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. Author manuscript; available in PMC: 2022 Dec 1.
Published in final edited form as: Environ Microbiol. 2021 Oct 21;23(12):7550–7562. doi: 10.1111/1462-2920.15817

ArsV and ArsW provide synergistic resistance to the antibiotic methylarsenite

Jian Chen 1,2, Jun Zhang 3, Yi-Fei Wu 3, Fang-Jie Zhao 3, Barry P Rosen 1,*
PMCID: PMC8865377  NIHMSID: NIHMS1778055  PMID: 34676971

Summary

Toxic organoarsenicals enter the environment from biogenic and anthropogenic activities such as microbial methylation of inorganic arsenic and pentavalent herbicides such as monosodium methylarsenate (MSMA or MAs(V)). Trivalent MAs(III) is considerably more toxic than arsenite or arsenate. Microbes have evolved mechanisms to detoxify organoarsenicals. We previously identified ArsV, a flavin-linked monooxygenase and demonstrated that it confers resistance to methylarsenite by oxidation to methylarsenate. The arsV gene is usually in an arsenic resistance (ars) operon controlled by an ArsR repressor and adjacent to a methylarsenite efflux gene, either arsK or a gene for a putative transporter. Here we show that Paracoccus sp. SY oxidizes methylarsenite. It has an ars operon with three genes, arsR, arsV and a transport gene termed arsW. Heterologous expression of arsV in Escherichia coli conferred resistance to MAs(III), while arsW did not. Co-expression of arsV and arsW increased resistance compared with either alone. The cells oxidized methylarsenite and accumulated less methylarsenate. Everted membrane vesicles from E. coli cells expressing arsW-accumulated methylarsenate. We propose that ArsV is a monooxygenase that oxidizes methylarsenite to methylarsenate, which is extruded by ArsW, one of only a few known pentavalent organoarsenical efflux permeases, a novel pathway of organoarsenical resistance.

Introduction

Arsenic occurs naturally in the environment in both inorganic and organic forms. Anthropogenic sources of organoarsenicals include pesticides such as monosodium methylarsenate (MSMA or MAs(V)), which is used on cotton fields and other non-food crops (Feng et al., 2005), and synthetic aromatic arsenicals such as roxarsone, which are used in poultry and swine production to treat coccidiosis and promote growth (Garbarino et al., 2003; Cortinas et al., 2006). Microbial communities drive global biogeochemical cycles of arsenic through diverse functions (Paez-Espino et al., 2009). Microbial arsenic metabolism transforms inorganic arsenic into a variety of less or more toxic organoarsenic species. Methylation of inorganic As(III) produces highly toxic trivalent methylated organoarsenicals, including methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III), catalysed by the ArsM As(III) S-adenosylmethionine (SAM) methyltransferase (Qin et al., 2006; Dheeman et al., 2014). Arsenic methylation has been proposed to play a significant role in arsenic biogeocycling (Zhu et al., 2014). MAs(III), the initial product in the ArsM catalytic cycle has been proposed to have been an primordial antibiotic before the great oxygenation event (GOE; Chen and Rosen, 2020). After the GOE, MAs(III) would have been oxidized to methylarsenate (MAs(V)), essentially detoxifying it (Le et al., 2000). Other members of aerobic microbial communities subsequently evolved pathways to re-reduce MAs(V), including the environmental isolate Burkholderia sp. MR1 (Yoshinaga et al., 2011), Shewanella putrefaciens 200 (Chen and Rosen, 2016) and Sinorhizobium meliloti RM1021 (Chen et al., 2018). Microbes that reduce MAs(V) gain the ability to utilize MAs(III) as an antibiotic to kill off its sensitive competitors that gives them a competitive growth advantage (Chen and Rosen, 2020).

In response, microbes have evolved diverse resistance mechanisms for detoxification of MAs(III). One mechanism of MAs(III) resistance is active efflux, which is catalysed by the MAs(III) efflux permeases ArsP (Chen et al., 2015b) and ArsK (Shi et al., 2018). A second mechanism is degradation of MAs(III) to As(III) catalysed by the ArsI C-As lyase (Yoshinaga and Rosen, 2014); A third mechanism is oxidation of MAs(III) to MAs(V), which can be catalysed by any of three unrelated enzymes, ArsH (Chen et al., 2015a), ArsU (Chen et al., 2021) and ArsV (Zhang et al., 2021). ArsH and ArsV encode flavoproteins that use NADPH and oxygen to oxidize MAs(III) to MAs(V). ArsU is a novel MAs(III) oxidase that uses neither FAD nor FMN. Unlike those enzymes and transporters, which are encoded by ars genes, NemA is not encoded in an ars operon but also catalyses oxidation of trivalent organoarsenicals (Shi et al., 2021). In many microbes, arsV gene is adjacent to either arsK or the gene for a putative membrane protein (Zhang et al., 2021), a member of the major facilitator superfamily (Reddy et al., 2012) that we term arsW.

How ArsW contributes to MAs(III) resistance is the focus of this study. We demonstrate that ArsW from Paracoccus sp. SY is an efflux permease that extrudes the MAs(V) product of ArsV oxidation to confer MAs(III) resistance. Paracoccus sp. SY is an MAs(III) resistant environmental isolate from arsenic-contaminated paddy soil (Zhang et al., 2015). It has an ars operon with three genes, arsRVW, where arsR encodes a member of the ArsR family of As(III)-responsive transcription factors (Qin et al., 2007) that likely controls expression of this operon. Escherichia coli has neither arsV nor arsW genes, and cells of E. coli expressing the Paracoccus sp. SY arsW gene did not exhibit MAs(III) resistance, indicating that, unlike ArsP and ArsK, ArsW is not an MAs(III) efflux permease. Heterologous expression of both the arsVW genes in E. coli conferred higher resistance to MAs(III) compared with arsV or arsW alone. Cells expressing both genes accumulated less MAs(III) than control cells without them, suggesting either reduced uptake of MAs(III) or efflux of MAs(V). Everted membrane vesicles prepared from E. coli cells expressing arsW accumulated MAs(V), indicating that ArsW is an MAs(V) efflux permease. We propose that in Paracoccus sp. SY ArsV oxidizes MAs(III) to MAs(V), which is then extruded from cells by ArsW, conferring MAs(III) resistance.

Results

Distribution of ArsVW two genes in ars operon

Many ars operons from soil bacteria carry arsRVK or arsRVW genes (Zhang et al., 2021). ArsK is an arsenic efflux protein with broad substrate specificity and can extrude As(III), Sb(III) and MAs(III) but not MAs(V) (Shi et al., 2018). Here, we examined the function of ArsW and its association with ArsV. Comparative analysis of the chromosomal ars operons from more than 80 bacterial species reveals that adjacent arsVW genes are commonly found in soil bacteria, primarily in alphaproteobacteria (Fig. 1A). Paracoccus sp. SY, which was used in this study, was isolated from an arsenic-contaminated paddy soil (Zhang et al., 2015). It is a chemoautotrophic As(III)-oxidizing bacterium that derives energy from oxidation of As(III) to As(V) under both aerobic and anaerobic conditions using either O2 or NO3− respectively, as electron acceptor.

Fig. 1.

Fig. 1.

Fig. 1.

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Distribution of arsVW genes in ars operons.

A. Linkage of arsV and arsW genes in ars operons. Shown are representative ars operons (accession numbers in parentheses) containing arsV and arsW genes (black fill). Paracoccus sp. SY (NZ_NWMQ01000040.1), Methylobacterium sp. SCN 67–24 (MEEG01000097.1), Rhodobacterales bacterium 65–51 (MKWD01000040.1), Bosea sp. 124 (NZ_PZZM01000001.1), Bradyrhizobium icense (NZ_CP016428.1), Kaistia sp. SCN 65–12 (MEDI01000015.1), Rhodobacter sphaeroides (QFQS01000001.1), Tabrizicola sp. DJC (NZ_QWEY01000008.1), Rhizobiales bacterium CCH3-A5 (LSIB00000000.1), Phreatobacter stygius (NZ_CP039690.1), Gemmobacter aquaticus (NZ_BMLP01000002.1), Enhydrobacter aerosaccus (NZ_FUWJ01000006.1), Bosea thiooxidans (NZ_LMAR01000082.1), Rhodospirillaceae bacterium (QOCQ00000000.1), A fipia massiliensis (LBIA02000001.1), Rhizobium sp. (SSFG01000113.1), Reyranella soli (BKAJ01000045.1), Niveispirillum cyanobacteriorum (CP025612.1), Azospirillum halopraeferens (NZ_AUCF01000033.1), Salipiger pacificus (NZ_CP022189.1), Roseomonas deserti (MLCO01000060.1), Acuticoccus kandeliae (NZ_QBBV01000002.1), Aminobacter sp. J44 (NZ_VLJG01000055.1).

B. Evolutionary relationship of PsArsW with membrane proteins encoded in ars operons. Six groups of MFS transporters are shown in a neighbour joining phylogenetic tree. The specificity for arsenicals of members of each group are proposed to differ. The group containing ArsW from Paracoccus sp. SY (*) shows a divergent evolutionary relationship with groups of MFS proteins.

To examine the evolutionary relatedness of ArsW with other members of the major facilitator superfamily, a phylogenetic analysis of MFS sequences in ars operons from 47 bacterial species was conducted (Fig. 1B). These permeases clustered in five groups. ArsK and ArsW are members of groups MFS2 and MFS3 respectively. ArsK has broad substrate specificity, but the substrates are all trivalent arsenicals (Shi et al., 2018). ArsJ in the MFS1 group transports the pentavalent organorarsenical 1-arseno-3-phosphoglycerate and is involved in As(V) resistance (Chen et al., 2016). MFS4 and MFS5 include uncharacterized transporters, and there are no experimental data that demonstrate their involvement in arsenic resistance. Unrelated groups of transport proteins include ArsP, which is a trivalent MAs(III) efflux permease and the unrelated arsenic efflux permeases ArsB and Acr3, which export inorganic As(III) from cells (Garbinski et al., 2019).

Paracoccus sp. SY confers resistance to MAs(III)

Since the arsV and arsW genes appear to be linked, arsenic resistance and biotransformation by Paracoccus sp. SY was examined by expression in E. coli W3110 and its arsenite-hypersensitive derivative AW3110 (ΔarsRBC; Carlin et al., 1995; Fig. 2). Cells of E. coli AW3110 are sensitive to both As(III) and MAs(III). Wild-type strain W3110 is resistant to As(III) due to expression of ArsB and ArsC but is sensitive to MAs(III) (Fig. 2A and Fig. 2B). Paracoccus sp. SY is resistant to both As(III) and MAs(III), which is likely due to expression of the acr3, arsV and arsH genes in the two chromosomal ars operons. To examine whether Paracoccus sp. SY resistance to As(III) or MAs(III) is related to either efflux or biotransformation, accumulation and modification of As(III) and MAs(III) were examined in cells (Fig. 2C and Fig. 2D). Small amounts of As(V) and MAs(V) were detected due to the oxidation by air during incubation. Paracoccus sp. SY oxidized more As(III) than E. coli due to presence of an aio gene cluster(KP881606)in the former. Paracoccus sp. SY efficiently oxidized MAs(III) to MAs(V). Cells nearly completely oxidized 5 μM MAs(III) to MAs(V) within 4 h (Fig. 2C). Cells Paracoccus sp. SY accumulated substantially more As(III) than E. coli. This may be due to oxidation to As(V) by the aio system; since Acr3 does not transport pentavalent species, the As(V) product might be trapped inside the cells. Cells of Paracoccus sp. SY accumulated lower amounts of methylated arsenic compared with E. coli (Fig. 2D). These results suggest that oxidation of MAs(III) to MAs(V) coupled with low cellular accumulation is responsible for MAs(III) detoxification in Paracoccus sp.SY.

Fig. 2.

Fig. 2.

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Organoarsenical resistance, oxidation and accumulation in Paracoccus sp. SY. Paracoccus sp. SY confers resistance to As(III) (A) and MAs(III) (B). Overnight cultures of Paracoccus sp. SY (o), Escherichia coli wild-type W3110 (□) and arsenite-hypersensitive strain AW3110 (Δars) (▽) were diluted 100-fold into fresh low phosphate medium containing the indicated concentrations of arsenic. After 16 h of growth at 30°C, the A600 nm was measured. Data are the mean ± SE(n =3).

C. Cells of Paracoccus sp. SY oxidizes MAs(III) to MAs(V).

D. Cells of Paracoccus sp. SY exhibit low MAs accumulation. Cultures of Paracoccus sp. SY were grown in low phosphate medium containing the indicated arsenicals at 30°C with shaking. Arsenic species were separated by high-pressure liquid chromatography (HPLC) with a C18 reverse column, and arsenic accumulation in cells was quantified by ICP-MS, as described in Experimental Procedures. Data are the mean ± SE (n = 3).

ArsV and ArsW act synergistically to confer resistance to MAs(III) by oxidation and reduce accumulation of MAs(V)

In the Paracoccus sp. SY genome, there are two clusters of ars genes (Fig. 3A). In one cluster is a putative arsRVW operon. ArsV (accession number WP_103174503) shown 50% percent identity with ArsV (WP_104668409) from Ensifer adhaerens ST2 (Zhang et al., 2021). ArsW (WP_103174504) shows 30% identity with ArsK (WP_104668410) from E. adhaerens ST2. The other cluster of ars genes contains a gene for another putative MFS transporter (WP_103175045) that is adjacent to an arsH gene. This putative transporter exhibits 40% identity and 52% similarity with ArsW, although it is not known if they have related functions. Genomics analysis reveals a number of putative MFS transporters in other ars operons.

Fig. 3.

Fig. 3.

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Expression of the arsV and arsW genes was induced by MAs(III).

A. Arsenic resistance gene clusters in the chromosome of Paracoccus sp. SY.

B. Transcriptional analysis of arsR3, arsV and arsW genes in the ars operon in response to 10 μM As(III) or 0.5 μM MAs(III). Differences are represented as **, P < 0.01.

C. Evolutionary relationship of ArsR3 with members of the ArsR/SmtB family of metal(loid) regulated transcriptional repressors. A neighbour joining phylogenetic tree shows that ArsR3 from Paracoccus sp. SY (*) clusters in group IV, where members exhibit selectivity for MAs(III).

The wide distribution of arsVW genes in the ars operon of a number of bacterial genomes suggests that the two genes may be co-transcribed. The transcript levels of arsRVW were determined in cells of Paracoccus sp. SY treated with either 10 μM As(III) or 0.5 μM MAs(III) (Fig. 3B). Transcription of arsR, arsV and arsW genes was not affected by addition of As(III). MAs(III) upregulated arsW 2.1-fold, arsV 1.6-fold and arsR 1.2-fold. Although upregulation was modest, the results demonstrate that expression of arsV and arsW are derepressed by MAs(III) but not As(III).

A phylogenetic comparison of ArsR3 with other ArsR sequences from 21 bacterial species was conducted (Fig. 3C). Proteins orthologous to the ArsR3 protein cluster in Group IV. This group includes the MAs(III)-selective Shewanella putrefaciens 200 SpArsR. SpArsR controls expression of MAs(III) resistance genes arsP and arsH (Chen et al., 2017a). SpArsR has two conserved cysteine residues, Cys101 and Cys102, that form a MAs(III) binding site. ArsR3 (WP_103174502) exhibits 41% identity and 57% similarity with SpArsR, with conserved cysteine residues Cys91 and Cys92 that correspond to the conserved cysteines in SpArsR. This suggests that ArsR3 is a MAs(III)-selective repressor that controls expression of the arsRVW operon in order to confer resistance to MAs(III) and possibly other trivalent organoarsenicals.

To further investigate the role of ArsV and ArsW in MAs(III) detoxification, the Paracoccus sp. SY arsV and arsW genes were cloned individually and together into vector plasmid pUC19 under control of the lac promoter, creating plasmids pUC-ArsV, pUC-ArsW and pUC-ArsV_ArsW. The three plasmids were individually expressed either in E. coli AW3110 (ΔarsRBC) or the arsenate sensitive strain WC3110 (ΔarsC; Mukhopadhyay et al., 2000). Cells of E. coli AW3110 with plasmid pUC-ArsV exhibited resistance to MAs(III) compared with the same strain with pUC-ArsW or vector only (Fig. 4B). Cells of E. coli AW3110 expressing both the arsV and arsW genes from plasmid pUC-ArsV_ArsW conferred significantly more resistance to MAs(III) compared with cells expressing either gene alone. Cells of E. coli AW3110 with either plasmid pUC-ArsV_ArsW or pUC-ArsV produced little resistance to As(III) (Fig. 4A) and essentially no resistance to As(V) (Fig. S1A). Expression of ArsW alone did not confer resistance to either MAs(III) or As(III). Paracoccus sp. SY ArsV (WP_103174503) shows 50% identity with EaArsV (WP_104668409), which has been shown to have MAs(III) oxidase activity (Zhang et al., 2021), and purified Paracoccus sp. SY ArsV similarly oxidizes MAs(III) with NADPH and FMN as electron donors (Fig. 4D). Purified ArsV also oxidizes As(III) to As(V) (Fig. 4C). Interestingly, cells of E. coli WC3110 (ΔarsC) with plasmid pUC-ArsV_ArsW incubated with As(III) accumulated more total arsenic than cells with plasmid pUC-ArsW or vector only (Fig. S1B). We interpret this result as oxidation of As(III) to As(V) by ArsV, which is trapped inside the cells because As(V) is not a substrate of ArsW. Since E. coli WC3110 lacks an arsC gene, the cells cannot reduce the As(V) produced by ArsV to As(III), the substrate of the E. coli As(III) efflux permease ArsB and so accumulates in high amounts, resulting in As(III) sensitivity.

Fig. 4.

Fig. 4.

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ArsV and ArsW confer synergistic resistance to MAs(III). Co-expression of Paracoccus sp. SY ArsV and ArsW confers AW3110 resistance to (A) MAs(III) but not to (B) As(III). Overnight cultures of Escherichia coli strain AW3110 bearing either vector plasmid pUC19 (o), pUC19-ArsV (□), pUC19-ArsW (◇) or pUC19-ArsV_ArsW (▽) were diluted 100-fold into fresh low phosphate medium containing the indicated concentrations of MAs(III). Expression of ArsV and ArsW was induced with 0.3 mM IPTG. Growth was measured after 16 h at 30°C. Data are the mean ± SE (n = 3). Oxidation of (C) As(III) and (D) MAs(III) by purified ArsV. Oxidation of MAs(III) and As(III) by purified ArsV was assayed as described in Experimental Procedures. Arsenicals were separated by HPLC using a C18 reverse phase column, and the amount of arsenic was estimated by ICP-MS.

ArsV oxidizes MAs(III) to MAs(V), which is extruded by ArsW

ArsW is an MFS membrane protein with 10 transmembrane segments, as predicted by the TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), suggesting that it could be an arsenical permease. ArsV oxidizes MAs(III) to MAs(V), and co-expression with ArsW gave increased resistance. These results clearly demonstrate that ArsV and ArsW act synergistically to detoxify MAs(III). One way that is could occur is if ArsW is an efflux permease for MAs(V), the product of ArsV oxidation of MAs(III). To test this hypothesis, the effect of heterologous expression of the Paracoccus sp. SY arsV and arsW genes in E. coli on accumulation of MAs(III) and As(III) was examined (Fig. 5A and Fig. S1B). Cells expressing both arsV and arsW accumulated considerably less MAs(III) compared with cells lacking the two genes. The combination of arsV and arsW genes reduced accumulation of MAs(III). This could be the result of decreased uptake of MAs(III) or increased efflux of MAs(V). To distinguish between these two possibilities, transport of MAs(V) and As(V) was assayed in everted membrane vesicles prepared from cells of E. coli AW3110 with pUC-ArsW (Fig. 5B). Uptake into everted membrane vesicles is the equivalent of efflux from cells (Rosen and Tsuchiya, 1979). Cells expressing ArsW accumulated MAs(V) but As(V) uptake was the same in vesicles with or without ArsW (Fig. 5B). MAs(V) uptake was dependent on addition of NADH, which generates a protonmotive force by electron transport chain oxidation (Fig. S2). Multiple sequence alignment of ArsW homologues with ArsK shows there a conserved cysteine residue in ArsW proteins that is absent in ArsK proteins (Fig. S3). However, side-directed mutagenesis of Cys166 in ArsW to a serine residue indicates that it is not required for the MAs(V) transport activity (Fig. S4). These results clearly demonstrate that ArsW is an efflux permease for pentavalent MAs(V).

Fig. 5.

Fig. 5.

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ArsV oxidizes MAs(III) to MAs(V), which is extruded by ArsW.

A. ArsV and ArsW reduce cellular MAs(III) accumulation. Uptake of MAs(III) by Escherichia coli AW3110 carrying either vector plasmid pUC19 (o), pUC19-ArsV (□), pUC19-ArsW (◇) or pUC19-ArsV_ArsW (▽) was assayed with 20 μM MAs(III), final concentration, was performed as described in Experimental Procedures. Data are the mean ± SE (n = 3).

B. Uptake of MAs(V) in everted membrane vesicles. Accumulation of MAs(V) (■, □) and As(V) (⬤, ○) in everted membrane vesicles prepared from E. coli AW3110 harbouring either vector plasmid pUC19 (open symbols) or vector plasmid pUC19-ArsW (filled symbols) was assayed as described in Experimental Procedures. The amount of arsenic was determined by ICP-MS. Data are the mean ± SE (n = 3).

Discussion

Evolution produces the diversity of life and is the result of interactions between organisms and environmental challenges over geological time periods (Hanson et al., 1999). Arsenic is released into the environment from geochemical and anthropogenic sources, which frequently results in serious contamination problems (Lemire et al., 2013). Arsenic in soil and water exerts considerable selective pressure on microbial communities, leading to the evolution and acquisition of arsenic resistance determinants. Environmental arsenic occurs most often in the inorganic form. However, methylated species such as highly toxic MAs(III) are formed microbially catalysed by ArsM As(III) S-adenosylmethionine methyltransferases (Qin et al., 2006; Qin et al., 2009). arsM genes are widely distributed in all kingdoms of life, and thus microbes have been methylating arsenic for several billion years (Chen et al., 2017b). In addition, pentavalent organoarsenicals have been used commercially as herbicides (MSMA) and growth promoters (roxarsone) for poultry and swine (Garbarino et al., 2003). These organoarsenicals can be reduced to their trivalent species by soil microbes, which increase their toxicity (Chen et al., 2014). In response to the continual exposure to toxic trivalent organoarsenicals, microbes have evolved diverse resistance mechanisms. Expression of arsenic resistance genes are usually transcriptionally regulated by members of the ArsR family of As(III)-responsive transcription factors (Ordonez et al., 2008). Some ArsR repressors have evolved selectivity for MAs(III) such as SpArsR, where the MAs(III) binding site appears to have arisen from an ancestral ArsR As(III) binding site (Chen et al., 2017a). These MAs(III)-responsive ArsRs control expression of a variety of MAs(III) detoxification pathways. For example, the ArsP and ArsK are efflux permeases that confer resistance by reducing the cellular concentration of MAs(III). ArsI is a C-As lyase that confers resistance by degrading MAs(III) to As(III) (Yoshinaga and Rosen, 2014). ArsH and ArsV are MAs(III) oxidases that use molecular oxygen to transform highly toxic MAs(III) to relatively nontoxic MAs(V).

Interestingly, the arsV gene is adjacent to either arsK or arsW genes in a number of bacterial genomes. The function of ArsW in resistance was previously unknown. In this study, we report that the arsV and arsW genes from the bacterium Paracoccus sp.SY together confer resistance to MAs(III). The arsRVW operon is regulated transcriptionally by MAs(III) but not As(III). Our results demonstrate that ArsV oxidizes MAs(III) to MAs(V), which is extruded from the cells by ArsW (Fig. 6). ArsW is an unusual MFS transporter because its substrate is a pentavalent and not trivalent organoarsenical. Nearly all efflux permeases encoded in ars operons have trivalent arsenicals as substrates. Only two exceptions have been identified to date: ArsJ, which has the pentavalent arsenical substrate 1-arseno-3-phosphoglycerate (Chen et al., 2016) and ArsQ, which is an efflux permease for the pentavalent arsenic-containing antibiotic arsinothricin (Galván et al., 2021).In summary, the arsRVW operon from Paracoccus sp. SY encodes a new and novel MAs(III) resistance, including the only identified pathway for efflux of MAs(V).

Fig. 6.

Fig. 6.

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Model of synergy between ArsV and ArsW in MAs(III) detoxification in Paracoccus sp. SY. The combination of ArsV and ArsW constitutes a novel pathway for MAs(III) detoxification in Paracoccus sp. SY. MAs(III) is proposed to be taken up by an aquaglyceroporin and induces expression of the arsRVW operon. ArsV catalyses oxidation of MAs(III) to MAs(V), which is then extruded from the cells by ArsW, conferring resistance.

Experimental procedures

Chemicals

Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich. MAs(V) was obtained from Thermo-Fisher Acros Organics Division, Waltham, MA, and Chem Service, West Chester, PA and reduced as described (Reay and Asher, 1977). The reduced species were simultaneously analysed for both arsenic and sulfur by HPLC (Series 2000; Perkin-Elmer, Waltham, MA) coupled with 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, 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), and WC3110(DE3) (ΔarsC), which is sensitive to As(V) (Sundaram et al., 2008) were used for complementation studies. E. coli BL21(DE3) (Novagen, Madison, WI) was used for protein expression. E. coli and Paracoccus sp. strain SY strains were grown aerobically at either 30°C or 37°C in either lysogeny broth (LB) medium (Sambrook et al., 1989), or low phosphate medium (Oden et al., 1994), as noted, supplemented with 125 μg ml−1 ampicillin or 34 μg ml−1 chloramphenicol, as required.

Phylogenetic analysis

Multiple alignment of ArsR and ArsW homologue sequences was performed using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). ArsR sequences with conserved cysteines and ArsW sequences in ars operons that are adjacent to arsV genes were selected for phylogenetic analysis. Acquisition of sequences was performed by searching a list of reference organisms or from the National Center for Biotechnology Information (NCBI) protein database using a BLASTP search (Johnson et al., 2008). Phylogenetic analysis was performed to infer the evolutionary relationship among the ArsR and ArsW proteins from various organisms. The phylogenetic trees were constructed using the Neighbour-Joining method using MEGA 6.0.1 (Tamura et al., 2013). The statistical significance of the branch patterns was estimated by conducting a 1000 bootstrap.

Plasmid construction and mutagenesis

For expression of Paracoccus sp. SY ArsV (WP_103174503) and ArsW (WP_103174504) in E. coli, PsarsV and PsarsW were cloned from genomic DNA of Paracoccus sp. SY (NZ_NWMQ01000040) using primers containing HindIII and BamHI restriction sites (Table S1). The PCR products were inserted into vector pUC19, which is controlled by the lac promoter, creating plasmids pUC-ArsV, pUC-ArsW and pUC-ArsV_W. For protein purification the arsV gene was cloned using NdeI and HindIII restriction sites and inserted into expression vector pET-29a (+) (Novagen) that adds a C-terminal six-histidine tag. Mutation of the cysteine residue in ArsW was introduced by site-directed mutagenesis using QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) with the primers containing the mutated sequences in Table S1. All sequences were confirmed by commercial DNA sequencing (Sequetech, Mountain View, CA).

Resistance assays

For metalloid resistance assays in liquid medium, competent cells of E. coli AW3110 or WC3110 were transformed with constructs bearing arsV, arsW, arsVW or derivatives. Cells of Paracoccus sp. SY and E. coli were grown overnight with shaking at 30°C in LB medium to an A600nm of 2.0. Overnight cultures were diluted 100-fold in low phosphate medium (Oden et al., 1994) containing the indicated various concentrations of either trivalent or pentavalent arsenicals plus 0.3 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and incubated at 30°C with shaking for an additional 16 h. Growth was estimated from the A600nm.

Arsenical accumulation in Paracoccus sp. SY and E. coli

For in vivo arsenical accumulation assays, Paracoccus sp. SY and E. coli cells were grown in low phosphate medium at 30°C to a cell density (A600nm) of 2. Cells were harvested and suspended in 1/5th volume of a buffer solution consisting of 75 mM HEPES-KOH, pH 7.5, 0.15 M KCl and 1 mM MgSO4. To initiate the transport reaction, arsenicals were added at a final concentration of 20 μM to 1 ml of cell suspension. Portions (0.1 ml) 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 the same buffer. 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) 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 an arsenic standard (Ultra Scientific, N. Kingstown, RI).

Assay of arsenicals biotransformation

Paracoccus sp. SY cells were cultured aerobically with shaking in LB medium overnight at 30°C. The cells were washed once and suspended in low phosphate medium without glucose at a cell density of A600nm = 3.0. Arsenicals was then added at 5 μM, final concentration, to the cell suspensions, which were incubated at 30°C with shaking for 4 h. Soluble arsenicals were speciated by HPLC coupled to ICP-MS using a Jupiter® 5 μm C18 300 Å reverse-phase column (250 mm × 4.6 mm; Phenomenex, Torrance, CA) eluted isocratically with a mobile phase consisting of 3 mM malonic acid, 5 mM tetrabutylammonium hydroxide and 5% methanol (vol/vol), pH 5.6, with a flow rate of 1 ml min−1 at 25°C. Some arsenic remained bound to cellular constituents and was not recovered.

Quantitative reverse transcription-PCR (qRT-PCR)

To determine whether transcription of the ars operon is induced by arsenicals, cells of Paracoccus sp. SY was cultured in 20*ST medium (Zhang et al., 2021) with or without 10 μM As(III) or 0.5 μM MAs(III). RNA was extracted from mid-exponential phase cells grown for 6 h after the addition of arsenicals. Total RNA was extracted using a bacterial RNA isolation kit (OMEGA) and treated with DNase I for 2 min to remove residual DNA. For each sample, 500 ng of RNA was reverse transcribed using HiScript Reverse Transcriptase (Vazyme), and cDNA was quantified using SYBR Green I with Taq Plus DNA Polymerase. Primers used for testing cotranscription are shown in Table S1. qRT-PCR was performed using a SYBR PrimeScript RT-PCR kit (TaKaRa) on a LightCycler (version 1.5) thermocycler. The gene expression data were normalized to the level of expression of the endogenous recA gene and are reported as relative values (Vandesompele et al., 2002).

ArsV purification

E. coli BL21(DE3) cells (Life Technologies) bearing plasmid pET29a-ArsV were grown in LB medium containing 50 μg ml−1 kanamycin with shaking at 37°C. Cells at an A600nm of 0.6 were induced by addition of 0.3 mM IPTG and cultured for an additional 4 h. The cells were harvested and suspended in 5 ml per gram of wet cells in buffer A (50 mM 4-morpholinepropanesulfonic acid, 20 mM imidazole, 0.5 M NaCl, 10 mM 2-mercaptoethanol and 20% glycerol (vol/vol), pH 7.5). The cells were broken by a single passage through a French pressure cell at 20000 psi and immediately treated with diisopropyl fluorophosphate (2.5 μl per gram wet cell). Membranes and unbroken cells were removed by centrifugation at 150 000 g for 1 h, and the supernatant solution was loaded onto a Ni2+-nitrilotriacetic acid column (Qiagen, Valencia, CA) at a flow rate of 0.5 ml min−1. The column was washed with more than 25 column volumes of buffer A. ArsV was eluted with buffer A containing 0.2 M imidazole, and the purity was analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Laemmli, 1970). Protein concentrations were estimated from the A280nm (ε = 38,850 M−1 cm−1; Gill and von Hippel, 1989). ArsV-containing fractions were divided into small portions, rapidly frozen and stored at −80°C until use.

Oxidation of arsenicals by purified ArsV

Oxidase activity of purified ArsV was assayed at 37°C in buffer B (25 mM Bis–Tris propane, pH 7.0, 0.3 mM NADPH and 25 μM FMN). MAs(III) or As(III) (10 μM) was incubated at 37°C in the presence or absence of 1 μM ArsV. Reactions were collected at the indicated time points, and protein was removed by centrifugation using a 3 kDa cutoff Amicon ultrafilter. The filtrate was speciated by HPLC-ICP-MS as described above.

Metalloid uptake assays in everted membrane vesicles

Transport assays in everted membrane vesicles were performed as described previously (Villadangos et al., 2012). Transport assays were performed in a buffer consisting of 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−1 membrane proteins, 20 μM of MAs(III) or As(III), in a final volume of 0.6 ml. The reaction was initiated by addition of 5 mM NADH. 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 the same buffer. Arsenic content was determined by ICP-MS following digestion of the filters with nitric acid as described above.

Supplementary Material

Supporting Information

Acknowledgements

This work was supported by NIH grant R35 GM136211 to B.P.R and a Natural Science Foundation of China grant 41967023 to J.C, and 31970108 to J.Z.

Abbreviations

GOE

great oxygenation event

HPLC

high-pressure liquid chromatography

ICP-MS

inductively coupled plasma mass spectroscopy

IPTG

β-d-1-thiogalactopyranoside

MAs(III)

methylarsenite

MAs(V)

methylarsenate

MFS

major facilitator superfamily

Footnotes

Supporting Information

Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:

Appendix S1: Supporting Information.

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Supplementary Materials

Supporting Information
NIHMS1778055-supplement-Supporting_Information.pdf (1.4MB, pdf)

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