FEMS Microbiology Letters, 362, 2015, 1–8 doi: 10.1093/femsle/fnu003 Advance Access Publication Date: 4 December 2014 Research Letter
R E S E A R C H L E T T E R – Physiology & Biochemistry
Identification and catalytic residues of the arsenite methyltransferase from a sulfate-reducing bacterium, Clostridium sp. BXM Pei-Pei Wang, Peng Bao and Guo-Xin Sun∗ State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China ∗ Corresponding author: State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China. Tel: 86-10-62849328; E-mail: [email protected] One sentence summary: Arsenite methyltransferase is responsible for As methylation and volatilization in sulfate-reducing bacterium. Editor: Robert Gunsalus
ABSTRACT Arsenic methylation is an important process frequently occurring in anaerobic environments. Anaerobic microorganisms have been implicated as the major contributors for As methylation. However, very little information is available regarding the enzymatic mechanism of As methylation by anaerobes. In this study, one novel sulfate-reducing bacterium isolate, Clostridium sp. BXM, which was isolated from a paddy soil in our laboratory, was demonstrated to have the ability of methylating As. One putative arsenite S-Adenosyl-Methionine methyltransferase (ArsM) gene, CsarsM was cloned from Clostridium sp. BXM. Heterologous expression of CsarsM conferred As resistance and the ability of methylating As to an As-sensitive strain of Escherichia coli. Purified methyltransferase CsArsM catalyzed the formation of methylated products from arsenite, further confirming its function of As methylation. Site-directed mutagenesis studies demonstrated that three conserved cysteine residues at positions 65, 153 and 203 in CsArsM are necessary for arsenite methylation, but only Cysteine 153 and Cysteine 203 are required for the methylation of monomethylarsenic to dimethylarsenic. These results provided the characterization of arsenic methyltransferase from anaerobic sulfate-reducing bacterium. Given that sulfate-reducing bacteria are ubiquitous in various wetlands including paddy soils, enzymatic methylation mediated by these anaerobes is proposed to contribute to the arsenic biogeochemical cycling. Key words: arsenic; methylation; sulfate-reducing bacterium; methyltransferase; conserved cysteine
INTRODUCTION Arsenic (As) is a ubiquitous contaminant in the environment (Duker, Carranza and Hale 2005). Anaerobic environments, such as sediments and wetlands, usually act as sinks or secondary sources of As due to natural and anthropogenic activities. Some reducing marine sediments might accumulate 3 mg g−1 As (Mandal and Suzuki 2002). The paddy soil system often accumulated relatively high concentration of As, particularly in south
Asia (Meharg and Rahman 2003). It was estimated that about 1360 tons of As are yearly added to arable soils in Bangladesh (Roberts et al., 2010). It has been well established that anaerobic environments play an important role in the transformation, mobilization and biogeochemical cycling of As (Brannon 1987; Ahmann et al., 1997; Bauer, Fulda and Blodau 2008; Roberts et al., 2010; Vriens et al., 2013). Arsenic methylation as an important process modulating As toxicity and biogeochemical cycling is
Received: 28 July 2014; Accepted: 28 October 2014 C FEMS 2014. All rights reserved. For permissions, please e-mail: [email protected]
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more prevalent under anaerobic conditions than aerobic conditions. Arsenic methylation and volatilization have been observed in many paddy soils (Mestrot et al., 2009, 2011; Huang et al., 2012; Jia et al., 2012). Anaerobic microorganisms inhabited in these ecosystems have been regarded as major contributors for As transformation including methylation (Bright et al., 1994). It has been reported that methylated As species in rice plants originated from soil microorganisms (Lomax et al., 2012; Jia et al., 2013). Several microbial isolates and a microbial coenzyme called methylcobalamin are capable of converting inorganic As to methylated forms (Zakharyan and Aposhian 1999; Qin et al., 2006, 2009; Thomas et al., 2011; Yin et al., 2011; Ye et al., 2014), but the identification of the microorganisms that are actually responsible for inorganic As methylation in anaerobic environments has rarely been clearly demonstrated (Lomax et al., 2012; Jia et al., 2013; Zhao et al., 2013). In the past few years, it has been identified that arsMs are the key microbial functional genes responsible for As methylation (Qin et al., 2006; Ye et al., 2012). However, these studies have mainly focused on aerobic bacteria or eukaryote, little attention was paid to anaerobic microorganisms. The identification of As-methylating microorganisms from anaerobic environments and their functional genes are of great importance in As biogeochemistry. Sulfate-reducing bacteria (SRB) are anaerobic microorganisms widely distributed in the environment, including paddy soils, sea water, thermal springs, sediments and human guts (Wakao and Furusaka 1973; Leloup et al., 2007; Liu et al., 2009; Rey et al., 2013). They are involved in numerous reactions of metal(loid)s (e.g. metal reduction and precipitation) (Lloyd et al., 1998, 2001) and often proposed as strategic microbes to treat water contaminated with a range of metals (Chuichulcherm et al., 2001; Teclu et al., 2008). Numerous reports have showed that SRB is the important methylator of mercury in natural sediments and soils (Compeau and Bartha 1985; King et al., 2000; Jay et al., 2002; Ekstrom, Morel and Benoit 2003), but it is not clear if SRB is a significant As methylator so far. In this study, SRB Clostridium sp. BXM previously isolated from a paddy soil in our laboratory was chosen as anaerobic sedimentary microorganism to study As methylation (Bao et al., 2012). Our main objectives were (1) to identity As methylation process in anaerobic microorganism Clostridium sp. BXM, (2) to provide insight into the enzymatic mechanism for As methylation in anaerobic SRB and (3) to identify catalytic residues of S-Adenosyl-Methionine methyltransferase (ArsM) enzyme and their function in As methylation.
MATERIALS AND METHODS Organisms and cultivations Clostridium sp. BXM was anaerobically grown in the medium (pH 7.2) contained (g l−1 ): MgSO4 r7H2 O 0.5, CaCl2 r2H2 O 0.1, Na2 SO4 0.75, NH4 Cl 0.25, KH2 PO4 0.3, yeast extract 1.0, vitamin C 0.2, Na2 S r9H2 O 0.36, sodium lactate 3.5, at 30◦ C in the dark. Standard anaerobic culturing techniques were used throughout the experiments (Widdel and Bak 1992; Bao et al., 2012). The medium was boiled and cooled to room temperature under an atmosphere of highly pure N2 gas. Anaerobic medium was dispensed into acid-cleaned serum bottles. Each serum bottle was sealed with a butyl rubber stopper, covered with aluminum foil and then autoclaved at 121◦ C for 20 min. During incubation, the cultures were brieffiy shaken by hand once a day. Escherichia coli strains were grown aerobically at 37◦ C in LB medium, supplemented with 50 μg ml−1 kanamycin/ampicillin,
or 0.3 mM IPTG as required. Strain DH5α (Promega) was used for plasmid construction and replication. Strain AW3110(DE3) (arsRBC, ArsR-repressor, ArsB-As(III) efflux pump, ArsC-As(V) reductase) (Carlin et al., 1995) was used for the expression and functional verification of arsM gene. Strain BL21(DE3) was used for protein purification.
Arsenic methylation by Clostridium sp. BXM Anaerobic medium in serum bottle was supplemented by 5.3 μM sodium arsenite (NaAsO2 ) or sodium arsenate (Na3 AsO4 ). The strain of Clostridium sp. BXM was inoculated into the medium with 2% (v/v) inoculum. After the growth of 20 days, the culture was centrifuged by 8000 rpm. The supernatant was filtrated by 0.45 μm disposable filter and oxidized by adding 10% (v/v) H2 O2 . All samples were kept in the refrigerator at 4◦ C until analysis. Every experimental group was assayed for three replicates.
Cloning of the arsM gene from Clostridium sp. BXM and its expression in E. coli Genomic DNA was isolated from the culture of Clostridium sp. BXM by using Genomic-tip 20/G and Buffer Set (Qiagen). Plasmid DNA was extracted with a QIAPrep Spin Miniprep Kit (Qiagen). To express the arsM from Clostridium. sp. BXM in E. coli, a 813 bp fragment containing the ATG start codon and excluding the TGA stop codon was PCR amplified from the genomic DNA of Clostridium sp. BXM by using the forward primer: 5 -CGGAATTCATGGATAATATTCGAGAGGG-3 (EcoRI restriction endonuclease site underlined) and the reverse primer: 5 -CCCTCGAGTGCCGGCTTGGCTGCCCGAAT-3 (XhoI restriction endonuclease site underlined). PCR (94◦ C for 5 min, 94◦ C for 30 s, 58◦ C for 30 s and 72◦ C for 1 min, 30 cycles) was performed with LA Taq DNA polymerase (Takara Bio). The PCR product was cloned into PMD19T simple vector (Takara Bio) to create the plasmid PMD19T-arsM. The sequence was verified by DNA sequencing. Then, after digestion with EcoRI and XhoI, arsM fragment was again inserted into pET28a vector to create the plasmid pET28a-arsM, in which arsM gene was controlled by T7 promoter. The sequence was still verified by DNA sequencing. As a consequence, the His-tagged ArsM was established composed of 314 amino acid residues, with a predicted molecular mass of 33.95 kDa. Plasmid pET28a or pET28a-arsM was transformed into E. coli strain AW3110(DE3), which was As-hypersensitive. Transformed cells were grown in LB medium containing 50 μg ml−1 kanamycin overnight at 37◦ C, and then diluted 50-fold into fresh LB medium containing 50 μg ml−1 kanamycin, 0.3 mM IPTG and a series of concentrations of As(III) (10, 20, 50 μM). After a growth of 20 h at 37◦ C, the culture was centrifuged at 8000 rpm. The supernatant was filtrated by 0.45 μm disposable filters and oxidized by 10% (v/v) H2 O2 . All the samples were kept in the refrigerator at 4◦ C until analysis. Every experimental group was assayed for three replicates. Single colonies of E. coli AW3110 expressing pET28a or pET28a-arsM were inoculated into LB medium (5 ml) contained 50 μg ml−1 kanamycin and cultured at 37◦ C overnight. Late exponential phase cells were diluted 100-fold into fresh LB medium (50 ml) containing 50 μg ml−1 kanamycin, 0.3 mM IPTG and a series of concentrations of As(III) (0, 20, 50, 100 μM). Cellular growths were monitored as absorbance at 600 nm via UV-vis spectrophotometer (New Century, Model T6) and continued until the cultures reached stationary phase.
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Arsenic volatilization by E. coli AW3110 expressing arsM Volatile As produced by E. coli expressing arsM was chemtrapped as described (Huang et al., 2012). Escherichia coli AW3110 expressing either pET28a-arsM or pET28a were cultured in triangular flasks (250 ml volume, contained 100 ml LB medium with a series of concentrations of As(III)). Every flask was capped with a T-junction stopper having a gas inlet and a gas outlet. The inlet was linked to a super silent adjustable air pump (ACO-9601, 2W power), to pump filtered air at a gas flow rate of 40 ml min−1 for refreshing the headspace of flask. The outlet of T-junction stopper was connected to a glass trapping tube filled with AgNO3 impregnated silica gel beads. After 48 h growth of E. coli, the total As absorbed by the trapping tube was eluted by 1% (v/v) HNO3 (5 ml) in a microwave digestion system (Mars II, CEM, US). The eluted solution was centrifuged at 6000 rpm. The supernatant was separated and filtrated by 0.45 μm disposable filter. The quantity of volatile As was tested by ICP-MS and the As species was analyzed by HPLC-ICP-MS.
Purification of ArsM enzyme and As(III) methylation by purified enzyme in vitro The strain E. coli BL21(DE3) expressing pET28-arsM was grown at 37◦ C in LB medium (200 ml) to an A600 of 0.4–0.6, at which point 0.3 mM IPTG was added to induce expression of His-tagged ArsM. After the 8 h growth, E. coli was harvested by centrifugation (8000 rpm.) at 4◦ C for 20 min. The wet cells were suspended in 30 ml of lysis buffer (buffer A: 50 mM NaH2 PO4 , 300 mM NaCl, 10 mM imidazole, pH 8.0) and lysed in ultrasonic cell disruption system (JY92–2D) at a program as follows: working for 5 s followed a rest for 5 s, it continued for 20 min at 4◦ C. Membranes and unbroken cells were removed by centrifugation at 8000 rpm for 30 min. The supernatant was separated, filtrated by 0.45 μm disposable sterile filter and then loaded onto a Ni(II)-NTA column (Qiagen, 5 ml) pre-equilibrated with buffer A. The column was then washed with 200 ml of wash buffer (buffer B: 50 mM NaH2 PO4 , 300 mM NaCl, 20 mM imidazole, pH 8.0) and finally eluted with 5 ml of elution buffer (buffer C: 50 mM NaH2 PO4 , 300 mM NaCl, 250 mM imidazole, pH 8.0) to gain the purified ArsM protein. Solution containing ArsM was removed to a 10 kDa ultrafiltration centrifuge tube (Sartorius) and concentrated by centrifugation at 8000 rpm for 1 h. Finally, the buffer C bearing ArsM protein was replaced by MOPS buffer (50 mM MOPS and 150 mM KCl, pH 7.4) using Micro Bio-SpinTM Chromatography Column (Bio-Rad). ArsM was identified by sodium-dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentration was estimated by the method of Bradford (Bio-Rad Protein Assay) (Bradford 1976), using BSA (Sigma) as a standard. In vitro assay of As methylation was performed in the MOPS buffer containing a certain concentration of ArsM protein, 8 mM GSH, 0.5 mM AdoMet and a series of concentrations of As(III) (5, 10, 20 μM). The reactions were kept in water bath at 37◦ C for the different time (0.5, 1.5, 3, 4.5, 6, 7.5, 20 h). The reactions were terminated by boiling for 5 min in the end. The reaction solutions were oxidized by 10% (v/v) H2 O2 overnight, filtered by 0.45 μm filters. After an appropriate dilution, all samples were analyzed for As species by HPLC-ICP-MS.
Preparation of CsArsM mutants Substitutions of Cys65, Cys153 and Cys203 of the CsArsM for serine were generated by site-directed mutagenesis through over-
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lapping extension PCR with the plasmid pET28a-arsM as the DNA template. The primers used for the site-directed mutagenesis are listed in Table S1 (Supporting Information). PCR products were double digested by EcoRI and XhoI, and cloned into the prepared EcoRI/XhoI-digested pET28a vector. All of the sequences of the arsM mutants were verified by DNA sequencing to ensure that no errors had been introduced during amplification. Escherichia coli BL21(DE3) was transformed using vectors carrying different mutations of arsM gene. Protein expression, purification, identification and concentration determination were performed as described above. Three mutants of ArsM (C65S, C153S, C203S) were constructed.
Arsenic speciation analysis Arsenic speciation was analyzed by HPLC-ICP-MS (7500a, Agilent Technologies) as described (Zhu et al., 2008). Chromatographic columns were obtained from Hamilton and consisted of a precolumn (11.2 mm, 12–20 μm) and a PRP-X100 10 μm anionexchange column (250 × 4.1 mm). The mobile phase consisted of 10 mM diammonium hydrogen phosphate [(NH4 )2 HPO4 ] and 10 mM ammonium nitrate [NH4 NO3 ], adjusted to pH 6.2. The mobile phase was pumped through the column at a flow rate of 1.0 ml min−1 .
RESULTS Arsenic methylation by SRB Clostridium sp. BXM When Clostridium sp. BXM was grown in the medium containing arsenite [As(III)] or arsenate [As(V)] for 20 days, monomethylarsenic (MMAs) and dimethylarsenic (DMAs) were detected (Fig. 1). About 9% of the total As in the medium has been methylated to MMAs as well as about 1% to DMAs (Fig. S1, Supporting Information). No methylated As was detected in the control (no bacterial inoculation). This result suggested that Clostridium sp. BXM have the capability of methylating As.
Cloning of the arsM gene from Clostridium sp. BXM By homologous comparison with the As(III) methyltransferases previously reported (Lin et al., 2002; Qin et al., 2006, 2009; Yin et al., 2011), a putative arsM gene (813 bp) was cloned from the genome of Clostridium sp. BXM, and designated as CsarsM. CsArsM protein encoded by CsarsM gene contained 270 residues, with a predicted molecular mass of 29.06 kDa. Comparative study based on protein sequences showed that CsArsM had the highest similarity with the ArsM from anaerobic bacterium Rhodopseudomonas palustris CGA009 (RpArsM) (53% identity and 84% similarity) (Qin et al., 2006). With those aerobic cyanobacterial ArsMs (Yin et al., 2011), CsArsM shared lower similarity (average 21% identity and 57% similarity). In addition, CsArsM also exhibited significant, but lower similarity to mammalian AS3MT methyltransferase (21% identity and 53% similarity) (Lin et al., 2002; Song et al., 2011), and to the ArsM of eukaryotic alga Cyanidioschyzon sp. isolate 5508 (20% identity and 51% similarity) (Qin et al., 2009).
CsArsM enzyme detoxified As(III) in vivo CsarsM gene was expressed in an As-sensitive E. coli AW3110 (arsRBC), which had no orthologous arsM gene. Expression of arsM clearly conferred the strain AW3110 with the resistance to As(III). Cells expressing CsarsM gene grew much better than that bearing empty plasmid pET28a when exposed to 50 μM As(III)
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Figure 2. The growth curves of E. coli AW3110 (arsRBC) expressing CsarsM or empty vector when exposed to different concentrations of NaAsO2 . Escherichia coli bearing empty vector pET28a or pET28a-arsM was cultured in LB medium with the indicated concentrations of NaAsO2 , 50 μg ml−1 kanamycin and 0.3 mM IPTG at 37◦ C. Cellular growths were monitored by spectrophotometer at absorbance of 600 nm (A600) at the indicated time points. The error bars indicate the standard errors of three triplicates.
Figure 1. Arsenic transformation by Clostridium sp. BXM. Clostridium sp. BXM was cultured with 5.3 μM As(III) or As(V) for 20 days. The medium without bacteria inoculation was as control. The As species in the medium were analyzed as described in ‘Experimental procedures’.
(Fig. 2). Although all bacteria exhibited a weak growth under 100 μM As(III), cells expressing CsarsM gene showed a little better growth than that bearing empty vector (Fig. 2). ArsM enzyme as the product of arsM gene maybe metabolize cellular As(III) to less toxic methylated products for facilitating growth. In other words, ArsM enzyme reduced the As toxicity in the cells, consistent with the idea that As methyltransferase has an important physiological role in detoxifying As.
As(III) methylation and volatilization by E. coli AW3110 expressing arsM DMAs and trimethylarsine oxide (TMAsO) except MMAs were observed in the medium of E. coli expressing CsarsM gene by comparison with the control (Fig. 3A and B). This result indicated that the enzyme encoded by CsarsM gene is indeed responsible for As(III) methylation. The time-course of As methylation by the strain AW3110 expressing CsarsM showed that TMAsO and DMAs have accumulated in the medium after 3 h incubation and increased with the culture time increasing, concomitant with a decrease of inorganic As (Fig. 3B). No detectable MMAs was observed throughout the experiment (within 20 h). Since the expression of CsarsM gene was controlled by T7 promoter, as well as with the induction of isopropyl β-D-thiogalactoside (IPTG), abundant CsArsM protein may be produced and could quickly convert MMAs to DMAs in the recombinant E. coli. About 10% of total As was transformed into DMAs and another 10% into TMAsO by E. coli in the presence of 10 μM As(III) (Fig. 3A). The total As in the medium of E. coli expressing CsarsM is a bit lower
than corresponding control (empty vector), probably attributed to As volatilization in the former. Escherichia coli AW3110 expressing CsarsM gene effectively volatilized As when exposed to different As(III) concentrations (Fig. 3C). The major gaseous product was TMAs which was oxidized and identified as TMAsO, together with tiny amount of dimethylarsine (DMAsH) detectable as oxidized DMAs (Fig. S2, Supporting Information). The amount of As volatilization in 100 μM As(III) was more than that in 50 μM As(III) (Fig. 3C), though the cellular growth in the former was much weaker than that in the latter (Fig. 2).
Methylation of As(III) by purified ArsM enzyme in vitro To further elucidate the function of CsarsM gene in As methylation, corresponding CsArsM protein was purified from recombinant E. coli cytosol for its As methylation activity analysis (Fig. 4A). Cofactor glutathione (GSH) as a necessary component was used to form As–GSH complex, which was optimal substrate for ArsM enzyme. S-Adenosyl-Methionine (AdoMet) was used as the methyl donor in ArsM-catalyzed As(III) methylation reaction in vitro. CsArsM enzyme converted As(III) to MMAs after 0.5 h, with small amount of DMAs appearing at 1.5 h (Fig. 4B). The concentrations of DMAs and MMAs kept increasing during the experiment process, concomitant with the decrease of inorganic As (Fig. 4B). At 20 h, DMAs became the dominant organic As species. Only little MMAs have been produced within 20 h compared to DMAs. TMAsO had not appeared until the time got to 20 h. About 45% of the total As was converted to the organic As by CsArsM in the presence of 5 μM As(III) (Fig. 4C). The total amount of methylated As increased with the increasing As(III) concentrations in the reaction system (Fig. 4C), although the percentage of that indeed decreased. In the presence of 20 μM As(III), about 23% of inorganic As was methylated by CsArsM (Fig. 4C). In the absence of GSH or AdoMet, no methylated species were observed (data not shown).
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Figure 3. Arsenic methylation and volatilization by E. coli expressing pET28aarsM. (A) As(III) methylation by E. coli AW3110 (arsRBC) expressing CsarsM. Escherichia coli expressing empty pET28a or pET28a-arsM was incubated in LB medium with the indicated concentrations of As(III) (10, 20, 50 μM), 50 μg ml−1 kanamycin and 0.3 mM IPTG at 37◦ C for 20 h. The As species in the supernatants were analyzed by HPLC-ICP-MS as described in ‘Experimental procedures’. C, control, which was E. coli expressing empty vector pET28a; arsM, E. coli expressing CsarsM. The error bars indicate the standard errors of three triplicates. (B) Time dynamics of As(III) methylation by E. coli AW3110 (arsRBC) expressing CsarsM. The strain was incubated in LB medium with 20 μM As(III), 50 μg ml−1 kanamycin and 0.3 mM IPTG at 37◦ C. At the indicated time points (3, 6, 9, 13, 16, 20 h), 1 ml samples of the bacterial culture were taken and centrifuged at 8000 rpm. The supernatants were appropriately diluted and analyzed for As species by HPLCICP-MS. (C) Volatilization of As by E. coli AW3110 (arsRBC) expressing CsarsM. E. coli with CsarsM was grown in LB medium with a series of As(III) concentrations, 50 μg ml−1 kanamycin and 0.3 mM IPTG at 37◦ C for 48 h. Volatile As produced was chemo-trapped as described in ‘Experimental procedures’ and the total quantities of volatile As were analyzed by ICP-MS. The error bars indicate the standard errors of three triplicates.
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Figure 4. Purification of CsArsM protein and As(III) methylation by purified CsArsM. (A) SDS-PAGE showing the purity of the soluble CsArsM. Lane 1, protein standards; Lane 2, cell lysis solution; Lane 3, collection of the washing buffer (20 mM imidazole) outflowing from Ni(II)-NTA column; Lane 4, collection of the washing buffer (50 mM imidazole) outflowing from Ni(II)-NTA column; Lane 5, concentrated His-tagged CsArsM protein. (B) Time dynamics of As(III) methylation by purified CsArsM enzyme. Each assay contained 10 μM As(III), 0.5 mM AdoMet, 8 mM GSH and 4 μM purified CsArsM in 500 μl MOPS buffer (pH 7.4). The reactions were kept at 37◦ C. Arsenic species of the samples were analyzed at the indicated time points by HPLC-ICP-MS as described in ‘Experimental procedures’. The error bars indicate the standard errors of three triplicates. (C) The final products of As(III) methylation by purified CsArsM enzyme at 37◦ C for 20 h. Each assay contained the indicated concentrations of As(III) (5, 10 or 20 μM), 0.5 mM AdoMet, 8 mM GSH and 10 μM purified CsArsM in 500 μl MOPS buffer (pH 7.4). Arsenic species were analyzed by HPLC-ICP-MS. The error bars indicate the standard errors of three triplicates.
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DISCUSSION
Figure 5. Methylation of As(III) or MMAs by wild-type or mutant CsArsM enzymes. Methylation of As(III) or MMAs was tested in a reaction system (pH 7.4) with 10 μM As(III) (A) or MMAs (B), 8 mM GSH, 0.5 mM AdoMet and 2 μM ArsM enzyme. The reactions were kept at 37◦ C for 20 h. The As species of the samples were analyzed by HPLC-ICP-MS.
Conserved cysteine (Cys) residues Cys65, Cys153 and Cys203 of CsArsM All ArsM proteins identified to date have three conserved Cys residues (Thomas et al., 2007; Hamdi et al., 2012; Ye et al., 2012), at positions 72, 174 and 224 in CmArsM of eukaryotic alga Cyanidioschyzon sp. 5508 (Marapakala, Qin and Rosen 2012), which were responsible for As methylation. The conserved Cys residues in CsArsM were Cys65, Cys153 and Cys203. To elucidate the function of three conserved Cys in As methylation, Cys65, Cys153 and Cys203 in CsArsM were substituted for serines, respectively, creating the mutants of C65S, C153S and C203S. All three mutants lost their activities of As(III) methylation completely (Fig. 5A). In contrast, methylation of MMAs was completed by the C65S mutant but not by the C153S or C203S (Fig. 5B). These results suggest that the three conserved Cys (Cys65, Cys153 and Cys203) are required for the first methylation step (from As(III) to MMAs), while only Cys153 and Cys203 are required for the second methylation step (from MMAs to DMAs) in the Challenger’s pathway (Challenger 1945; Cullen and Reimer 1989). Note that Cys29, Cys30 and Cys262 in CsArsM were not conserved in other ArsM orthologues and their single mutations did not affect enzymatic activity of As methylation (data not shown).
Due to the critical role of microorganisms in As biogeochemical cycling in wetland and water environments, it is important to understand As transformation by anaerobic bacteria (Tsai, Singh and Chen 2009). The methylation of As is one of the key processes governing the fate of As in the environment, and thus detailed understanding of the mechanism of As methyltransferase is crucial. This study provided the insight into a newly identified ArsM from anaerobic SRB, which play crucial roles in As transformation of anaerobic environments (Kirk et al., 2004; Oremland, Stolz and Hollibaugh 2004). CsArsM enzyme encoded by CsarsM gene cloned from SRB Clostridium sp. BXM conferred the ability of methylating and volatilizing As to As-sensitive strain of E. coli, concomitant with an increase of cellular tolerance to As. These results suggest that ArsM-mediated As methylation in SRB is a mechanism for relieving cellular toxicity of As. By the site-directed mutagenesis studies, we confirmed that three residues of CsArsM, Cys65, Cys153 and Cys203 are required for As(III) methylation, while only Cys153 and Cys203 are necessary for MMAs methylation. These Cys are conserved in all ArsM and AS3MT proteins identified to date (Thomas et al., 2007; Ye et al., 2011, 2012). For example, they are homologous to Cys72, Cys174 and Cys224 of CmArsM from eukaryotic alga Cyanidioschyzon sp. 5508 (Marapakala et al., 2012). It was reported that the substitution of Cys72, Cys174 or Cys224 led to loss of As(III) methylation, while the substitution of Cys174 or Cys224 abolished the methylation of monomethylarsenite [MMAs(III)]. These Cys affected the binding of As(III) or MMAs(III) to CmArsM enzyme during the methylation process. Similarly, the ArsM from Danio rerio has been identiffied to possess two conserved residues, Cys160 and Cys210, which were suggested to be related to As(III) binding (Hamdi et al., 2012). To the recombinant human AS3MT enzyme (Geng et al., 2009), Cys72, Cys156 and Cys206 have been proved to be functionally conserved, any substitution of which abolished enzymatic activity in As methylation (Song et al., 2009, 2011). Inorganic As predominately undergoes successive oxidative and reductive methylation reactions: As(III) → MMAs → DMAs → TMAs (Challenger 1945). Dimethylated As is the major product of purified CsArsM, with relatively less amounts of MMAs and TMAsO produced (Fig. 4C). DMAs can bind to only a single thiol of ArsM and readily dissociates from enzyme (Kitchin and Wallace 2006). This would cause that the third methylation step (from DMAs to TMAs) occurs difficultly and less TMAsO is produced. The product MMAs of the first methylation step can bind to two thiols of ArsM enzyme. The resulting complex is relatively stable and prone to the second step of methylation (Kitchin and Wallace 2006; Kitchin 2011), causing less MMAs accumulation and more DMAs production. DMAs accumulates as the principal product in the reaction system. Arsenic methylation in soils increased with the decrease of redox potential (Frohne et al., 2011), suggesting that it may occur more easily in anaerobic microorganisms than aerobic microorganisms (Wang et al., 2014). Wetlands, a typical submerged soil with low redox potential, have been recognized as important sinks for As, and typically play an important role in As transformation and mobilization (Vriens et al., 2013). Our results demonstrating the ability of As methylation by SRB may have implications to other anaerobic microorganisms. Elucidating such processes is critical in predicting the metabolic pathways and fate of As in the environment. For example, As methylation in soil influences its accumulation by rice (Jia et al., 2013), and methylated As in rice grains was greatly increased when rice was grown in
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anaerobic soil compared with the corresponding aerobic soil (Xu et al., 2008; Arao et al., 2009). This phenomenon was due to anaerobic microbial activities in paddy soil. It has been implicated that SRB is among the key functional microorganisms in paddy soil and they may contribute to the generation of methylated As (Liu et al., 2009; Lomax et al., 2012). The activities of SRB in paddy soil may reduce the ecological and health effects of As pollution by transforming As from the more hazardous forms to less toxic or volatile methylated forms. SRB have shown the potential for the use in the remediation of environments contaminated with toxic metal(loid)s (Jong and Parry 2003; Teclu et al., 2008). Considering its ubiquity in sediments and wetlands, it seems that SRB could play a significant role in As remediation and biogeochemical cycling in widespread anaerobic environments.
SUPPLEMENTARY DATA Supplementary data is available at FEMSLE online.
FUNDING This project was financially supported by the Natural Science Foundation of China (No. 41371459), the State Key Program of Natural Science Foundation of China (No. 41330853) and the National High Technology Research and Development Program of China (863 Program, 2013AA06A209). Conflict of interest statement. None declared.
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Lomax C, Liu WJ, Wu L, et al. Methylated arsenic species in plants originate from soil microorganisms. New Phytol 2012;193:665–72. Mandal BK, Suzuki KT. Arsenic round the world: a review. Talanta 2002;58:201–35. Marapakala K, Qin J, Rosen BP. Identification of catalytic residues in the As(III) S-adenosylmethionine methyltransferase. Biochemistry 2012;51:944–51. Meharg AA, Rahman MM. Arsenic contamination of Bangladesh paddy field soils: implications for rice contribution to arsenic consumption. Environ Sci Technol 2003;37:229–34. Mestrot A, Feldmann J, Krupp EM, et al. Field fluxes and speciation of arsines emanating from soils. Environ Sci Technol 2011;45:1798–804. Mestrot A, Uroic MK, Plantevin T, et al. Quantitative and qualitative trapping of arsines deployed to assess loss of volatile arsenic from paddy soil. Environ Sci Technol 2009;43:8270–5. Oremland RS, Stolz JF, Hollibaugh JT. The microbial arsenic cycle in Mono Lake, California. FEMS Microbiol Ecol 2004;48:15–27. Qin J, Lehr CR, Yuan C, et al. Biotransformation of arsenic by a Yellowstone thermoacidophilic eukaryotic alga. P Natl Acad Sci USA 2009;106:5213–7. Qin J, Rosen BP, Zhang Y, et al. Arsenic detoxification and evolution of trimethylarsine gas by a microbial arsenite Sadenosylmethionine methyltransferase. P Natl Acad Sci USA 2006;103:2075–80. Rey FE, Gonzalez MD, Cheng J, et al. Metabolic niche of a prominent sulfate-reducing human gut bacterium. P Natl Acad Sci USA 2013;110:13582–87. Roberts LC, Hug SJ, Dittmar J, et al. Arsenic release from paddy soils during monsoon flooding. Nat Geosci 2010;3:53–9. Song X, Geng Z, Li X, et al. Functional and structural evaluation of cysteine residues in the human arsenic (+3 oxidation state) methyltransferase (hAS3MT). Biochimie 2011;93:369–75. Song X, Geng Z, Zhu J, et al. Structure-function roles of four cysteine residues in the human arsenic (+3 oxidation state) methyltransferase (hAS3MT) by site-directed mutagenesis. Chem-Biol Interact 2009;179:321–8. Teclu D, Tivchev G, Laing M, et al. Bioremoval of arsenic species from contaminated waters by sulphate-reducing bacteria. Water Res 2008;42:4885–93.
Thomas DJ, Li JX, Waters SB, et al. Arsenic (+3 oxidation state) methyltransferase and the methylation of arsenicals. Exp Biol Med 2007;232:3–13. Thomas F, Diaz-Bone RA, Wuerfel O, et al. Connection between multimetal(loid) methylation in methanoarchaea and central intermediates of methanogenesis. Appl Environ Microb 2011;77:8669–75. Tsai SL, Singh S, Chen W. Arsenic metabolism by microbes in nature and the impact on arsenic remediation. Curr Opin Biotechnol 2009;20:659–67. Vriens B, Lenz M, Charlet L, et al. Natural wetland emissions of methylated trace elements. Nat Commun 2013:5:3035. Wakao N, Furusaka C. Distribution of sulfate-reducing bacteria in paddy-field soil. Soil Sci Plant Nutr 1973;19:47–52. Wang P, Sun G, Jia Y, et al. Completing arsenic biogeochemical cycle: microbial volatilization of arsines in environment. J Environ Sci 2014;26:1–11. Widdel F, Bak F. Gram-negative mesophilic sulfate reducing bacteria. In: Balows A, Truper HG, Dworkin M, et al. (eds). The ¨ Prokaryotes. New York: Springer, 1992, 3352–78. Xu XY, Mcgrath SP, Meharg AA, et al. Growing rice aerobically markedly decreases arsenic accumulation. Environ Sci Technol 2008;42:5574–9. Ye J, Chang Y, Yan Y, et al. Identiffication and characterization of the arsenite methyltransferase from a protozoan, Tetrahymena pyriformis. Aquat Toxicol 2014;149:50–7. Ye J, Rensing C, Rosen BP, et al. Arsenic biomethylation by photosynthetic organisms. Trends Plant Sci 2012;17:155–62. Yin XX, Chen J, Qin J, et al. Biotransformation and volatilization of arsenic by three photosynthetic cyanobacteria. Plant Physiol 2011;156:1631–8. Zakharyan RA, Aposhian HV. Arsenite methylation by methylvitamin B12 and glutathione does not require an enzyme. Toxicol Appl Pharm 1999;154:287–91. Zhao FJ, Harris E, Jia Y, et al. Arsenic methylation in soils and its relationship with microbial arsM abundance and diversity, and as speciation in rice. Environ Sci Technol 2013;47: 7147–54. Zhu YG, Sun GX, Lei M, et al. High percentage inorganic arsenic content of mining impacted and nonimpacted Chinese rice. Environ Sci Technol 2008;42:5008–13.
R E S E A R C H L E T T E R – Physiology & Biochemistry
Identification and catalytic residues of the arsenite methyltransferase from a sulfate-reducing bacterium, Clostridium sp. BXM Pei-Pei Wang, Peng Bao and Guo-Xin Sun∗ State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China ∗ Corresponding author: State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China. Tel: 86-10-62849328; E-mail: [email protected] One sentence summary: Arsenite methyltransferase is responsible for As methylation and volatilization in sulfate-reducing bacterium. Editor: Robert Gunsalus
ABSTRACT Arsenic methylation is an important process frequently occurring in anaerobic environments. Anaerobic microorganisms have been implicated as the major contributors for As methylation. However, very little information is available regarding the enzymatic mechanism of As methylation by anaerobes. In this study, one novel sulfate-reducing bacterium isolate, Clostridium sp. BXM, which was isolated from a paddy soil in our laboratory, was demonstrated to have the ability of methylating As. One putative arsenite S-Adenosyl-Methionine methyltransferase (ArsM) gene, CsarsM was cloned from Clostridium sp. BXM. Heterologous expression of CsarsM conferred As resistance and the ability of methylating As to an As-sensitive strain of Escherichia coli. Purified methyltransferase CsArsM catalyzed the formation of methylated products from arsenite, further confirming its function of As methylation. Site-directed mutagenesis studies demonstrated that three conserved cysteine residues at positions 65, 153 and 203 in CsArsM are necessary for arsenite methylation, but only Cysteine 153 and Cysteine 203 are required for the methylation of monomethylarsenic to dimethylarsenic. These results provided the characterization of arsenic methyltransferase from anaerobic sulfate-reducing bacterium. Given that sulfate-reducing bacteria are ubiquitous in various wetlands including paddy soils, enzymatic methylation mediated by these anaerobes is proposed to contribute to the arsenic biogeochemical cycling. Key words: arsenic; methylation; sulfate-reducing bacterium; methyltransferase; conserved cysteine
INTRODUCTION Arsenic (As) is a ubiquitous contaminant in the environment (Duker, Carranza and Hale 2005). Anaerobic environments, such as sediments and wetlands, usually act as sinks or secondary sources of As due to natural and anthropogenic activities. Some reducing marine sediments might accumulate 3 mg g−1 As (Mandal and Suzuki 2002). The paddy soil system often accumulated relatively high concentration of As, particularly in south
Asia (Meharg and Rahman 2003). It was estimated that about 1360 tons of As are yearly added to arable soils in Bangladesh (Roberts et al., 2010). It has been well established that anaerobic environments play an important role in the transformation, mobilization and biogeochemical cycling of As (Brannon 1987; Ahmann et al., 1997; Bauer, Fulda and Blodau 2008; Roberts et al., 2010; Vriens et al., 2013). Arsenic methylation as an important process modulating As toxicity and biogeochemical cycling is
Received: 28 July 2014; Accepted: 28 October 2014 C FEMS 2014. All rights reserved. For permissions, please e-mail: [email protected]
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more prevalent under anaerobic conditions than aerobic conditions. Arsenic methylation and volatilization have been observed in many paddy soils (Mestrot et al., 2009, 2011; Huang et al., 2012; Jia et al., 2012). Anaerobic microorganisms inhabited in these ecosystems have been regarded as major contributors for As transformation including methylation (Bright et al., 1994). It has been reported that methylated As species in rice plants originated from soil microorganisms (Lomax et al., 2012; Jia et al., 2013). Several microbial isolates and a microbial coenzyme called methylcobalamin are capable of converting inorganic As to methylated forms (Zakharyan and Aposhian 1999; Qin et al., 2006, 2009; Thomas et al., 2011; Yin et al., 2011; Ye et al., 2014), but the identification of the microorganisms that are actually responsible for inorganic As methylation in anaerobic environments has rarely been clearly demonstrated (Lomax et al., 2012; Jia et al., 2013; Zhao et al., 2013). In the past few years, it has been identified that arsMs are the key microbial functional genes responsible for As methylation (Qin et al., 2006; Ye et al., 2012). However, these studies have mainly focused on aerobic bacteria or eukaryote, little attention was paid to anaerobic microorganisms. The identification of As-methylating microorganisms from anaerobic environments and their functional genes are of great importance in As biogeochemistry. Sulfate-reducing bacteria (SRB) are anaerobic microorganisms widely distributed in the environment, including paddy soils, sea water, thermal springs, sediments and human guts (Wakao and Furusaka 1973; Leloup et al., 2007; Liu et al., 2009; Rey et al., 2013). They are involved in numerous reactions of metal(loid)s (e.g. metal reduction and precipitation) (Lloyd et al., 1998, 2001) and often proposed as strategic microbes to treat water contaminated with a range of metals (Chuichulcherm et al., 2001; Teclu et al., 2008). Numerous reports have showed that SRB is the important methylator of mercury in natural sediments and soils (Compeau and Bartha 1985; King et al., 2000; Jay et al., 2002; Ekstrom, Morel and Benoit 2003), but it is not clear if SRB is a significant As methylator so far. In this study, SRB Clostridium sp. BXM previously isolated from a paddy soil in our laboratory was chosen as anaerobic sedimentary microorganism to study As methylation (Bao et al., 2012). Our main objectives were (1) to identity As methylation process in anaerobic microorganism Clostridium sp. BXM, (2) to provide insight into the enzymatic mechanism for As methylation in anaerobic SRB and (3) to identify catalytic residues of S-Adenosyl-Methionine methyltransferase (ArsM) enzyme and their function in As methylation.
MATERIALS AND METHODS Organisms and cultivations Clostridium sp. BXM was anaerobically grown in the medium (pH 7.2) contained (g l−1 ): MgSO4 r7H2 O 0.5, CaCl2 r2H2 O 0.1, Na2 SO4 0.75, NH4 Cl 0.25, KH2 PO4 0.3, yeast extract 1.0, vitamin C 0.2, Na2 S r9H2 O 0.36, sodium lactate 3.5, at 30◦ C in the dark. Standard anaerobic culturing techniques were used throughout the experiments (Widdel and Bak 1992; Bao et al., 2012). The medium was boiled and cooled to room temperature under an atmosphere of highly pure N2 gas. Anaerobic medium was dispensed into acid-cleaned serum bottles. Each serum bottle was sealed with a butyl rubber stopper, covered with aluminum foil and then autoclaved at 121◦ C for 20 min. During incubation, the cultures were brieffiy shaken by hand once a day. Escherichia coli strains were grown aerobically at 37◦ C in LB medium, supplemented with 50 μg ml−1 kanamycin/ampicillin,
or 0.3 mM IPTG as required. Strain DH5α (Promega) was used for plasmid construction and replication. Strain AW3110(DE3) (arsRBC, ArsR-repressor, ArsB-As(III) efflux pump, ArsC-As(V) reductase) (Carlin et al., 1995) was used for the expression and functional verification of arsM gene. Strain BL21(DE3) was used for protein purification.
Arsenic methylation by Clostridium sp. BXM Anaerobic medium in serum bottle was supplemented by 5.3 μM sodium arsenite (NaAsO2 ) or sodium arsenate (Na3 AsO4 ). The strain of Clostridium sp. BXM was inoculated into the medium with 2% (v/v) inoculum. After the growth of 20 days, the culture was centrifuged by 8000 rpm. The supernatant was filtrated by 0.45 μm disposable filter and oxidized by adding 10% (v/v) H2 O2 . All samples were kept in the refrigerator at 4◦ C until analysis. Every experimental group was assayed for three replicates.
Cloning of the arsM gene from Clostridium sp. BXM and its expression in E. coli Genomic DNA was isolated from the culture of Clostridium sp. BXM by using Genomic-tip 20/G and Buffer Set (Qiagen). Plasmid DNA was extracted with a QIAPrep Spin Miniprep Kit (Qiagen). To express the arsM from Clostridium. sp. BXM in E. coli, a 813 bp fragment containing the ATG start codon and excluding the TGA stop codon was PCR amplified from the genomic DNA of Clostridium sp. BXM by using the forward primer: 5 -CGGAATTCATGGATAATATTCGAGAGGG-3 (EcoRI restriction endonuclease site underlined) and the reverse primer: 5 -CCCTCGAGTGCCGGCTTGGCTGCCCGAAT-3 (XhoI restriction endonuclease site underlined). PCR (94◦ C for 5 min, 94◦ C for 30 s, 58◦ C for 30 s and 72◦ C for 1 min, 30 cycles) was performed with LA Taq DNA polymerase (Takara Bio). The PCR product was cloned into PMD19T simple vector (Takara Bio) to create the plasmid PMD19T-arsM. The sequence was verified by DNA sequencing. Then, after digestion with EcoRI and XhoI, arsM fragment was again inserted into pET28a vector to create the plasmid pET28a-arsM, in which arsM gene was controlled by T7 promoter. The sequence was still verified by DNA sequencing. As a consequence, the His-tagged ArsM was established composed of 314 amino acid residues, with a predicted molecular mass of 33.95 kDa. Plasmid pET28a or pET28a-arsM was transformed into E. coli strain AW3110(DE3), which was As-hypersensitive. Transformed cells were grown in LB medium containing 50 μg ml−1 kanamycin overnight at 37◦ C, and then diluted 50-fold into fresh LB medium containing 50 μg ml−1 kanamycin, 0.3 mM IPTG and a series of concentrations of As(III) (10, 20, 50 μM). After a growth of 20 h at 37◦ C, the culture was centrifuged at 8000 rpm. The supernatant was filtrated by 0.45 μm disposable filters and oxidized by 10% (v/v) H2 O2 . All the samples were kept in the refrigerator at 4◦ C until analysis. Every experimental group was assayed for three replicates. Single colonies of E. coli AW3110 expressing pET28a or pET28a-arsM were inoculated into LB medium (5 ml) contained 50 μg ml−1 kanamycin and cultured at 37◦ C overnight. Late exponential phase cells were diluted 100-fold into fresh LB medium (50 ml) containing 50 μg ml−1 kanamycin, 0.3 mM IPTG and a series of concentrations of As(III) (0, 20, 50, 100 μM). Cellular growths were monitored as absorbance at 600 nm via UV-vis spectrophotometer (New Century, Model T6) and continued until the cultures reached stationary phase.
Wang et al.
Arsenic volatilization by E. coli AW3110 expressing arsM Volatile As produced by E. coli expressing arsM was chemtrapped as described (Huang et al., 2012). Escherichia coli AW3110 expressing either pET28a-arsM or pET28a were cultured in triangular flasks (250 ml volume, contained 100 ml LB medium with a series of concentrations of As(III)). Every flask was capped with a T-junction stopper having a gas inlet and a gas outlet. The inlet was linked to a super silent adjustable air pump (ACO-9601, 2W power), to pump filtered air at a gas flow rate of 40 ml min−1 for refreshing the headspace of flask. The outlet of T-junction stopper was connected to a glass trapping tube filled with AgNO3 impregnated silica gel beads. After 48 h growth of E. coli, the total As absorbed by the trapping tube was eluted by 1% (v/v) HNO3 (5 ml) in a microwave digestion system (Mars II, CEM, US). The eluted solution was centrifuged at 6000 rpm. The supernatant was separated and filtrated by 0.45 μm disposable filter. The quantity of volatile As was tested by ICP-MS and the As species was analyzed by HPLC-ICP-MS.
Purification of ArsM enzyme and As(III) methylation by purified enzyme in vitro The strain E. coli BL21(DE3) expressing pET28-arsM was grown at 37◦ C in LB medium (200 ml) to an A600 of 0.4–0.6, at which point 0.3 mM IPTG was added to induce expression of His-tagged ArsM. After the 8 h growth, E. coli was harvested by centrifugation (8000 rpm.) at 4◦ C for 20 min. The wet cells were suspended in 30 ml of lysis buffer (buffer A: 50 mM NaH2 PO4 , 300 mM NaCl, 10 mM imidazole, pH 8.0) and lysed in ultrasonic cell disruption system (JY92–2D) at a program as follows: working for 5 s followed a rest for 5 s, it continued for 20 min at 4◦ C. Membranes and unbroken cells were removed by centrifugation at 8000 rpm for 30 min. The supernatant was separated, filtrated by 0.45 μm disposable sterile filter and then loaded onto a Ni(II)-NTA column (Qiagen, 5 ml) pre-equilibrated with buffer A. The column was then washed with 200 ml of wash buffer (buffer B: 50 mM NaH2 PO4 , 300 mM NaCl, 20 mM imidazole, pH 8.0) and finally eluted with 5 ml of elution buffer (buffer C: 50 mM NaH2 PO4 , 300 mM NaCl, 250 mM imidazole, pH 8.0) to gain the purified ArsM protein. Solution containing ArsM was removed to a 10 kDa ultrafiltration centrifuge tube (Sartorius) and concentrated by centrifugation at 8000 rpm for 1 h. Finally, the buffer C bearing ArsM protein was replaced by MOPS buffer (50 mM MOPS and 150 mM KCl, pH 7.4) using Micro Bio-SpinTM Chromatography Column (Bio-Rad). ArsM was identified by sodium-dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentration was estimated by the method of Bradford (Bio-Rad Protein Assay) (Bradford 1976), using BSA (Sigma) as a standard. In vitro assay of As methylation was performed in the MOPS buffer containing a certain concentration of ArsM protein, 8 mM GSH, 0.5 mM AdoMet and a series of concentrations of As(III) (5, 10, 20 μM). The reactions were kept in water bath at 37◦ C for the different time (0.5, 1.5, 3, 4.5, 6, 7.5, 20 h). The reactions were terminated by boiling for 5 min in the end. The reaction solutions were oxidized by 10% (v/v) H2 O2 overnight, filtered by 0.45 μm filters. After an appropriate dilution, all samples were analyzed for As species by HPLC-ICP-MS.
Preparation of CsArsM mutants Substitutions of Cys65, Cys153 and Cys203 of the CsArsM for serine were generated by site-directed mutagenesis through over-
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lapping extension PCR with the plasmid pET28a-arsM as the DNA template. The primers used for the site-directed mutagenesis are listed in Table S1 (Supporting Information). PCR products were double digested by EcoRI and XhoI, and cloned into the prepared EcoRI/XhoI-digested pET28a vector. All of the sequences of the arsM mutants were verified by DNA sequencing to ensure that no errors had been introduced during amplification. Escherichia coli BL21(DE3) was transformed using vectors carrying different mutations of arsM gene. Protein expression, purification, identification and concentration determination were performed as described above. Three mutants of ArsM (C65S, C153S, C203S) were constructed.
Arsenic speciation analysis Arsenic speciation was analyzed by HPLC-ICP-MS (7500a, Agilent Technologies) as described (Zhu et al., 2008). Chromatographic columns were obtained from Hamilton and consisted of a precolumn (11.2 mm, 12–20 μm) and a PRP-X100 10 μm anionexchange column (250 × 4.1 mm). The mobile phase consisted of 10 mM diammonium hydrogen phosphate [(NH4 )2 HPO4 ] and 10 mM ammonium nitrate [NH4 NO3 ], adjusted to pH 6.2. The mobile phase was pumped through the column at a flow rate of 1.0 ml min−1 .
RESULTS Arsenic methylation by SRB Clostridium sp. BXM When Clostridium sp. BXM was grown in the medium containing arsenite [As(III)] or arsenate [As(V)] for 20 days, monomethylarsenic (MMAs) and dimethylarsenic (DMAs) were detected (Fig. 1). About 9% of the total As in the medium has been methylated to MMAs as well as about 1% to DMAs (Fig. S1, Supporting Information). No methylated As was detected in the control (no bacterial inoculation). This result suggested that Clostridium sp. BXM have the capability of methylating As.
Cloning of the arsM gene from Clostridium sp. BXM By homologous comparison with the As(III) methyltransferases previously reported (Lin et al., 2002; Qin et al., 2006, 2009; Yin et al., 2011), a putative arsM gene (813 bp) was cloned from the genome of Clostridium sp. BXM, and designated as CsarsM. CsArsM protein encoded by CsarsM gene contained 270 residues, with a predicted molecular mass of 29.06 kDa. Comparative study based on protein sequences showed that CsArsM had the highest similarity with the ArsM from anaerobic bacterium Rhodopseudomonas palustris CGA009 (RpArsM) (53% identity and 84% similarity) (Qin et al., 2006). With those aerobic cyanobacterial ArsMs (Yin et al., 2011), CsArsM shared lower similarity (average 21% identity and 57% similarity). In addition, CsArsM also exhibited significant, but lower similarity to mammalian AS3MT methyltransferase (21% identity and 53% similarity) (Lin et al., 2002; Song et al., 2011), and to the ArsM of eukaryotic alga Cyanidioschyzon sp. isolate 5508 (20% identity and 51% similarity) (Qin et al., 2009).
CsArsM enzyme detoxified As(III) in vivo CsarsM gene was expressed in an As-sensitive E. coli AW3110 (arsRBC), which had no orthologous arsM gene. Expression of arsM clearly conferred the strain AW3110 with the resistance to As(III). Cells expressing CsarsM gene grew much better than that bearing empty plasmid pET28a when exposed to 50 μM As(III)
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Figure 2. The growth curves of E. coli AW3110 (arsRBC) expressing CsarsM or empty vector when exposed to different concentrations of NaAsO2 . Escherichia coli bearing empty vector pET28a or pET28a-arsM was cultured in LB medium with the indicated concentrations of NaAsO2 , 50 μg ml−1 kanamycin and 0.3 mM IPTG at 37◦ C. Cellular growths were monitored by spectrophotometer at absorbance of 600 nm (A600) at the indicated time points. The error bars indicate the standard errors of three triplicates.
Figure 1. Arsenic transformation by Clostridium sp. BXM. Clostridium sp. BXM was cultured with 5.3 μM As(III) or As(V) for 20 days. The medium without bacteria inoculation was as control. The As species in the medium were analyzed as described in ‘Experimental procedures’.
(Fig. 2). Although all bacteria exhibited a weak growth under 100 μM As(III), cells expressing CsarsM gene showed a little better growth than that bearing empty vector (Fig. 2). ArsM enzyme as the product of arsM gene maybe metabolize cellular As(III) to less toxic methylated products for facilitating growth. In other words, ArsM enzyme reduced the As toxicity in the cells, consistent with the idea that As methyltransferase has an important physiological role in detoxifying As.
As(III) methylation and volatilization by E. coli AW3110 expressing arsM DMAs and trimethylarsine oxide (TMAsO) except MMAs were observed in the medium of E. coli expressing CsarsM gene by comparison with the control (Fig. 3A and B). This result indicated that the enzyme encoded by CsarsM gene is indeed responsible for As(III) methylation. The time-course of As methylation by the strain AW3110 expressing CsarsM showed that TMAsO and DMAs have accumulated in the medium after 3 h incubation and increased with the culture time increasing, concomitant with a decrease of inorganic As (Fig. 3B). No detectable MMAs was observed throughout the experiment (within 20 h). Since the expression of CsarsM gene was controlled by T7 promoter, as well as with the induction of isopropyl β-D-thiogalactoside (IPTG), abundant CsArsM protein may be produced and could quickly convert MMAs to DMAs in the recombinant E. coli. About 10% of total As was transformed into DMAs and another 10% into TMAsO by E. coli in the presence of 10 μM As(III) (Fig. 3A). The total As in the medium of E. coli expressing CsarsM is a bit lower
than corresponding control (empty vector), probably attributed to As volatilization in the former. Escherichia coli AW3110 expressing CsarsM gene effectively volatilized As when exposed to different As(III) concentrations (Fig. 3C). The major gaseous product was TMAs which was oxidized and identified as TMAsO, together with tiny amount of dimethylarsine (DMAsH) detectable as oxidized DMAs (Fig. S2, Supporting Information). The amount of As volatilization in 100 μM As(III) was more than that in 50 μM As(III) (Fig. 3C), though the cellular growth in the former was much weaker than that in the latter (Fig. 2).
Methylation of As(III) by purified ArsM enzyme in vitro To further elucidate the function of CsarsM gene in As methylation, corresponding CsArsM protein was purified from recombinant E. coli cytosol for its As methylation activity analysis (Fig. 4A). Cofactor glutathione (GSH) as a necessary component was used to form As–GSH complex, which was optimal substrate for ArsM enzyme. S-Adenosyl-Methionine (AdoMet) was used as the methyl donor in ArsM-catalyzed As(III) methylation reaction in vitro. CsArsM enzyme converted As(III) to MMAs after 0.5 h, with small amount of DMAs appearing at 1.5 h (Fig. 4B). The concentrations of DMAs and MMAs kept increasing during the experiment process, concomitant with the decrease of inorganic As (Fig. 4B). At 20 h, DMAs became the dominant organic As species. Only little MMAs have been produced within 20 h compared to DMAs. TMAsO had not appeared until the time got to 20 h. About 45% of the total As was converted to the organic As by CsArsM in the presence of 5 μM As(III) (Fig. 4C). The total amount of methylated As increased with the increasing As(III) concentrations in the reaction system (Fig. 4C), although the percentage of that indeed decreased. In the presence of 20 μM As(III), about 23% of inorganic As was methylated by CsArsM (Fig. 4C). In the absence of GSH or AdoMet, no methylated species were observed (data not shown).
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Figure 3. Arsenic methylation and volatilization by E. coli expressing pET28aarsM. (A) As(III) methylation by E. coli AW3110 (arsRBC) expressing CsarsM. Escherichia coli expressing empty pET28a or pET28a-arsM was incubated in LB medium with the indicated concentrations of As(III) (10, 20, 50 μM), 50 μg ml−1 kanamycin and 0.3 mM IPTG at 37◦ C for 20 h. The As species in the supernatants were analyzed by HPLC-ICP-MS as described in ‘Experimental procedures’. C, control, which was E. coli expressing empty vector pET28a; arsM, E. coli expressing CsarsM. The error bars indicate the standard errors of three triplicates. (B) Time dynamics of As(III) methylation by E. coli AW3110 (arsRBC) expressing CsarsM. The strain was incubated in LB medium with 20 μM As(III), 50 μg ml−1 kanamycin and 0.3 mM IPTG at 37◦ C. At the indicated time points (3, 6, 9, 13, 16, 20 h), 1 ml samples of the bacterial culture were taken and centrifuged at 8000 rpm. The supernatants were appropriately diluted and analyzed for As species by HPLCICP-MS. (C) Volatilization of As by E. coli AW3110 (arsRBC) expressing CsarsM. E. coli with CsarsM was grown in LB medium with a series of As(III) concentrations, 50 μg ml−1 kanamycin and 0.3 mM IPTG at 37◦ C for 48 h. Volatile As produced was chemo-trapped as described in ‘Experimental procedures’ and the total quantities of volatile As were analyzed by ICP-MS. The error bars indicate the standard errors of three triplicates.
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Figure 4. Purification of CsArsM protein and As(III) methylation by purified CsArsM. (A) SDS-PAGE showing the purity of the soluble CsArsM. Lane 1, protein standards; Lane 2, cell lysis solution; Lane 3, collection of the washing buffer (20 mM imidazole) outflowing from Ni(II)-NTA column; Lane 4, collection of the washing buffer (50 mM imidazole) outflowing from Ni(II)-NTA column; Lane 5, concentrated His-tagged CsArsM protein. (B) Time dynamics of As(III) methylation by purified CsArsM enzyme. Each assay contained 10 μM As(III), 0.5 mM AdoMet, 8 mM GSH and 4 μM purified CsArsM in 500 μl MOPS buffer (pH 7.4). The reactions were kept at 37◦ C. Arsenic species of the samples were analyzed at the indicated time points by HPLC-ICP-MS as described in ‘Experimental procedures’. The error bars indicate the standard errors of three triplicates. (C) The final products of As(III) methylation by purified CsArsM enzyme at 37◦ C for 20 h. Each assay contained the indicated concentrations of As(III) (5, 10 or 20 μM), 0.5 mM AdoMet, 8 mM GSH and 10 μM purified CsArsM in 500 μl MOPS buffer (pH 7.4). Arsenic species were analyzed by HPLC-ICP-MS. The error bars indicate the standard errors of three triplicates.
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DISCUSSION
Figure 5. Methylation of As(III) or MMAs by wild-type or mutant CsArsM enzymes. Methylation of As(III) or MMAs was tested in a reaction system (pH 7.4) with 10 μM As(III) (A) or MMAs (B), 8 mM GSH, 0.5 mM AdoMet and 2 μM ArsM enzyme. The reactions were kept at 37◦ C for 20 h. The As species of the samples were analyzed by HPLC-ICP-MS.
Conserved cysteine (Cys) residues Cys65, Cys153 and Cys203 of CsArsM All ArsM proteins identified to date have three conserved Cys residues (Thomas et al., 2007; Hamdi et al., 2012; Ye et al., 2012), at positions 72, 174 and 224 in CmArsM of eukaryotic alga Cyanidioschyzon sp. 5508 (Marapakala, Qin and Rosen 2012), which were responsible for As methylation. The conserved Cys residues in CsArsM were Cys65, Cys153 and Cys203. To elucidate the function of three conserved Cys in As methylation, Cys65, Cys153 and Cys203 in CsArsM were substituted for serines, respectively, creating the mutants of C65S, C153S and C203S. All three mutants lost their activities of As(III) methylation completely (Fig. 5A). In contrast, methylation of MMAs was completed by the C65S mutant but not by the C153S or C203S (Fig. 5B). These results suggest that the three conserved Cys (Cys65, Cys153 and Cys203) are required for the first methylation step (from As(III) to MMAs), while only Cys153 and Cys203 are required for the second methylation step (from MMAs to DMAs) in the Challenger’s pathway (Challenger 1945; Cullen and Reimer 1989). Note that Cys29, Cys30 and Cys262 in CsArsM were not conserved in other ArsM orthologues and their single mutations did not affect enzymatic activity of As methylation (data not shown).
Due to the critical role of microorganisms in As biogeochemical cycling in wetland and water environments, it is important to understand As transformation by anaerobic bacteria (Tsai, Singh and Chen 2009). The methylation of As is one of the key processes governing the fate of As in the environment, and thus detailed understanding of the mechanism of As methyltransferase is crucial. This study provided the insight into a newly identified ArsM from anaerobic SRB, which play crucial roles in As transformation of anaerobic environments (Kirk et al., 2004; Oremland, Stolz and Hollibaugh 2004). CsArsM enzyme encoded by CsarsM gene cloned from SRB Clostridium sp. BXM conferred the ability of methylating and volatilizing As to As-sensitive strain of E. coli, concomitant with an increase of cellular tolerance to As. These results suggest that ArsM-mediated As methylation in SRB is a mechanism for relieving cellular toxicity of As. By the site-directed mutagenesis studies, we confirmed that three residues of CsArsM, Cys65, Cys153 and Cys203 are required for As(III) methylation, while only Cys153 and Cys203 are necessary for MMAs methylation. These Cys are conserved in all ArsM and AS3MT proteins identified to date (Thomas et al., 2007; Ye et al., 2011, 2012). For example, they are homologous to Cys72, Cys174 and Cys224 of CmArsM from eukaryotic alga Cyanidioschyzon sp. 5508 (Marapakala et al., 2012). It was reported that the substitution of Cys72, Cys174 or Cys224 led to loss of As(III) methylation, while the substitution of Cys174 or Cys224 abolished the methylation of monomethylarsenite [MMAs(III)]. These Cys affected the binding of As(III) or MMAs(III) to CmArsM enzyme during the methylation process. Similarly, the ArsM from Danio rerio has been identiffied to possess two conserved residues, Cys160 and Cys210, which were suggested to be related to As(III) binding (Hamdi et al., 2012). To the recombinant human AS3MT enzyme (Geng et al., 2009), Cys72, Cys156 and Cys206 have been proved to be functionally conserved, any substitution of which abolished enzymatic activity in As methylation (Song et al., 2009, 2011). Inorganic As predominately undergoes successive oxidative and reductive methylation reactions: As(III) → MMAs → DMAs → TMAs (Challenger 1945). Dimethylated As is the major product of purified CsArsM, with relatively less amounts of MMAs and TMAsO produced (Fig. 4C). DMAs can bind to only a single thiol of ArsM and readily dissociates from enzyme (Kitchin and Wallace 2006). This would cause that the third methylation step (from DMAs to TMAs) occurs difficultly and less TMAsO is produced. The product MMAs of the first methylation step can bind to two thiols of ArsM enzyme. The resulting complex is relatively stable and prone to the second step of methylation (Kitchin and Wallace 2006; Kitchin 2011), causing less MMAs accumulation and more DMAs production. DMAs accumulates as the principal product in the reaction system. Arsenic methylation in soils increased with the decrease of redox potential (Frohne et al., 2011), suggesting that it may occur more easily in anaerobic microorganisms than aerobic microorganisms (Wang et al., 2014). Wetlands, a typical submerged soil with low redox potential, have been recognized as important sinks for As, and typically play an important role in As transformation and mobilization (Vriens et al., 2013). Our results demonstrating the ability of As methylation by SRB may have implications to other anaerobic microorganisms. Elucidating such processes is critical in predicting the metabolic pathways and fate of As in the environment. For example, As methylation in soil influences its accumulation by rice (Jia et al., 2013), and methylated As in rice grains was greatly increased when rice was grown in
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anaerobic soil compared with the corresponding aerobic soil (Xu et al., 2008; Arao et al., 2009). This phenomenon was due to anaerobic microbial activities in paddy soil. It has been implicated that SRB is among the key functional microorganisms in paddy soil and they may contribute to the generation of methylated As (Liu et al., 2009; Lomax et al., 2012). The activities of SRB in paddy soil may reduce the ecological and health effects of As pollution by transforming As from the more hazardous forms to less toxic or volatile methylated forms. SRB have shown the potential for the use in the remediation of environments contaminated with toxic metal(loid)s (Jong and Parry 2003; Teclu et al., 2008). Considering its ubiquity in sediments and wetlands, it seems that SRB could play a significant role in As remediation and biogeochemical cycling in widespread anaerobic environments.
SUPPLEMENTARY DATA Supplementary data is available at FEMSLE online.
FUNDING This project was financially supported by the Natural Science Foundation of China (No. 41371459), the State Key Program of Natural Science Foundation of China (No. 41330853) and the National High Technology Research and Development Program of China (863 Program, 2013AA06A209). Conflict of interest statement. None declared.
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