Clostridium sp. BXM - FEMS Microbiology Letters

FEMS Microbiology Letters Advance Access published December 4, 2014
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Identification and catalytic residues of the arsenite
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methyltransferase from a sulfate-reducing bacterium,
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Clostridium sp. BXM
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Pei-Pei Wang1, Peng Bao1 and Guo-Xin Sun1*
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Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
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*Corresponding author. Phone: 86-10-62849328, E-mail: [email protected]
State Key Laboratory of Urban and Regional Ecology, Research Center for
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Abbreviations: ArsM, arsenite S-Adenosyl-Methionine methyltransferase; As,
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arsenic; SRB, sulfate-reducing bacterium; As(III), arsenite; Cys, cysteine; MMAs,
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monomethylarsenic;
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trimethylarsine oxide; IPTG, isopropyl β-D-thiogalactoside; TMAs, trimethylarsine;
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DMAsH, dimethylarsine; GSH, glutathione; AdoMet, S-Adenosyl-Methionine;
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MMAs(III), monomethylarsenite.
DMAs,
dimethylarsenic;
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As(V),
arsenate;
TMAsO,
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Abstract
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Arsenic methylation is an important process frequently occurring in anaerobic
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environments. Anaerobic microorganisms have been implicated as the major
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contributors for As methylation. However, very little information is available
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regarding the enzymatic mechanism of As methylation by anaerobes. In this study,
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one novel sulfate-reducing bacterium isolate, Clostridium sp. BXM, which was
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isolated from a paddy soil in our laboratory, was demonstrated to have the ability of
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methylating As. One putative arsenite S-Adenosyl-Methionine methyltransferase
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(ArsM) gene, CsarsM was cloned from Clostridium sp. BXM. Heterologous
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expression of CsarsM conferred As resistance and the ability of methylating As to an
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As-sensitive strain of E. coli. Purified methyltransferase CsArsM catalyzed the
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formation of methylated products from arsenite, further confirming its function of As
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methylation. Site-directed mutagenesis studies demonstrated that three conserved
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cysteine residues at positions 65, 153, and 203 in CsArsM are necessary for arsenite
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methylation, but only Cysteine 153 and Cysteine 203 are required for the methylation
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of monomethylarsenic to dimethylarsenic. These results provided the characterization
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of arsenic methyltransferase from anaerobic sulfate-reducing bacterium. Given that
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sulfate-reducing bacteria are ubiquitous in various wetlands including paddy soils,
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enzymatic methylation mediated by these anaerobes is proposed to contribute to the
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arsenic biogeochemical cycling.
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Keywords: arsenic; methylation; sulfate-reducing bacterium; methyltransferase;
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conserved cysteine
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Introduction
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Arsenic (As) is a ubiquitous contaminant in the environment (Duker et al., 2005).
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Anaerobic environments, such as sediments and wetlands, usually act as sinks or
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secondary sources of As due to natural and anthropogenic activities. Some reducing
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marine sediments might accumulate 3 mg g-1 As (Mandal and Suzuki, 2002). The
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paddy soil system often accumulated relatively high concentration of As, particularly
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in south Asia (Meharg and Rahman, 2003). It was estimated that about 1360 tons of
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As are yearly added to arable soils in Bangladesh (Roberts et al., 2010). It has been
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well established that anaerobic environments play an important role in the
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transformation, mobilization and biogeochemical cycling of As (Brannon, 1987;
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Ahmann et al., 1997; Bauer et al., 2008; Roberts et al., 2010; Vriens et al., 2013).
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Arsenic methylation as an important process modulating As toxicity and
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biogeochemical cycling is more prevalent under anaerobic conditions than aerobic
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conditions. Arsenic methylation and volatilization have been observed in many paddy
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soils (Mestrot et al., 2009; Mestrot et al., 2011; Huang et al., 2012; Jia et al., 2012).
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Anaerobic microorganisms inhabited in these ecosystems have been regarded as
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major contributors for As transformation including methylation (Bright et al., 1994).
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It has been reported that methylated As species in rice plants originated from soil
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microorganisms (Lomax et al., 2012; Jia et al., 2013).
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Several microbial isolates and a microbial coenzyme called methylcobalamin are
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capable of converting inorganic As to methylated forms (Zakharyan and Aposhian,
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1999; Qin et al., 2006; Qin et al., 2009; Thomas et al., 2011; Yin et al., 2011; Ye et al.,
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2014), but the identification of the microorganisms that are actually responsible for
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inorganic As methylation in anaerobic environments has rarely been clearly
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demonstrated (Lomax et al., 2012; Jia et al., 2013; Zhao et al., 2013). In the past few
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years, it has been identified that arsMs are the key microbial functional genes
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responsible for As methylation (Qin et al., 2006; Ye et al., 2012). However, these
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studies have mainly focused on aerobic bacteria or eukaryote, little attention was paid
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to anaerobic microorganisms. The identification of As-methylating microorganisms
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from anaerobic environments and their functional genes are of great importance in As
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biogeochemistry.
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Sulfate-reducing bacteria (SRB) are anaerobic microorganisms widely distributed
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in the environment, including paddy soils, sea water, thermal springs, sediments and
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human guts (Wakao and Furusaka, 1973; Leloup et al., 2007; Liu et al., 2009; Rey et
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al., 2013). They are involved in numerous reactions of metal(loid)s (e.g., metal
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reduction and precipitation) (Lloyd et al., 1998; Lloyd et al., 2001) and often
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proposed as strategic microbes to treat water contaminated with a range of metals
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(Chuichulcherm et al., 2001; Teclu et al., 2008). Numerous reports have showed that
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SRB is the important methylator of mercury in natural sediments and soils (Compeau
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and Bartha, 1985; King et al., 2000; Jay et al., 2002; Ekstrom et al., 2003), but it is
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not clear if SRB is a significant As methylator so far. In this study, SRB Clostridium
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sp. BXM previously isolated from a paddy soil in our laboratory was chosen as
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anaerobic sedimentary microorganism to study As methylation (Bao et al., 2012). Our
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main objectives were (1) to identity As methylation process in anaerobic
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microorganism Clostridium sp. BXM, (2) to provide insight into the enzymatic
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mechanism for As methylation in anaerobic SRB, (3) to identify catalytic residues of
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ArsM enzyme and their function in As methylation.
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Materials and methods
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Organisms and cultivations
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Clostridium sp. BXM was anaerobically grown in the medium (pH 7.2) contained
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(g l-1): MgSO4•7H2O 0.5, CaCl2•2H2O 0.1, Na2SO4 0.75, NH4Cl 0.25, KH2PO4 0.3,
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yeast extract 1.0, vitamin C 0.2, Na2S•9H2O 0.36, sodium lactate 3.5, at 30°C in the
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dark. Standard anaerobic culturing techniques were used throughout the experiments
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(Widdel and Bak, 1992; Bao et al., 2012). The medium was boiled and cooled to
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room temperature under an atmosphere of highly pure N2 gas. Anaerobic medium was
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dispensed into acid-cleaned serum bottles. Each serum bottle was sealed with a butyl
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rubber stopper, covered with aluminum foil, and then autoclaved at 121°C for 20 min.
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During incubation, the cultures were brieﬂy shaken by hand once a day.
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E. coli strains were grown aerobically at 37°C in LB medium, supplemented with
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50 μg ml-1 kanamycin/ampicillin, or 0.3 mM IPTG as required. Strain DH5α
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(Promega) was used for plasmid construction and replication. Strain AW3110(DE3)
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[ΔarsRBC; ArsR-repressor; ArsB-As(III) efflux pump; ArsC-As(V) reductase] (Carlin
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et al., 1995) was used for the expression and functional verification of arsM gene.
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Strain BL21(DE3) was used for protein purification.
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Arsenic methylation by Clostridium sp. BXM
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Anaerobic medium in serum bottle was supplemented by 5.3 μM sodium arsenite
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(NaAsO2) or sodium arsenate (Na3AsO4). The strain of Clostridium sp. BXM was
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inoculated into the medium with 2% (v/v) inoculum. After the growth of 20 days, the
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culture was centrifuged by 8000 r.p.m. The supernatant was filtrated by 0.45 μm
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disposable filter, and oxidized by adding 10% (v/v) H2O2. All samples were kept in
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the refrigerator at 4°C until analysis. Every experimental group was assayed for three
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replicates.
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Cloning of the arsM gene from Clostridium sp. BXM and its expression in E. coli
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Genomic DNA was isolated from the culture of Clostridium sp. BXM by using
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Genomic-Tip 20/G and Buffer set (Qiagen). Plasmid DNA was extracted with a
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QIAPrep Spin Miniprep kit (Qiagen). To express the arsM from Clostridium. sp.
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BXM in E. coli, a 813 bp fragment containing the ATG start codon and excluding the
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TGA stop codon was PCR amplified from the genomic DNA of Clostridium sp. BXM
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by using the forward primer: 5’-CGGAATTCATGGATAATATTCGAGAGGG-3’
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(EcoRI restriction endonuclease site underlined) and the reverse primer:
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5’-CCCTCGAGTGCCGGCTTGGCTGCCCGAAT-3’ (XhoI restriction endonuclease
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site underlined). PCR (94°C for 5 min, 94°C for 30 s, 58°C for 30 s, and 72°C for 1
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min, 30 cycles) was performed with LA Taq DNA polymerase (Takara Bio). The PCR
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product was cloned into PMD19T simple vector (Takara Bio) to create the plasmid
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PMD19T-arsM. The sequence was verified by DNA sequencing. Then, after digestion
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with EcoRI and XhoI, arsM fragment was again inserted into pET28a vector to create
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the plasmid pET28a-arsM, in which arsM gene was controlled by T7 promoter. The
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sequence was still verified by DNA sequencing. As a consequence, the His-tagged
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ArsM was established composed of 314 amino acid residues, with a predicted
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molecular mass of 33.95 kDa.
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Plasmid pET28a or pET28a-arsM was transformed into E. coli strain
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AW3110(DE3), which was As-hypersensitive. Transformed cells were grown in LB
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medium containing 50 μg ml-1 kanamycin overnight at 37°C, then diluted 50-fold into
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fresh LB medium containing 50 μg ml-1 kanamycin, 0.3 mM IPTG, and a series of
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concentrations of As(III) (10, 20, 50 μM). After a growth of 20 h at 37°C, the culture
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was centrifuged at 8000 r.p.m. The supernatant was filtrated by 0.45 μm disposable
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filters, and oxidized by 10% (v/v) H2O2. All the samples were kept in the refrigerator
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at 4°C until analysis. Every experimental group was assayed for three replicates.
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Single colonies of E. coli AW3110 expressing pET28a or pET28a-arsM were
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inoculated into LB medium (5 ml) contained 50 μg ml-1 kanamycin and cultured at
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37°C overnight. Late exponential phase cells were diluted 100-fold into fresh LB
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medium (50 ml) containing 50 μg ml-1 kanamycin, 0.3 mM IPTG and a series of
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concentrations of As(III) (0, 20, 50, 100 μM). Cellular growths were monitored as
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absorbance at 600 nm via UV-vis spectrophotometer (New Century, Model T6) and
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continued until the cultures reached stationary phase.
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Arsenic volatilization by E. coli AW3110 expressing arsM
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Volatile As produced by E. coli expressing arsM was chem-trapped as described
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(Huang et al., 2012). E. coli AW3110 expressing either pET28a-arsM or pET28a were
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cultured in triangular flasks [250 ml volume; contained 100 ml LB medium with a
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series of concentrations of As(III)]. Every flask was capped with a T-junction stopper
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having a gas inlet and a gas outlet. The inlet was linked to a super silent adjustable air
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pump (ACO-9601; 2-W power), to pump filtered air at a gas flow rate of 40 ml min-1
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for refreshing the headspace of flask. The outlet of T-junction stopper was connected
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to a glass trapping tube filled with AgNO3-impregnated silica gel beads. After 48 h
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growth of E. coli, the total As absorbed by the trapping tube was eluted by 1% (v/v)
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HNO3 (5 ml) in a microwave digestion system (Mars II, CEM, US). The eluted
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solution was centrifuged at 6000 r.p.m. The supernatant was separated and filtrated by
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0.45 μm disposable filter. The quantity of volatile As was tested by ICP-MS and the
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As species was analyzed by HPLC-ICP-MS.
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Purification of ArsM enzyme and As(III) methylation by purified enzyme in vitro
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The strain E. coli BL21(DE3) expressing pET28-arsM was grown at 37°C in LB
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medium (200 ml) to an A600 of 0.4-0.6, at which point 0.3 mM IPTG was added to
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induce expression of His-tagged ArsM. After the 8 h growth, E. coli was harvested by
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centrifugation (8000 r.p.m.) at 4°C for 20 min. The wet cells were suspended in 30 ml
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of lysis buffer (buffer A: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0)
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and lysed in ultrasonic cell disruption system (JY92-2D) at a program as follows:
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working for 5 s followed a rest for 5 s, it continued for 20 min at 4°C. Membranes and
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unbroken cells were removed by centrifugation at 8000 r.p.m. for 30 min. The
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supernatant was separated, filtrated by 0.45 μm disposable sterile filter, and then
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loaded onto a Ni(II)-NTA column (Qiagen; 5 ml) pre-equilibrated with buffer A. The
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column was then washed with 200 ml of wash buffer (buffer B: 50 mM NaH2PO4,
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300 mM NaCl, 20 mM imidazole, pH 8.0) and finally eluted with 5 ml of elution
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buffer (buffer C: 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) to
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gain the purified ArsM protein. Solution containing ArsM was removed to a 10-kDa
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ultrafiltration centrifuge tube (Sartorius) and concentrated by centrifugation at 8000
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r.p.m. for 1 h. Finally, the buffer C bearing ArsM protein was replaced by MOPS
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buffer (50 mM MOPS and 150 mM KCl, pH 7.4) using Micro Bio-SpinTM
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Chromatography Column (Bio-Rad). ArsM was identified by sodium-dodecyl-sulfate
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polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentration was estimated
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by the method of Bradford (Bio-Rad Protein Assay) (Bradford, 1976), using BSA
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(Sigma) as a standard.
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In vitro assay of As methylation was performed in the MOPS buffer containing a
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certain concentration of ArsM protein, 8 mM GSH, 0.5 mM AdoMet and a series of
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concentrations of As(III) (5, 10, 20 μM). The reactions were kept in water bath at
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37°C for the different time (0.5, 1.5, 3, 4.5, 6, 7.5, 20 h). The reactions were
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terminated by boiling for 5 min in the end. The reaction solutions were oxidized by
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10% (v/v) H2O2 overnight, filtered by 0.45 μm filters. After a appropriate dilution, all
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samples were analyzed for As species by HPLC-ICP-MS.
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Preparation of CsArsM mutants
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Substitutions of Cys65, Cys153, and Cys203 of the CsArsM for serine were
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generated by site-directed mutagenesis through overlapping extension PCR with the
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plasmid pET28a-arsM as the DNA template. The primers used for the site-directed
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mutagenesis are listed in Table S1. PCR products were double-digested by EcoRI and
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XhoI, and cloned into the prepared EcoRI/XhoI-digested pET28a vector. All of the
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sequences of the arsM mutants were verified by DNA sequencing to ensure that no
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errors had been introduced during amplification. E. coli BL21(DE3) was transformed
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using vectors carrying different mutations of arsM gene. Protein expression,
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purification, identification and concentration determination were performed as
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described above. Three mutants of ArsM (C65S, C153S, C203S) were constructed.
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Arsenic speciation analysis
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Arsenic speciation was analyzed by HPLC-ICP-MS (7500a; Agilent Technologies)
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as described (Zhu et al., 2008). Chromatographic columns were obtained from
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Hamilton and consisted of a precolumn (11.2 mm; 12-20 μm) and a PRP-X100 10-μm
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anion-exchange column (250 x 4.1 mm). The mobile phase consisted of 10 mM
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diammonium hydrogen phosphate [(NH4)2HPO4] and 10 mM ammonium nitrate
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[NH4NO3], adjusted to pH 6.2. The mobile phase was pumped through the column at
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a flow rate of 1.0 ml min-1.
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Results
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Arsenic methylation by SRB Clostridium sp. BXM
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When Clostridium sp. BXM was grown in the medium containing arsenite [As(III)]
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or arsenate [As(V)] for 20 days, monomethylarsenic (MMAs) and dimethylarsenic
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(DMAs) were detected (Fig. 1). About 9% of the total As in the medium has been
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methylated to MMAs as well as about 1% to DMAs (Fig. S1). No methylated As was
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detected in the control (no bacterial inoculation). This result suggested that
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Clostridium sp. BXM have the capability of methylating As.
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Cloning of the arsM gene from Clostridium sp. BXM
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By homologous comparison with the As(III) methyltransferases previously reported
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(Lin et al., 2002; Qin et al., 2006; Qin et al., 2009; Yin et al., 2011), a putative arsM
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gene (813 bp) was cloned from the genome of Clostridium sp. BXM, and designated
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as CsarsM. CsArsM protein encoded by CsarsM gene contained 270 residues, with a
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predicted molecular mass of 29.06 kDa.
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Comparative study based on protein sequences showed that CsArsM had the
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highest similarity with the ArsM from anaerobic bacterium Rhodopseudomonas
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palustris CGA009 (RpArsM) (53% identity and 84% similarity) (Qin et al., 2006).
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With those aerobic cyanobacterial ArsMs (Yin et al., 2011), CsArsM shared lower
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similarity (average 21% identity and 57% similarity). In addition, CsArsM also
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exhibited significant, but lower similarity to mammalian AS3MT methyltransferase
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(21% identity and 53% similarity) (Lin et al., 2002; Song et al., 2011), and to the
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ArsM of eukaryotic alga Cyanidioschyzon sp. isolate 5508 (20% identity and 51%
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similarity) (Qin et al., 2009).
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CsArsM enzyme detoxified As(III) in vivo
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CsarsM gene was expressed in an As-sensitive E. coli AW3110 (ΔarsRBC), which
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had no orthologous arsM gene. Expression of arsM clearly conferred the strain
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AW3110 with the resistance to As(III). Cells expressing CsarsM gene grew much
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better than that bearing empty plasmid pET28a when exposed to 50 μM As(III) (Fig.
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2). Although all bacteria exhibited a weak growth under 100 μM As(III), cells
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expressing CsarsM gene showed a little better growth than that bearing empty vector
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(Fig. 2). ArsM enzyme as the product of arsM gene maybe metabolize cellular As(III)
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to less toxic methylated products for facilitating growth. In other words, ArsM
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enzyme reduced the As toxicity in the cells, consistent with the idea that As
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methyltransferase has an important physiological role in detoxifying As.
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As(III) methylation and volatilization by E. coli AW3110 expressing arsM
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DMAs and trimethylarsine oxide (TMAsO) except MMAs were observed in the
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medium of E. coli expressing CsarsM gene by comparison with the control (Fig. 3A,
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B). This result indicated that the enzyme encoded by CsarsM gene is indeed
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responsible for As(III) methylation. The time-course of As methylation by the strain
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AW3110 expressing CsarsM showed that TMAsO and DMAs have accumulated in
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the medium after 3 h incubation and increased with the culture time increasing,
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concomitant with a decrease of inorganic As (Fig. 3B). No detectable MMAs was
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observed throughout the experiment (within 20 h). Since the expression of CsarsM
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gene was controlled by T7 promoter, as well as with the induction of isopropyl
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β-D-thiogalactoside (IPTG), abundant CsArsM protein may be produced and could
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quickly convert MMAs to DMAs in the recombinant E. coli. About 10% of total As
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was transformed into DMAs and another 10% into TMAsO by E. coli in the presence
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of 10 μM As(III) (Fig. 3A). The total As in the medium of E. coli expressing CsarsM
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is a bit lower than corresponding control (empty vector), probably attributed to As
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volatilization in the former.
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E. coli AW3110 expressing CsarsM gene effectively volatilized As when exposed
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to different As(III) concentrations (Fig. 3C). The major gaseous product was TMAs
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which was oxidized and identified as TMAsO, together with tiny amount of
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dimethylarsine (DMAsH) detectable as oxidized DMAs (Fig. S2). The amount of As
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volatilization in 100 μM As(III) was more than that in 50 μM As(III) (Fig. 3C),
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though the cellular growth in the former was much weaker than that in the latter (Fig.
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2).
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Methylation of As(III) by purified ArsM enzyme in vitro
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To further elucidate the function of CsarsM gene in As methylation, corresponding
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CsArsM protein was purified from recombinant E. coli cytosol for its As methylation
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activity analysis (Fig. 4A). Cofactor glutathione (GSH) as a necessary component was
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used to form As–GSH complex, which was optimal substrate for ArsM enzyme.
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S-Adenosyl-Methionine (AdoMet) was used as the methyl donor in ArsM-catalyzed
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As(III) methylation reaction in vitro. CsArsM enzyme converted As(III) to MMAs
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after 0.5 h, with small amount of DMAs appearing at 1.5 h (Fig. 4B). The
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concentrations of DMAs and MMAs kept increasing during the experiment process,
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concomitant with the decrease of inorganic As (Fig. 4B). At 20 h, DMAs became the
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dominant organic As species. Only little MMAs have been produced within 20 h
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compared to DMAs. TMAsO had not appeared until the time got to 20 h.
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About 45% of the total As was converted to the organic As by CsArsM in the
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presence of 5 μM As(III) (Fig. 4C). The total amount of methylated As increased with
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the increasing As(III) concentrations in the reaction system (Fig. 4C), although the
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percentage of that indeed decreased. In the presence of 20 μM As(III), about 23% of
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inorganic As was methylated by CsArsM (Fig. 4C). In the absence of GSH or AdoMet,
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no methylated species were observed (data not shown).
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Conserved cysteine (Cys) residues Cys65, Cys153, and Cys203 of CsArsM
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All ArsM proteins identified to date have three conserved cysteine residues
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(Thomas et al., 2007; Hamdi et al., 2012; Ye et al., 2012), at positions 72, 174, and
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224 in CmArsM of eukaryotic alga Cyanidioschyzon sp. 5508 (Marapakala et al.,
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2012), which were responsible for As methylation. The conserved cysteine residues in
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CsArsM were Cys65, Cys153, and Cys203. To elucidate the function of three
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conserved cysteines in As methylation, Cys65, Cys153, and Cys203 in CsArsM were
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substituted for serines, respectively, creating the mutants of C65S, C153S and C203S.
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All three mutants lost their activities of As(III) methylation completely (Fig. 5A). In
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contrast, methylation of MMAs was completed by the C65S mutant but not by the
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C153S or C203S (Fig. 5B). These results suggest that the three conserved cysteines
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(Cys65, Cys153 and Cys203) are required for the first methylation step [from As(III)
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to MMAs], while only Cys153 and Cys203 are required for the second methylation
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step (from MMAs to DMAs) in the Challenger’s pathway (Challenger, 1945; Cullen
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and Reimer, 1989). Note that Cys29, Cys30 and Cys262 in CsArsM were not
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conserved in other ArsM orthologues and their single mutations did not affect
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enzymatic activity of As methylation (data not shown).
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Discussion
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Due to the critical role of microorganisms in As biogeochemical cycling in wetland
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and water environments, it is important to understand As transformation by anaerobic
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bacteria (Tsai et al., 2009). The methylation of As is one of the key processes
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governing the fate of As in the environment, and thus detailed understanding of the
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mechanism of As methyltransferase is crucial. This study provided the insight into a
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newly identified arsenite S-Adenosyl-Methionine methyltransferase from anaerobic
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SRB, which play crucial roles in As transformation of anaerobic environments (Kirk
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et al., 2004; Oremland et al., 2004). CsArsM enzyme encoded by CsarsM gene
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cloned from SRB Clostridium sp. BXM conferred the ability of methylating and
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volatilizing As to As-sensitive strain of E. coli, concomitant with an increase of
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cellular tolerance to As. These results suggest that ArsM-mediated As methylation in
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SRB is a mechanism for relieving cellular toxicity of As.
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By the site-directed mutagenesis studies, we confirmed that three residues of
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CsArsM, Cys65, Cys153, and Cys203 are required for As(III) methylation, while only
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Cys153 and Cys203 are necessary for MMAs methylation. These cysteines are
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conserved in all ArsM and AS3MT proteins identified to date (Thomas et al., 2007;
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Ye et al., 2011; Ye et al., 2012). For example, they are homologous to Cys72, Cys174,
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and Cys224 of CmArsM from eukaryotic alga Cyanidioschyzon sp. 5508 (Marapakala
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et al., 2012). It was reported that the substitution of Cys72, Cys174, or Cys224 led to
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loss of As(III) methylation, while the substitution of Cys174 or Cys224 abolished the
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methylation of monomethylarsenite [MMAs(III)]. These cysteines affected the
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binding of As(III) or MMAs(III) to CmArsM enzyme during the methylation process.
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Similarly, the ArsM from Danio rerio has been identiﬁed to possess two conserved
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residues, Cys160 and Cys210, which were suggested to be related to As(III) binding
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(Hamdi et al., 2012). To the recombinant human AS3MT enzyme (Geng et al., 2009),
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Cys72, Cys156 and Cys206 have been proved to be functionally conserved, any
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substitution of which abolished enzymatic activity in As methylation (Song et al.,
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2009, 2011).
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Inorganic As predominately undergoes successive oxidative and reductive
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methylation reactions: As(III) → MMAs → DMAs → TMAs (Challenger, 1945).
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Dimethylated As is the major product of purified CsArsM, with relatively less
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amounts of MMAs and TMAsO produced (Fig. 4C). DMAs can bind to only a single
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thiol of ArsM and readily dissociates from enzyme (Kitchin and Wallace, 2006). This
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would cause that the third methylation step (from DMAs to TMAs) occurs difficultly
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and less TMAsO is produced. The product MMAs of the first methylation step can
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bind to two thiols of ArsM enzyme. The resulting complex is relatively stable and
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prone to the second step of methylation (Kitchin and Wallace, 2006; Kitchin, 2011),
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causing less MMAs accumulation and more DMAs production. DMAs accumulates
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as the principal product in the reaction system.
343
Arsenic methylation in soils increased with the decrease of redox potential (Frohne
344
et al., 2011), suggesting that it may occur more easily in anaerobic microorganisms
345
than aerobic microorganisms (Wang et al., 2014). Wetlands, a typical submerged soil
346
with low redox potential, have been recognized as important sinks for As, and
347
typically play an important role in As transformation and mobilization (Vriens et al.,
16
348
2013). Our results demonstrating the ability of As methylation by SRB may have
349
implications to other anaerobic microorganisms. Elucidating such processes is critical
350
in predicting the metabolic pathways and fate of As in the environment. For example,
351
As methylation in soil influences its accumulation by rice (Jia et al., 2013) and
352
methylated As in rice grains was greatly increased when rice was grown in anaerobic
353
soil compared with the corresponding aerobic soil (Xu et al., 2008; Arao et al., 2009).
354
This phenomenon was due to anaerobic microbial activities in paddy soil. It has been
355
implicated that SRB is among the key functional microorganisms in paddy soil and
356
they may contribute to the generation of methylated As (Liu et al., 2009; Lomax et al.,
357
2012). The activities of SRB in paddy soil may reduce the ecological and health
358
effects of As pollution by transforming As from the more hazardous forms to less
359
toxic or volatile methylated forms. SRB have shown the potential for the use in the
360
remediation of environments contaminated with toxic metal(loid)s (Jong and Parry,
361
2003; Teclu et al., 2008). Considering its ubiquity in sediments and wetlands, it seems
362
that SRB could play a significant role in As remediation and biogeochemical cycling
363
in widespread anaerobic environments.
364
365
Acknowledgements
366
This project was financially supported by the Natural Science Foundation of China
367
(No. 41371459), the State Key Program of Natural Science Foundation of China (No.
368
41330853) and the National High Technology Research and Development Program of
369
China (863 Program, 2013AA06A209).
17
370
371
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25
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Figure legends
540
Fig. 1. Arsenic transformation by Clostridium sp. BXM. Clostridium sp. BXM was
541
cultured with 5.3 μM As(III) or As(V) for 20 days. The medium without bacteria
542
inoculation was as control. The As species in the medium were analyzed as described
543
in “Experimental procedures”.
544
545
Fig. 2. The growth curves of E. coli AW3110 (ΔarsRBC) expressing CsarsM or empty
546
vector when exposed to different concentrations of NaAsO2. E. coli bearing empty
547
vector pET28a or pET28a-arsM was cultured in LB medium with the indicated
548
concentrations of NaAsO2, 50 μg ml-1 kanamycin and 0.3 mM IPTG at 37°C. Cellular
549
growths were monitored by spectrophotometer at absorbance of 600 nm (A600) at the
550
indicated time points. The error bars indicate the standard errors of three triplicates.
551
552
Fig. 3. Arsenic methylation and volatilization by E. coli expressing pET28a-arsM. (A)
553
As(III) methylation by E. coli AW3110 (ΔarsRBC) expressing CsarsM. E. coli
554
expressing empty pET28a or pET28a-arsM was incubated in LB medium with the
555
indicated concentrations of As(III) (10, 20, 50 μM), 50 μg ml-1 kanamycin and 0.3
556
mM IPTG at 37°C for 20 h. The As species in the supernatants were analyzed by
557
HPLC-ICP-MS as described in “Experimental procedures”. C, control, which was E.
558
coli expressing empty vector pET28a; arsM, E. coli expressing CsarsM. The error
559
bars indicate the standard errors of three triplicates. (B) Time dynamics of As(III)
560
methylation by E. coli AW3110 (ΔarsRBC) expressing CsarsM. The strain was
26
561
incubated in LB medium with 20 μM As(III), 50 μg ml-1 kanamycin and 0.3 mM
562
IPTG at 37°C. At the indicated time points (3, 6, 9, 13, 16, 20 h), 1 ml samples of the
563
bacterial culture were taken and centrifuged at 8000 r.p.m. The supernatants were
564
appropriately diluted and analyzed for As species by HPLC-ICP-MS. (C)
565
Volatilization of As by E. coli AW3110 (ΔarsRBC) expressing CsarsM. E. coli with
566
CsarsM was grown in LB medium with a series of As(III) concentrations, 50 μg ml-1
567
kanamycin and 0.3 mM IPTG at 37°C for 48 h. Volatile As produced was
568
chemo-trapped as described in “Experimental procedures” and the total quantities of
569
volatile As were analyzed by ICP-MS. The error bars indicate the standard errors of
570
three triplicates.
571
572
Fig. 4. Purification of CsArsM protein and As(III) methylation by purified CsArsM.
573
(A) SDS-PAGE showing the purity of the soluble CsArsM. Lane 1, protein standards;
574
Lane 2, cell lysis solution; Lane 3, collection of the washing buffer (20 mM imidazole)
575
outflowing from Ni(II)-NTA column; Lane 4, collection of the washing buffer (50
576
mM imidazole) outflowing from Ni(II)-NTA column; Lane 5, concentrated
577
His-tagged CsArsM protein. (B) Time dynamics of As(III) methylation by purified
578
CsArsM enzyme. Each assay contained 10 μM As(III), 0.5 mM AdoMet, 8 mM GSH,
579
and 4 μM purified CsArsM in 500 μl MOPS buffer (pH 7.4). The reactions were kept
580
at 37°C. Arsenic species of the samples were analyzed at the indicated time points by
581
HPLC-ICP-MS as described in “Experimental procedures”. The error bars indicate
582
the standard errors of three triplicates. (C) The final products of As(III) methylation
27
583
by purified CsArsM enzyme at 37°C for 20 h. Each assay contained the indicated
584
concentrations of As(III) (5, 10, or 20 μM), 0.5 mM AdoMet, 8 mM GSH, and 10 μM
585
purified CsArsM in 500 μl MOPS buffer (pH 7.4). Arsenic species were analyzed by
586
HPLC-ICP-MS. The error bars indicate the standard errors of three triplicates.
587
588
Fig. 5. Methylation of As(III) or MMAs by wild-type or mutant CsArsM enzymes.
589
Methylation of As(III) or MMAs was tested in a reaction system (pH 7.4) with 10 μM
590
As(III) (A) or MMAs (B), 8 mM GSH, 0.5 mM AdoMet and 2 μM ArsM enzyme.
591
The reactions were kept at 37 °C for 20 h. The As species of the samples were
592
analyzed by HPLC-ICP-MS.
593
28
594
595
29
596
597
30
598
599
31
600
601
32
602
603
33
604
Graphical abstract
605
606
34
607
Paper-table of content2
608
609
610
35
611
612
Identification and its catalytic residues of the arsenite
613
methyltransferase from a sulfate-reducing bacterium,
614
Clostridium sp. BXM
615
616
Pei-Pei Wang, Yong-Guan Zhu, Peng Bao, and Guo-Xin Sun*
617
618
*Corresponding author, E-mail: [email protected].
619
620
621
Fig. S1. The As speciation produced by Clostridium sp. BXM. Clostridium sp. BXM
622
was exposed to 5.3 μM As(III) for 20 days. The As species in the medium were
623
analyzed as described in “Experimental procedures”. The error bars indicate the
624
standard errors of three triplicates.
625
626
36
627
628
629
Fig. S2. The species of volatile As formed by E. coli AW3110 (ΔarsRBC) expressing
630
CsarsM. The E. coli expressing empty vector pET28a was as control. E. coli was
631
grown in LB medium with 100 μM As(III), 50 μg ml-1 kanamycin and 0.3 mM IPTG
632
at 37°C for 48 h. The volatile As produced was trapped and analyzed for species by
633
HPLC-ICP-MS as described in “Experimental procedures”.
634
635
636
37
637
638
Table S1 Primers used for the site-directed mutagenesis in enzyme CsArsM
C65S
C153S
C203S
Whole
Primer
Sequence
+
5'CAATTCGTTTGGGTCTGGTAACCCCACTGC3'
-
5'GCAGTGGGGTTACCAGACCCAAACGAATTG3'
+
5'TATTATTTCAAACTCTGTAATTAATCTTTC3'
-
5'GAAAGATTAATTACAGAGTTTGAAATAATA3'
+
5'TTGCTTGGGCCGGATCTATTGCTGGAGCAA3'
-
5'TTGCTCCAGCAATAGATCCGGCCCAAGCAA3'
+
5'CGGAATTCATGGATAATATTCGAGAGGGAGTAA3'
-
5'CCCTCGAGTGCCGGCTTGGCTGCCCGAATAAA3'
639
640
38

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