Qin et al., 2006).

Purification of ArsM and in Vitro Methylation Assays

Proteins were expressed in E. coli strain BL21(DE3) bearing plasmid pET28a-

arsM and purified by Ni(II)-NTA chromatography by elution with a buffer

consisting of 50 mM MOPS, pH 7.5, containing 20% (w/v) glycerol, 0.5 M NaCl,

200 mM imidazole, and 10 mM 2-mercaptoethanol. Fractions containing SsArsM

were concentrated by centrifugation using a 30-kD cutoff Amicon Ultrafilter

(Millipore). ArsMs were identified by SDS-PAGE. Fractions containing purified

ArsMs were pooled and concentrated. Purified proteins were stored at 270�Cuntil analysis. Protein concentrations were estimated by themethod of Bradford

(1976) using bovine serum albumin (Sigma-Aldrich) as a standard.

As(III) methylation with purified NsArsM and SsArsM was performed in

a buffer consisting of 50 mM K2HPO2, pH 7.4, containing 8 mM GSH, 0.3 mM

S-adenosyl-Met chloride, 10 mM As(III), and either 5 mM NsArsM or SsArsM

at 37�C for 12 h (Qin et al., 2006). The samples were filtered through 3-kDcutoff Amicon Ultrafilters (Millipore). The As species were analyzed by

HPLC-ICP-MS with either a reverse-phase C18 column (Jupiter 300) 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 (Cui et al., 2004) or with an anion-exchange column (PRP-

X100; Hamilton) 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. Methylation reactions (4 mL)

were performed in capped 20-mL vials, and TMAs(III) was trapped by H2O2oxidation as described above.

ACKNOWLEDGMENTS

We thank Miroslav Styblo (University of North Carolina, Chapel Hill, NC)

for MAs(III) and Timothy McDermott (Montana State University, Bozeman,

MT) for TMAsO(V).

Received April 24, 2011; accepted May 5, 2011; published May 11, 2011.





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