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. Author manuscript; available in PMC: 2016 Nov 1.
Published in final edited form as: Mol Microbiol. 2015 Aug 22;98(4):625–635. doi: 10.1111/mmi.13145

ArsP: a methylarsenite efflux permease

Jian Chen 1, Mahendra Madegowda 1, Hiranmoy Bhattacharjee 1, Barry P Rosen 1,*
PMCID: PMC4681449  NIHMSID: NIHMS741532  PMID: 26234817

Abstract

Trivalent organoarsenic compounds are far more toxic than either pentavalent organoarsenicals or inorganic arsenite. Many microbes methylate inorganic arsenite (As(III)) to more toxic and carcinogenic methylarsenite (MAs(III)). Additionally, monosodium methylarsenate (MSMA or MAs(V)) has been used widely as an herbicide and is reduced by microbial communities to MAs(III). Roxarsone (3-nitro-4-hydroxybenzenearsonic acid) is a pentavalent aromatic arsenical that is used as antimicrobial growth promoter for poultry and swine, and its active form is the trivalent species Rox(III). A bacterial permease, ArsP, from Campylobacter jejuni, was recently shown to confer resistance to roxarsone. In this study C. jejuni arsP was expressed in Escherichia coli and shown to confer resistance to MAs(III) and Rox(III) but not to inorganic As(III) or pentavalent organoarsenicals. Cells of E. coli expressing arsP did not accumulate trivalent organoarsenicals. Everted membrane vesicles from those cells accumulated MAs(III)>Rox(III) with energy supplied by NADH oxidation, reflecting efflux from cells. The vesicles did not transport As(III), MAs(V) or pentavalent roxarsone. Mutation or modification of the two conserved cysteine residues resulted in loss of transport activity, suggesting that they play a role in ArsP function. Thus ArsP is the first identified efflux system specific for trivalent organoarsenicals.

Keywords: Methylarsenite, herbicide resistance, roxarsone, antimicrobial growth promoter, efflux permease

Introduction

Arsenic is a ubiquitous toxic metalloid that enters the environment primarily from geochemical sources and to a lesser extent from anthropogenic sources (Zhu et al., 2014). Humans are continuously exposed to this Group I carcinogen in food, water, air and soil (Naujokas et al., 2013). Exposure is most often to inorganic arsenicals, either arsenate (As(V)) or arsenite (As(III)). As a result, nearly every organism has evolved pathways for detoxification of inorganic arsenic (Liu et al., 2013). In bacteria these resistance genes are organized in ars operons (Yang et al., 2012). The most common ars genes encode efflux transporters specific for As(III) (arsB or acr3) or reductases (arsC) that reduce As(V) to As(III) for subsequent efflux (Yang et al., 2012, Mukhopadhyay & Rosen, 2002).

Recently additional genes encoding enzymes of arsenic biotransformations have been identified. One such is ArsM, an As(III) S-adenosylmethionine methyltransferase, that methylates As(III) into more toxic and carcinogenic methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)) (Qin et al., 2009, Qin et al., 2006). These are rapidly oxidized in air to the relatively nontoxic pentavalent species MAs(V) and DMAs(V), so organisms with arsM detoxify As(III). In addition to biologically-generated methylated arsenicals, the pentavalent methylated arsenicals, monosodium methanearsonate (MSMA) and disodium methanearsonate (DSMA or cacodylate), have been or are still used as herbicides (http://www2.epa.gov/ingredients-used-pesticide-products/monosodium-methanearsonate-msma-organic-arsenical). Some bacteria reduce MAs(V) to MAs(III), which acts like an antibiotic to kill off other members of the microbial communities (Yoshinaga et al., 2011). In response to this environmental pressure, other bacteria have evolved other ars genes that detoxify MAs(III). ArsI is a newly identified C-As bond lyase, a member of the dioxygenase superfamily, that detoxifies MAs(III) by cleaving it into As(III) and H2CO (Yoshinaga & Rosen, 2014). A second independently-evolved MAs(III) detoxification enzyme is ArsH, an NADPH-FMN dependent oxidoreductase that oxidizes MAs(III) to MAs(V) (Chen et al., 2015).

Recently, a putative membrane permease (CjArsP), encoded in a four-gene ars operon in Campylobacter jejuni, was shown to confer resistance to cells exposed to roxarsone (Shen et al., 2014). Roxarsone, a pentavalent aromatic arsenical, has been widely used in poultry production to treat coccidiosis and for growth promotion (Cortinas et al., 2006). Although Pfizer has voluntarily suspended sales of roxarsone in the U.S., it is still widely produced in countries such as China and India. We have previously shown that roxarsone, a pentavalent aromatic arsenical, is not toxic to bacteria, but that the reduced trivalent species is highly toxic (Chen et al., 2014). Consequently, we propose that the true substrate of CjArsP is Rox(III), not Rox(V). For that reason, in this study we investigated the transport properties of CjArsP. Heterologous expression of CjarsP in the arsenite-hypersensitive E. coli strain AW3110, in which the chromosomal arsRBC operon had been deleted (Carlin et al., 1995), confers resistance to MAs(III), Rox(III) and reduced forms of the related aromatic arsenical growth promoters phenylarsenite (PhAs(III)), nitarsone (4-nitrophenyl)arsenite (Nit(III)) and p-aminophenyl arsenite (pASA(III)), but not As(III) or pentavalent organoarsenicals. Cells of E. coli AW3110 without CjarsP accumulated the trivalent organoarsenicals, while cells expressing CjarsP did not. Everted membrane vesicles prepared from the CjarsP-expressing strain accumulated MAs(III) with the energy supplied by NADH oxidation. No transport of As(III), MAs(V) or pentavalent aromatic arsenicals was observed. MAs(III) was taken up to much higher levels than Rox(III) and PhAs(III), indicating that CjArsP is selective for the methylated compound. CjArsP has two cysteine residues (Cys65 and Cys67) conserved in ArsP orthologs. Mutagenesis of either resulted in loss of resistance and transport activity, indicating that the vicinal cysteine pair plays a role in ArsP function. These results demonstrate that ArsP is a pathway for efflux and detoxification of trivalent organoarsenicals. Thus prokaryotes have independent transport pathways for detoxification of inorganic As(III) (ArsB and Acr3) and organoarsenicals (ArsP).

Results

The arsP gene from C. jejuni confers resistance to trivalent organoarsenicals

Cells of C. jejuni expressing the arsP gene have been shown to exhibit resistance to pentavalent roxarsone and nitarsone (Shen et al., 2014). In this study the CjarsP gene was cloned downstream of the trc promoter in plasmid pTrcHis2A and expressed in the arsenite-hypersensitive E. coli strain AW3110, in which the chromosomal arsRBC operon was deleted (Carlin et al., 1995). E. coli strain AW3110 lacking the chromosomal ars operon is sensitive to 50 µM As(III) (Fig. 1A) but not to pentavalent MAs(V) (Chen et al., 2014). Expression of either R773 arsB or Corynebacterium glutamicum acr3 complemented the arsenite (As(III)) hypersensitivity of AW3110 cells. In contrast, E. coli AW3110 expressing C. jejuni arsP (CjarsP) remained sensitive to As(III), but exhibited resistance to MAs(III) (Fig. 1B). AW3110 cells exhibited sensitivity to as little as 10 µM MAs(III), while cells expressing CjarsP were resistant to MAs(III) either in liquid (Fig. 1B) or solid medium (Fig. S1). Expression of neither arsB nor acr3 complemented the sensitivity of AW3110 cells to MAs(III) (Fig. 1B).

Fig 1. CjarsP confers resistance to trivalent organoarsenicals but not inorganic As(III).

Fig 1

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Overnight cultures of E. coli strain AW3110 bearing either pTrcHis2A-CjArsP (CjarsP) (▽), pKK223-ArsB (R773 arsB) (□), pTrcHis2A-Acr3 (Cgacr3) (◊) or vector plasmid pTrcHis2A (○) were diluted 100-fold into fresh M9 medium containing the indicated concentrations of arsenical as indicated. A, As(III); B, MAs(III); C, Rox(III); D, PhAs(III); E, Nit(III); and F, pASA(III). Expression of the ars genes were induced with 0.1 mM IPTG. After 24 h of growth at 30 °C, the absorbance at 600 nm was measured. Data are the mean ± SE (n = 3).

E. coli AW3110 is naturally resistant to pentavalent aromatic arsenicals (Chen et al., 2014) but is sensitive to trivalent aromatic arsenicals (Fig. 1 and S1). In addition to trivalent methylated arsenic, cells of E. coli AW3110 expressing CjarsP were resistant in liquid medium to the trivalent aromatic arsenicals Rox(III) (Fig. 1C), PhAs(III) (Fig. 1D), Nit(III) (Fig. 1E) and pASA(III) (Fig. 1F). On solid medium resistance to MAs(III) was obvious (Fig. S1A), but resistance to synthetic aromatic arsenicals was not as clear (Fig. S1B–E). Neither arsB nor acr3 expression complemented sensitivity of AW3110 cells to any of the trivalent aromatic arsenicals (Fig. 1C–F). Cells expressing CjarsP exhibited the greatest resistance to MAs(III) compared with synthetic aromatic organoarsenicals, suggesting that MAs(III), which can be produced by microbes, is the preferred physiological substrate of CjArsP. Since the ars operon was deleted in E. coli AW3110, CjarsP expression alone is sufficient to confer resistance to the active trivalent form of the herbicide MSMA and to the aromatic arsenical antimicrobial growth promoters. These results clearly demonstrate that ArsP is an MAs(III)-selective resistance, while ArsB and Acr3 are specific for inorganic As(III).

Role of Cys65 and Cys67 in organoarsenical extrusion by ArsP

CjArsP has five cysteine residues located at position 65, 67, 169, 170 and 277 of its primary sequence. Of those, Cys65 and Cys67 are present in a highly conserved motif (TPFCSCSXXP); the other three cysteine residues are not conserved (Fig. S2). Blast analysis of CjArsP with the NCBI data base yielded 7856 entries in 4582 bacterial and archaeal species. Nearly all have the two conserved cysteine residues. An exception is Cupriavidus metallidurans CH34 ArsP, which lacks the conserved motif (Fig. S2). To examine whether this motif has a functional role, the arsP genes from Shewanella putrefaciens and Cupriavidus metallidurans CH34 were also cloned downstream of the trc promoter in plasmid pTrcHis2A and expressed individually in E. coli strain AW3110. CjArsP exhibits 34% sequence identity with SpArsP and only 15% identity with CmArsP. AW3110 cells expressing S. putrefaciens arsP (SparsP) were resistant to MAs(III), PhAs(III), Rox(III), Nit(III) and pASA(III) (Fig. 2). In contrast, cells expressing C. metallidurans arsP (CmarsP) were as sensitive to organoarsenicals as cells lacking an arsP gene, which indicates that the C. metallidurans protein is not an ArsP ortholog. These results suggest that presence of the conserved motif differentiates ArsP orthologs from related permeases.

Fig 2. CjArsP and SpArsP but not CmArsP confer trivalent organoarsenical tolerance.

Fig 2

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Overnight cultures of E. coli strain AW3110 bearing either pTrcHis2A-CjArsP (CjarsP) (light grey), pTrcHis2A-SpArsP (SpArsP) (white), pTrcHis2A-CmArsP (CmarsP) (dark grey) or vector plasmid pTrcHis2A (black) were diluted 100-fold into fresh M9 medium containing the indicated concentrations of arsenicals. Expression of the arsP genes were induced with 0.1 mM IPTG. Growth was measured after 24 h at 30 °C. Data are the mean ± SE (n = 3).

To examine whether the two cysteine residues, Cys65 or Cys67, in the conserved motif are involved in ArsP activity, each was changed individually to serine by site-directed mutagenesis. Expression of neither the CjarsPC65S nor the CjarsPC67S mutant gene conferred resistance to MAs(III) (Fig. 3). The level of expression of wild type and altered CjarsPs in AW3110 cells was estimated by immunoblot analysis using anti-His antibodies. Each altered protein was produced in approximately the same amount and migrated with the same mobility as wild type CjArsP indicating that the mutations did not affect expression (Fig. S3).

Fig 3. Effect of alteration of the conserved cysteine residues on MAs(III) resistance.

Fig 3

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Resistance analyses in E. coli AW3110 cells expressing either pTrcHis2A-CjArsP (wild type), pTrcHis2A-C65S CjArsP (C65S), pTrcHis2A-C67S CjArsP (C67S) or vector plasmid pTrcHis2A were performed in M9 medium in the absence (black) or presence (white) of 20 µM MAs(III). Expression of arsP genes were induced with 0.1 mM IPTG. Growth was measured after 24 h at 30 °C. Data are the mean ± SE (n = 3).

CjArsP catalyzes trivalent organoarsenical efflux

Cells expressing CjarsP accumulated considerably less MAs(III) compared to cells lacking the gene (Fig. 4). Cells expressing CjarsPC65S nor the CjarsPC67S genes were unable to keep MAs(III) out of the cells. No uptake of pentavalent organoarsenicals was observed in E. coli AW3110, so it was not possible to determine whether cells of E. coli AW3110 expressing CjarsP can extrude pentavalent species. However, cells expressing CjarsP exhibited reduced accumulation of Rox(III), PhAs(III), Nit(III) or pASA(III) (Fig. S4). These results suggest that ArsP is a trivalent organoarsenical efflux permease.

Fig. 4. Effect of alteration of the conserved cysteine residues on MAs(III) transport.

Fig. 4

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Uptake of 20 µM MAs(III) by cells of E. coli AW3110 expressing wild type and mutant CjarsP genes was performed as described in Experimental Procedures. Plasmids: (▽), pTrcHis2A-CjArsP (wild type); (□), pTrcHis2A-CjArsPC65S; (◊), pTrcHis2A-CjArsPC67S or (○), vector plasmid pTrcHis2A. Data are the mean ± SE (n = 3).

While those results are consistent with active extrusion of trivalent organoarsenicals, direct evidence could be obtained by measuring uptake into everted membrane vesicles. The use of everted membrane vesicles to assay efflux permeases coupled to the proton motive force has been well-documented (Adler et al., 1977, Rosen & Tsuchiya, 1979, Tsuchiya & Rosen, 1975). The orientation of the membrane in these vesicles in opposite that of intact cells, which renders the cytosolic side of the membrane accessible to respiratory substrates such as NADH, and solutes that are extruded from intact cells are accumulated in the vesicles. Everted membrane vesicles from AW3110 expressing the arsB gene have been shown to accumulate As(III) and Sb(III) (Meng et al., 2004). Everted membrane vesicles from cells expressing CjarsP accumulated MAs(III) when NADH was supplied as a respiratory substrate (Fig. 5). No uptake was observed without NADH or in vesicles from cells with vector only. Addition of the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) produced a rapid loss of accumulated MAs(III). Vesicles from cells expressing CjarsP also accumulated either Rox(III) or PhAs(III) (Fig. 6) but not As(III), MAs(V) or pentavalent aromatic arsenicals (Fig. S5). These results support the hypothesis that ArsP catalyzes efflux of MAs(III) and trivalent aromatic arsenicals but not inorganic As(III) or pentavalent organoarsenicals, and that efflux is coupled to the electrochemical proton gradient established by NADH respiration.

Fig. 5. Energy-dependent accumulation of MAs(III) in everted membrane vesicles.

Fig. 5

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Accumulation of 20 µM MAs(III) in everted membrane vesicles prepared from E. coli strain AW3110 harboring either plasmid pTrcHis2A-CjArsP or vector plasmid pTrcHis2A was assayed as described in Experimental Procedures. pTrcHis2A-CjArsP with (◊) or without (□) 5 mM NADH; pTrcHis2A-CjArsP with 5 mM NADH to which 10 µM FCCP was added at the indicated time (Δ); vector plasmid pTrcHis2A with (▽) or without (○) 5 mM NADH. Data are the mean ± SE (n = 3).

Fig. 6. Uptake of trivalent organoarsenicals into everted membrane vesicles.

Fig. 6

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Accumulation of 20 µM each of the indicated organoarsenical in everted membrane vesicles prepared from E. coli strain AW3110 harboring either plasmid pTrcHis2A-CjArsP ((●), MAs(III); (▼), Rox(III); (○), PhAs(III)) or vector plasmid pTrcHis2A ((Δ), MAs(III); (□), Rox(III); (■), PhAs(III)) was assayed as described in Experimental Procedures. The assays were performed at a final concentration of in the presence of 5 mM NADH. Arsenic uptake was measured by ICP-MS. Data are the mean ± SE (n = 3).

Membrane topology and a homology model of CjArsP

From topological algorithms, Shen et al (Shen et al., 2014) predicted that ArsP has eight transmembrane spanning helices (TMs). We analyzed ArsP membrane topology using the TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) and arrived at a similar conclusion of 8 TMs. In contrast to their topology model, we predict that the N- and C-termini and the central hydrophilic loop are intracellular, with four short loops connecting the transmembrane helices in the periplasm (Fig. 7A), with the conserved Cys65SerCys67 in the middle of TM2. It may be that their prediction that the N- and C-termini are extracellular is due to their assignment of ArsP as a member of the Duf318 family of β-barrel outer membrane porins, but our topology model prediction is primarily α-helical without significant β-strand character.

Fig. 7. ArsP transmembrane topology and homology structural model.

Fig. 7

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A. The CjArsP transmembrane topology model was calculated with TMHMM and illustrated with Protter (Omasits et al.). Eight TMs are predicted, with conserved residues Cys65 and Cys67 (yellow) in TM2. B. A homology model of CjArsP from residues 3–315 was constructed based on the structure of the E. coli GlpT glycerol-3-phosphate/phosphate exchanger (PDB entry:1PW4) (Huang et al., 2003). Transmembrane helix 2 is colored in green. C. Expanded view of TM2 with Cys65 and Cys67 shown in stick form. The distance between the two sulfur atoms is calculated to be 10.5 Å.

We queried the NCBI structural database for structural homologues of CjArsP and found that it aligned well with the core (TMs 3–10) of the E. coli glycerol-3-phosphate transporter (GlpT; PBD 1PW4) (Huang et al., 2003) over 277 residues with an RMSD of 0.81 Å. A structural homology model was constructed that is consistent with 8 TMs, with both the N- and C-termini in the cytosol, and the Cys65SerCys67 in TM2 (Fig. 7B). The observed distance between the sulfur atoms of the two cysteines is approximately 10.5 Å (Fig. 7C). We theorize that these two thiolates form the binding site for MAs(III) and trivalent aromatic arsenicals. Based on the single-binding site, alternating-access mechanism proposed for permeases such as GlpT (Huang et al., 2003), we propose that that ArsP assumes two different conformations during the transport cycle. 1) In the inward-facing conformation the two cysteine residues are close enought to each other to form a two-coordinate binding site MAs(III), and an outward-facing in which MAs(III) is released into the periplasm (Fig. 8). 2) In the inward-facing conformation, the two cysteines form the binding site, with an expected distance between the arsenic and sulfur atoms in an MAs(III)S2 site of 2.2–2.3 Å, and about 3.2 Å between the two sulfur atoms. The helix in the outward-facing conformation would be similar to that in the homology model, where the two cysteines move apart by 10.5 Å. This conformational change would facilitate release of MAs(III).

Fig. 8. Proposed transport model.

Fig. 8

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ArsP employs a binding site that is exposed alternately to the two sides of the membrane. Helices are represented by cylinders. The positions of Cys65 and Cys67 in TM2 are indicated. A. ArsP assumes a conformation in which the two cysteine residues in TM2 are exposed to the cytosol. TM2 is distorted by proline residues on each side of the two cysteine residues to allow the two sulfur atoms to form an S2 site with MAs(III). B. A conformational change reorients the binding site so that it is exposed to the periplasm. TM2 becomes more helical, disrupting the MAsS2 complex and releasing MAs(III) into the periplasm.

Discussion

C. jejuni is a Gram-negative microaerophilic bacterium that is a major cause of human gastroenteritis. It is commonly found in the feces of animals, especially chicken litter. Aromatic arsenicals, including roxarsone, nitarsone and arsanilic acid have been used as growth promoters for poultry and swine, primarily due to their antimicrobial activity to prevent coccidiosis and other infections. Consequently organoarsenical contamination of poultry litter is not uncommon. The litter is often used as fertilizer on agricultural fields, and the arsenic is taken up by plants or released into soil and surface waters (Garbarino et al., 2003, Rutherford et al., 2003, Stolz et al., 2007). It is not surprising, therefore, to find resistance to roxarsone and other aromatic arsenicals in organisms such as C. jejuni that inhabit chicken litter. Recently Shen et al (Shen et al., 2014) demonstrated that the arsP gene of C. jejuni is responsible for roxarsone resistance. They showed that ArsP is a permease that results in lowered roxarsone in the cells of C. jejuni. In this study we extend those studies to reveal that ArsP is an efflux permease for trivalent organoarsenicals, including MAs(III) and trivalent forms of the aromatic arsenicals, and not their pentavalent forms. Even though the pentavalent forms are used commercially as herbicides and growth promoters, the reduced forms are, in fact, the actual antimicrobial agents (Chen et al., 2014).

What is the original physiological substrate of ArsP? It is unlikely to be roxarsone, which has been used in animal husbandry for less than a century. ArsP exhibits selectivity for MAs(III) over trivalent aromatic arsenicals, and bacteria have been metabolizing arsenic for at least 2.7 billion years (Sforna et al., 2014). We speculate that ArsP evolved to detoxify MAs(III) generated by other bacteria. Environmentally MAs(III) is generated by As(III) S-adenosylmethionine methyltransferases, termed ArsM in prokaryotic and eukaryotic microbes and AS3MT in animals (Thomas & Rosen, 2013). Although arsenic methylation has long been considered to be a pathway to detoxify inorganic arsenic (Challenger, 1947), more recently the initial product of ArsM and AS3MT has been demonstrated to be the much more toxic MAs(III) (Drobna et al., 2005). It is only after exposure to air that it is transformed into relatively nontoxic MAs(V). We propose that organisms produced MAs(III) as a primitive antibiotic before the atmosphere became oxidizing. Even though MAs(III) oxidizes when exposed to air in the present-day oxidizing atmosphere, various soil organisms re-reduce it, continually regenerating the toxic species (Yoshinaga et al., 2011). This provides a selective pressure for evolution of MAs(III) resistance genes. Two such have been identified to date. ArsI is a C-As lyase that detoxifies MAs(III) by cleaving the carbon-arsenic bond (Yoshinaga & Rosen, 2014). ArsH is an NADPH-FMN oxidoreductase that detoxifies MAs(III) by oxidizing it to MAs(V) (Chen et al., 2015). Both genes confer resistance to MAs(III) and are wide-spread in soil microbes. ArsP is here recognized as an additional member of the panoply of MAs(III) detoxifying proteins that confers resistance by active extrusion from the cell. One difference between ArsP and the others, ArsI and ArsH, is that ArsP does not depend on oxygen for activity. Since ArsI and ArsH both use molecular oxygen in their catalytic mechanism, it is likely that they evolved after the appearance of atmospheric O2. We propose that ArsP co-evolved with ArsM during the late anoxic Archean Eon. Some members of these primordial microbial communities would produce the MAs(III) antibiotic, while others would detoxify it. Even though in present-day organisms ArsM and ArsP are not linked, it is possible that the original antibiotic producer evolved ArsP as a mechanism to protect itself from its own toxic product, simultaneously pumping out the antibiotic to kill off its neighbors. This is similar to the efflux systems that antibiotic-producing bacteria employ to rid themselves of the antibiotics they produce. For example, Streptomyces peucetius produces doxorubicin and daunorubicin and pumps them out of the cells with the DrrAB efflux pump (Kaur, 1997).

ArsP is specific for MAs(III) efflux and does not transport inorganic As(III). ArsB and Acr3, the product of two other ars genes, are efflux permeases for inorganic As(III), and neither transports MAs(III). These results illustrate that there are two separate arsenic detoxification pathways, one for inorganic arsenic and the other for organic arsenic. Although both are encoded by ars operons, they do not share gene products, and we propose that they are the result of independent evolution.

Experimental Procedures

Chemicals

Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich. Roxarsone (Rox(V)) and MAs(V) were obtained from ThermoFisher Acros Organics Division, Waltham, MA, and Chem Service, Inc., West Chester, PA, respectively. Phenylarsenite (PhAs(III) or PAO), nitarsone (Nit(V)) and p-aminophenyl arsenate (pASA(V) or arsanilic acid) were purchased from Sigma-Aldrich, St. Louis, MO. Pentavalent arsenicals were reduced as described (Reay & Asher, 1977). The reduced species were simultaneously analyzed for both arsenic and sulfur by high pressure liquid chromatography (HPLC) coupled with inductively coupled mass spectroscopy (ICP-MS) (ELAN DRC-e; PerkinElmer, Waltham, MA) (Qin et al., 2006), which showed that the trivalent organoarsenicals were not thiol adducts.

Strains, media and growth Conditions

E. coli Stellar™ (Clontech Laboratories, Inc., Mountain View, CA) (F−, endA1, supE44, thi-1, recA1, relA1, gyrA96, phoA, Φ80d lacZΔ M15, Δ(lacZYA-argF)U169, Δ(mrr-hsdRMS-mcrBC), ΔmcrA, λ−) was used for plasmid DNA construction and replication. E. coli AW3110 (Δars::cam F− IN(rrn-rrnE) (Carlin et al., 1995), which is hypersensitive to As(III), was used for complementation studies. E. coli strains were grown aerobically at either 30 °C or 37 °C in either Luria-Bertani (LB) medium or M9 medium (Sambrook et al., 1989), as noted, supplemented with 125 µg/ml ampicillin or 34 µg/ml chloramphenicol, as required.

Cloning of CjarsP

The CjarsP gene (Accession number ACG76368) was amplified by polymerase chain reaction (PCR) from Campylobacter jejuni RM1221 chromosomal DNA (a gift from Qijing Zhang, Iowa State University) using the primer pairs 5’-GAGGAATAAACCATGCAGTCTTTTTTAAATACTTTCAAAGAGTTTTT-3’ and 5’-GATGATGATGGTCGACAATTAAATTAATAAAAATTCCAAAAGATATAGCC-3’. PCR was performed in a volume of 50 µl containing 0.5 µM primers, 200 µM deoxynucleoside triphosphates (dNTPs), and 1 U of Phusion® High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA). After initial heating at 94 °C for 120 s, 30 cycles of PCR amplification were performed, each consisting of four steps: denaturation at 94 °C for 20 s; annealing at 52 °C for 10 s followed by 48 °C for 10 s; and extension at 60 °C for 60 s, and finally 60 °C for ten minutes. The amplified product was gel purified and ligated to NcoI/SalI-digested pTrcHis2A vector (Life Technologies, Carlsbad, CA) using the In-Fusion® HD Cloning Plus kit (Clontech Laboratories, Inc.). The resulting plasmid (pTrcHis2A-CjArsP) encodes ArsP fused with a six-histidine residue tag at its C-terminus.

The arsP gene from genomic DNA of Cupriavidus metallidurans CH34 (a gift from Christopher Rensing, University of Copenhagen) was amplified similarly using primer pairs 5’-GAGGAATAAACCATGGAGCCGAGCAGTAGCGTGA-3’ and 5’-GATGATGATGGTCGACTGTTGTGCTGGTCTTGAGC-3’. The arsP gene from Shewanella putrefaciens 200 (a gift from Flynn Picardal, Indiana University) was amplified using primer pairs 5’-GAGGAATAAACCATGAATCCTGAAACCCTAGCCAT-3’ and 5’-GATGATGATGGTCGACGCTAAATACATAGCTATAAAGAAACC-3’. The cycling conditions were 98 °C for 30 s for the initial denaturation, followed by 30 cycles of 98 °C for 10 s and 72 °C for 45 s, and then 72 °C for 10 min. The resulting PCR products and NcoI/SalI-digested pTrcHis2A vector were ligated using an In-Fusion® HD Cloning Plus kit to create plasmid pTrcHis2A-CmArsP and pTrcHis2A-SpArsP; each ArsP has a six-histidine residue tag at its C-terminus. Each construct was transformed into Stellar™ competent cells (Clontech Laboratories, Inc.) and the transformants were selected on LB agar plates containing 125 µg/ml ampicillin.

Comparison of ArsP sequences

Multiple alignment of ArsP sequence was performed using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). 7856 entries in 4582 bacterial and archaeal species were found in the NCBI data base, and six are compared in Fig. S2.

Mutagenesis of the CjarsP gene

Mutations in C. jejuni arsP were introduced by site-directed mutagenesis using QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). The mutagenic oligonucleotides used for both strands and the respective changes introduced (underlined) are as follows: C65S, 5'-TAGGTGCTTTAACGCCATTTAGTTCCTGTTCTACCATAC-3' and 5’-GTATGGTAGAACAGGAACTAAATGGCGTTAAAGCACCTA-3'; C67S, 5'-CTTTAACGCCATTTTGTTCCAGTTCTACCATACCGCTTTTA-3' and 5'-TAAAAGCGGTATGGTAGAACTGGAACAAAATGGCGTTAAAG-3'. Each mutation was confirmed by commercial DNA sequencing (Sequetech, Mountain View, CA).

Resistance assays

For metalloid resistance assays in liquid medium, competent cells of AW3110 were transformed with constructs bearing arsP genes. Cells were grown overnight with shaking at 37 °C in LB medium with 125 µg/ml ampicillin. Overnight cultures were diluted 100-fold in M9 medium containing various concentrations of either trivalent or pentavalent arsenicals plus 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated at 30 °C with shaking for another 24 h. Growth was estimated from the absorbance at 600 nm. For inhibition assays on solid medium, 0.1 ml of fresh cultures of E. coli (1 × 108 cells) were spread over the surface of minimal agar plates supplemented with 0.2% glucose (w/v) as the carbon source and 0.1 mM IPTG as inducer. The plates were allowed to dry, and then sterile filter paper disks were applied. 10 µl of each organoarsenical was dispensed on the disks, and the plates were incubated overnight at 30 °C.

Transport assays

For in vivo uptake assays, E. coli cells were grown to A600nm = 2 at 37 °C with aeration in M9 medium. The cells were harvested and suspended in buffer A (75 mM HEPES-KOH, pH 7.5, 0.15 M KCl, and 1 mM MgSO4 at A600nm = 10. To initiate the transport reaction, either 20 µM As(III), 20 µM MAs(III)/(V), 10 µM Rox(III)(V), 5 µM PhAs(III)/(V), 10 µM Nit(III)/(V) or 10 µM pASA(III)/V(V) was added to 1 ml of cell suspension. Portions (0.1 ml) from the cell suspension were withdrawn at the indicated times, filtered through nitrocellulose filters (0.2 µm pore diameter; EMD Millipore, Billerica, MA), and washed twice at room temperature with 5 ml of buffer A. The filters were digested with 0.3 ml of concentrated HNO3 (68–70%) overnight at room temperature. The dissolved filters were incubated for 10 min at 70 °C, allowed to cool to room temperature and diluted with HPLC-grade water (Sigma-Aldrich, St. Louis, MO) to produce a final HNO3 concentration of 2%. Arsenic was quantified by ICP-MS. Standard solutions were made in the range of 0.5–50 ppb in 2% nitric acid using arsenic standard (Ultra Scientific, N. Kingstown, RI). Protein content was determined using a Pierce™ BCA Protein Assay Kit (Life Technologies).

Transport assays in everted membrane vesicles were performed as described earlier (Villadangos et al., 2012). E. coli cultures were grown in 1 L of LB at 37 °C to A600nm = 2. Cells were harvested, and the pellet suspended in buffer B (75 mM HEPES-KOH, pH 7.5, 0.15 M KCl, 1 mM MgSO4 and 0.25 M sucrose) and lysed using a Model 505 Sonic Dismembrator at 30% amplitude (ThermoFisher, Waltham, MA). The lysate was treated with diisopropyl fluorophosphate and DNase I and then centrifuged at 20,000 × g for 20 min at 4 °C to remove unbroken cells and cell debris. The supernatant suspension was centrifuged at 150,000 × g for 1 h, and the membrane fraction was suspended in buffer B. The everted membrane vesicles were rapidly frozen in liquid nitrogen and stored at −80 °C until use. Transport assays were performed in buffer C (75 mM HEPES-KOH, pH 7.5, 0.15 M K2SO4, 1 mM MgSO4 and 0.25 M sucrose). The reaction mixture contained 1 mg/ml membrane proteins, 20 µM of MAs(III), Rox(III) or PhAs(III), respectively, in a final volume of 0.6 ml of buffer C. The reaction was initiated by addition of 5 mM NADH. The uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) was added as indicated. Portions (0.1 ml) were withdrawn at the indicated times, filtered through 0.2 µm pore size nitrocellulose filters and washed twice with 5 ml of buffer C. The arsenic content was determined by ICP-MS, as described above.

Immunological detection of ArsP

Western blot analysis was used to detect expression of wild type and mutant ArsPs. Proteins in everted membrane vesicles were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) on 12% acrylamide gels and transferred to a Schleicher & Schuell Protran® nitrocellulose transfer membrane (PerkinElmer, Waltham, MA). Western blot analysis was performed according to the manufacturer’s directions using a Western Lighting Ultra Chemiluminescence Substrate Kit and an antimouse IgG to the six-histidine tag (PerkinElmer, Waltham, MA).

Topological analysis and homology modeling of the ArsP structure

The prediction of the transmembrane helices in CjArsP was calculated by the on-line TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Based on this prediction, the intramembrane topological plot was constructed with Protter (http://wlab.ethz.ch/protter/) to analyze and illustrate the sequence, topology and annotations (Omasits et al.).

The CjArsP sequence was used for the prediction of the tertiary structure by CPHmodels 3.2 (http://www.cbs.dtu.dk/services/CPHmodels/), a comparative protein homology modeling server (Nielsen et al., 2010). The three dimensional structural model was predicted on the basis of profile-profile alignment guided by secondary structure and exposure predictions. The profile query for CjArsP yielded the E. coli GlpT glycerol-3-phosphate/phosphate exchanger (PDB entry:1PW4) as a best aligned structural template with a high reliable Z-score of 14.4 and 99.4% of query sequence coverage length. The root-mean-square-deviation (RMSD) of the CjArsP model with 1PW4 is 0.81 Å over 277 aligned residues. Finally the quality of the three dimensional structure was assessed by PROCHECK (Laskowski et al., 1996). The model was constructed by using PYMOL, Molecular Graphics System, Version 1.3, Schrodinger LLC (http://www.pymol.org/).

Supplementary Material

Supp Figure S1-S5

Acknowledgements

This work was supported by National Institutes of Health Grant R37 GM55425. M.M. was supported by a Raman Postdoctoral Fellowship from the University Grants Commission, Government of India. We thank Qijing Zhang, Iowa State University, Christopher Rensing, University of Copenhagen and Flynn Picardal, Indiana University for gifts of DNA.

Abbreviations

FCCP

Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone

MAs(III)

methylarsenite

MAs(V)

methylarsenate

PhAs(III)

phenylarsenite

Rox(V)

roxarsone, 3-nitro-4-hydroxybenzenearsonic acid

Rox(III)

roxarsone with reduced As(III)

Nit(V)

nitarsone, 4-nitrobenzenearsonic acid

Nit(III)

nitarsone with reduced As(III)

pASA(V)

4-arsanilic acid

pASA(III)

4-arsanilic acid with reduced As(III)

HPLC

high pressure liquid chromatography

ICP-MS

inductively coupled plasma-mass spectroscopy

TM

transmembrane spanning helix

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Supplementary Materials

Supp Figure S1-S5
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