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. Author manuscript; available in PMC: 2024 Apr 1.
Published in final edited form as: Mol Microbiol. 2023 Feb 24;119(4):505–514. doi: 10.1111/mmi.15045

The ArsQ permease and transport of the antibiotic arsinothricin

Ngozi P Paul 1, Thiruselvam Viswanathan 1, Jian Chen 1, Masafumi Yoshinaga 1, Barry P Rosen 1,*
PMCID: PMC10101903  NIHMSID: NIHMS1884169  PMID: 36785875

Abstract

The pentavalent organoarsenical arsinothricin (AST) is a natural product synthesized by the rhizosphere bacterium Burkholderia gladioli GSRB05. AST is a broad-spectrum antibiotic effective against human pathogens such as carbapenem-resistant Enterobacter cloacae. It is a non-proteogenic amino acid and glutamate mimetic that inhibits bacterial glutamine synthetase. The AST biosynthetic pathway is composed of a three-gene cluster, arsQML. ArsL catalyzes synthesis of reduced trivalent hydroxyarsinothricin (R-AST-OH), which is methylated by ArsM to the reduced trivalent form of AST (R-AST). In the culture medium of B. gladioli both trivalent species appear as the corresponding pentavalent arsenicals, likely due to oxidation in air. ArsQ is an efflux permease that is proposed to transport AST or related species out of the cells, but the chemical nature of the actual transport substrate is unclear. In this study B. gladioli arsQ was expressed in Escherichia coli and shown to confer resistance to AST and its derivatives. Cells of E. coli accumulate R-AST, and exponentially-growning cells expressing arsQ take up less R-AST. The cells exhibit little transport of their pentavalent forms. Transport was independent of cellular energy and appears to be equilibrative. A homology model of ArsQ suggests that Ser320 is in the substrate binding site. A S320A mutant exhibits reduced R-AST-OH transport, suggesting that it plays a role in ArsQ function. The ArsQ permease is proposed to be an energy-independent uniporter responsible for downhill transport of the trivalent form of AST out of cells, which is oxidized extracellularly to the active form of the antibiotic.

Keywords: Arsinothricin, antibiotic, resistance, efflux permease

Graphical Abstract

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Abbreviated summary

The organoarsenical arsinothricin (AST) is a broad-spectrum antibiotic synthesized by Burkhoderia gladioli. Shown is a homology structural model of ArsQ, a permease responsible for downhill transport of the trivalent form of the AST out of cells, which is oxidized extracellularly to the active form of the antibiotic.

Introduction

Arsenic is the most prevalent environmental toxic substance. It comes primarily from geochemical sources, with lower amounts contributed by human activities (Zhu et al., 2014). Arsenic enters our food supply from water, air and soil (Naujokas et al., 2013). It has no nutritional value and is both a carcinogen and a toxin. In fact, arsenic has been called the King of Poisons (Paul et al., 2022). Soon after the origin of life, microbes adapted to the presence of environmental arsenic through the evolution of pathways for its detoxification (Liu et al., 2013), with resistance genes organized in ars operons (Yang et al., 2012). Even more striking is the ability of microbes to utilize this toxic metalloid to gain a competitive advantage over other microbes. Early in evolution, bacteria evolved the arsM gene encoding the ArsM As(III) S-adenosinemethionine (SAM) methyltransferase, which catalyzes methylation of inorganic arsenic to form highly toxic methylarsenite (MAs(III) (Chen et al., 2019), which has antibiotic properties in extant microbial communities (Yoshinaga et al., 2011).

A more recent example of the adaptation of arsenic as a weapon in microbial warfare is synthesis of the natural product arsinothricin (2-amino-4-(hydroxymethylarsinoyl)butanoic acid or AST) by the soil bacterium Burkholderia gladioli GSRB05. AST has broad-spectrum antibiotic action and is effective against both gram-positive and gram-negative bacteria, including some of the most dangerous human pathogens (Nadar et al., 2019). AST is a non-proteogenic amino acid with a chemical structure similar to that of glutamate, where the γ-carboxyl group of glutamate is replaced with a methylarsenate moiety through a C-As bond. AST is unusual in that the arsenic atom has a pentavalent oxidation state. Pentavalent arsenicals in general have relatively low toxicity, and yet AST is as toxic as trivalent methylarsenite, one of the most toxic organoarsenicals. This is because the predicted mechanism of action of pentavalent AST is through inhibition of the enzyme glutamine synthetase, an essential enzyme in most bacteria. This is in contrast with trivalent arsenicals that disrupt metabolism by reaction with thiol groups in proteins and formation of complexes with small molecular weight thiols such as glutathione (Shen et al., 2013).

Recently the AST biosynthetic gene cluster of B. gladioli GSRB05 was shown to consist of three genes, arsQML, that are transcriptionally controlled by the product of the arsR gene. ArsL catalyzes formation of R-AST-OH, the reduced trivalent form of hydroxyarsinothricin (AST-OH), the unmethylated precursor of AST, from inorganic As(III) and the 3-amino-3-carboxypropyl (ACP) group of S-adenosylmethionine (SAM) (Fig. S1). R-AST-OH is methylated to reduced trivalent AST (R-AST) by ArsM, an As(III) SAM methyltransferase. ArsQ is proposed to be an efflux permease that extrudes AST-related species from the cells. Efflux is a common final step in bacteria that produce antibiotics because the transporter puts the antibiotic into the medium, where it kills susceptible bacteria and simultaneously protects the producer from its own antimicrobial agent. In this study we characterized ArsQ to identify its substrate(s) and determine its transport properties. We propose that the physiological substrate of ArsQ is the reduced trivalent form of the antibiotic, R-AST that is oxidized in air to the pentavalent form, AST. B. gladioli GSRB05 arsQ was cloned and heterologously expressed in the As(III)-hypersensitive strain E. coli AW3110, in which the chromosomal arsRBC operon had been deleted (Carlin et al., 1995). ArsQ confers resistance to AST and reduced R-AST. Cells of E. coli are not very sensitive to AST-OH or R-AST-OH, but ArsQ increases resistance. Cells of E. coli expressing arsQ selectively transport R-AST-OH and R-AST. Transport is independent of cellular energy and appears to be equilibrative. Examination of a homology model of ArsQ based on the structure of the divalent anion sodium symporter (DASS) family member VcINDY (PDB ID: 5ULD) suggested that Ser320 contributes to the substrate binding site. Cells of a S320A mutant exhibited reduced uptake of R-AST-OH, consistent with a role in ArsQ function. We propose that the ArsQ permease is an energy-independent bidirectional uniporter that is responsible for downhill transport of the trivalent form of AST in AST-producing B. gladioli GSRB05. R-AST is oxidized extracellularly to the active pentavalent form of the antibiotic.

Results

ArsQ confers resistance to a broad range of arsenicals.

Four families of organoarsenical efflux permeases, ArsJ (Chen et al., 2016), ArsK (Shi et al., 2018), ArsP (Chen et al., 2015) and ArsW (Chen et al., 2021), have previously been identified (Fig. 1). Here we identify B. gladioli GSRB05 ArsQ as a member of a different group of organoarsenical permeases (Fig. 1). Previously, it appeared that ArsQ transports AST, but the true substrate was not identified in that study (Galvan et al., 2021). Here the substrate specificity of ArsQ was examined by transgenic expression in the As(III)-hypersensitive strain E. coli AW3110 in which the chromosomal arsRBC operon had been deleted (Carlin et al., 1995). E. coli cells expressing arsQ have been shown to confer resistance to AST (Galvan et al., 2021).

Figure 1. Evolutionary relatedness of ArsQ to other organoarsenical efflux permeases.

Figure 1.

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The five phylogenetic trees show that ArsQ forms an evolutionarily separate family from the four other known families of organoarsenical efflux permeases. B. gladioli ArsQ is indicated (►). ArsK and ArsP both transport and confer resistance to trivalent MAs(III). ArsW transports pentavalent MAs(V) and confers resistance to MAs(III) by coupling to the ArsV MAs(III) oxidase (Zhang et al., 2022, Chen et al., 2021). ArsJ is an efflux permease for 1-arseno-3-phosphoglycerate and couples to glyceraldehyde-3-phosphate dehydrogenase to confer resistance to arsenate (Chen et al., 2016). NCBI accession numbers are indicated.

In this study, we extend the resistance studies to include trivalent R-AST and R-AST-OH, as well as pentavalent AST and AST-OH (Fig. 2). In general bacteria are resistant to low concentrations of pentavalent arsenicals. The antibiotic activity of AST is due to its ability to inhibit glutamine synthetase, an essential enzyme in nitrogen metabolism (Nadar et al., 2019), while trivalent arsenicals exhibit toxicity by binding to protein and small molecule thiols. Both pentavalent AST and trivalent R-AST produced substantial inhibition of growth of cells of E. coli AW3110 at a concentration of 10 μM, and expression of arsQ conferred resistance to both. In contrast, compared with AST, E. coli cells are relatively resistant to the AST precursor R-AST-OH and its pentavalent form AST-OH at concentrations as high as 50 μM, but, even so, resistance is increased by arsQ expression. AST did not confer resistance to MAs(III) (Fig. S2). indicating that ArsQ is specific for substrates in the AST biosynthetic pathway. AST-OH does not inhibit glutamine synthetase, which may explain its lower growth inhibition. The results from heterologous expression in E. coli are consistent with a physiological role of ArsQ in efflux of reduced AST in the producer, B. gladioli GSRB05.

Figure 2. ArsQ confers resistance to arsenicals in cells of E. coli AW3110.

Figure 2.

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Resistance in cells of E. coli AW3110 heterologously expressing ArsQ (white fill) was compared with cells with vector plasmid pTrcHisA (black fill). Growth was assayed after overnight incubation at 37 °C in the presence of 10 μM AST, 10 μM R-AST, 50 μM AST-OH or 50 μM R-AST-OH. Growth was normalized to the density of the control cells without arsenicals. Data are the mean ± SD (n=3).

ArsQ facilitates uptake of R-AST-OH and R-AST in cells of E. coli.

To examine the transport properties of ArsQ, uptake of AST and related organoarsenicals was examined in E. coli cells expressing arsQ. Stationary phase cells expressing arsQ accumulated considerably more R-AST-OH and R-AST compared to cells without the arsQ gene (Fig. 3). Little uptake of pentavalent AST or AST-OH was observed by E. coli cells with and without arsQ expression. Similarly, ArsQ did not catalyze uptake of MAs(III) or trivalent roxarsone (Rox(III)) (Fig. S3). The uptake data suggests that R-AST-OH and R-AST are preferred substrates of ArsQ, with little uptake of the pentavalent species or other organoarsenicals. It was not clear how cells are sensitive to AST without substantial uptake. Resistance was assayed over 24 h or longer, while transport was assayed over a period of minutes, so enough AST may permeate over the longer time period to inhibit growth. We also considered the possibility that the endogenous E. coli uptake systems that take up R-AST adventitiously are not very active or well expressed in stationary phase cells, so additional uptake experiments were performed with cells in the exponential phase of growth and at a lower concentration of R-AST (10 μM). These cells accumulated approximately 3-fold more R-AST compared with stationary phase cells (Fig. S4A). Expression of the arsQ gene in these cells reduced accumulation (Fig. S4B), which confers resistance. As described below, these results are consistent with the function of ArsQ to facilitate bidirectional movement of R-AST.

Figure 3. ArsQ facilitates uptake of R-AST-OH and R-AST in cells of E. coli AW3110.

Figure 3.

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Uptake of the following substrates was assayed in cells expressing ArsQ or vector plasmid pTrcHis2A: (□,■) R-AST-OH; (◊,◆), R-AST; (○,●) AST; (▼,Δ) AST-OH. All substrates were added at 25 μM, final concentration. (Note that (○) is visible only at 2.5 min and otherwise is obscured by the (●) symbols). Data are the mean ± SD (n=3).

GlpF does not transport R-AST or R-AST-OH

GlpF is a channel protein in the inner membrane of E. coli that is responsible for facilitated diffusion of polyols into the cells (Sweet et al., 1990). It also serves as the major entry route of the trivalent As(III) (Yang et al., 2005) and Sb(III) (Sanders et al., 1997). In solution, arsenite is present as the trivalent trihydroxylated species As(OH)3, which is likely recognized by GlpF as the inorganic equivalent of a polyol. Since our data suggest that ArsQ is specific for the transport of trivalent R-AST-OH and R-AST, we examined the possibility that GlpF also facilitates uptake of R-AST or R-AST-OH. Uptake of R-AST-OH and R-AST was assayed in cells of E. coli strain OSBR1, a derivative of strain AW3110 in which glpF was inactivated and compared with cells of E. coli AW3110 expressing arsQ. Cells of both E. coli OSBR1 and AW3110 exhibited little accumulation of R-AST-OH (Fig. 4A) or R-AST (Fig. 4B) compared with cells of E. coli AW3110 expressing ArsQ. The level of accumulation of R-AST and R-AST-OH were similar in the two strains. In comparison, cells of E. coli OSBR1 accumulated little As(III) compared with cells of E. coli AW3110, as shown previously (Meng et al., 2004), whether or not the cells expressed ArsQ (Fig. 4C). These results demonstrate that ArsQ catalyzes uptake of R-AST-OH or R-AST, but GlpF does not.

Figure 4. GlpF does not transport R-AST or R-AST-OH.

Figure 4.

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Uptake of (A) R-AST-OH, (B) R-AST, (C) inorganic As(III) was assayed in cells of E. coli AW3110 expressing ArsQ (▽) or vector plasmid pTrcHis2A (○), or in E. coli strain OSBR1, a derivative of strain AW3110 in which glpF was inactivated (□). All substrates were added at 25 μM. Data are the mean ± SD (n=3).

ArsQ is an energy-independent permease

The resistance data suggest that ArsQ catalyzes efflux of R-AST-OH or R-AST (Fig. 2), as shown in expontially-growing cells expressing ArsQ (Fig. S4B). On the other hand, stationary phase cells expressing ArsQ catalyze uptake of R-AST-OH or R-AST (Fig. 3). Taken together, these two sets of data suggest that ArsQ catalyzes bidirectional transport of R-AST-OH or R-AST. To examine the energy dependence of ArsQ, the effect of the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) on uptake of R-AST-OH was assayed in cells of E. coli with or without arsQ (Fig. 5A). Uptake of R-AST-OH in ArsQ-expressing cells was insensitive to FCCP. As a control, ArsQ transport activity was compared with that of the known transmembrane arsenite antiporter Acr3, which is coupled to the protonmotive force (Fu et al., 2009, Villadangos et al., 2012). The acr3 gene from Alkaliphilus metalliredigens was expressed in E. coli AW3110, and uptake of As(III) into cells of E. coli AW3110 was reduced by FCCP (Fig. 5B). Clearly Acr3 transport activity is uncoupled by FCCP, while ArsQ activity is not. These results demonstrate that ArsQ is not coupled to cellular energy and is a uniporter that facilitates bidirectional movement of R-AST-OH and R-AST.

Figure 5. R-AST and R-AST-OH uptake is not energy dependent.

Figure 5.

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(A) Uptake of R-AST-OH was assayed in stationary phase cells of E. coli AW3110 expressing ArsQ in the (□) presence or (▽) absence of FCCP or (o) vector plasmid pTrcHis2A. (B) Uptake of inorganic As(III) was assayed in stationary phase cells of E. coli AW3110 expressing Acr3 in the (□).presence or (▽) absence of FCCP or (o) vector plasmid pTrcHis2A. Substrates were added at 25 μM, and FCCP was added at 10 μM, final concentrations. Data are the mean ± SD (n=3).

An ArsQ structural model

Structural information will be useful to understand the mechanism of ArsQ transport and substrate specificity. At this time, there are no structures for ArsQ family proteins, but a transmembrane topological analysis predicts that ArsQ has 11 transmembrane segments (TMs) (Fig. S5). In the NCBI database, ArsQ is annotated as a putative member of the GntP family of permeases that transport gluconate (Peekhaus et al., 1997). However, no protein structural data are available for any member of the GntP family. A search for structural homologs of ArsQ identified the divalent anion sodium symporter (DASS) family of divalent anion/Na+ symporters that transport di- and tricarboxylic acids or sulfate coupled to a Na+ gradient (Nie et al., 2017). An alignment of ArsQ with the 445-residue divalent anion/Na+ symporter VcINDY (PDB ID: 5ULD) from Vibrio cholerae (Nie et al., 2017), which also has 11 TMs, indicates that the two proteins have 19% identity and 31% overall similarity (Fig. 6). A homology structural model based on the VcINDY structure with bound terephthalate (Sauer et al., 2020) was constructed (Fig. 7A and B). The model includes ArsQ residues 24 to 381 and shows 10 TMs (Fig. 7C). Topological connectivity indicates that TM3, 8 and 10 are discontinuous and composed of two shorter helices (Fig. S6). Predicted TM11 includes residues 283-402, but the model does not extend to those residues, so TM11 is not visible in the structural model.

Figure 6. Sequence alignment of ArsQ and VcINDY.

Figure 6.

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ArsQ from Burkholderia gladioli GSRB05 (Accession number: WP_219608245.1) was aligned with the Na+/succinate transporter VcINDY, from Vibrio cholerae (Accession number: WP_071919799.1). Identical residues (*) are highlighted in black, and related residues (:,.) are highlighted in gray. Ser320 in ArsQ and Ser377 in VcINDY are highlighted in red. The multiple alignment was calculated with CLUSTAL W.

Figure 7. ArsQ homology structural model.

Figure 7.

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The predicted ArsQ structural model is shown as cartoon representation. Transmembrane segments (TMs) are shown as α-helices in different colors from the side (A) or top (B) relative to the plasma membrane. C, TMs are shown in cylindrical representation in a membrane to illustrate topological connectivity. Models were visualized with PyMOL.

A putative contribution of Ser320 in ArsQ catalysis

In the VcINDY structure the terephthalate binding site includes Ser377, which is considered crucial for substrate binding and transport. That residue corresponds to Ser320 in ArsQ (Fig. 6). To examine whether Ser320 plays a role in ArsQ activity, it was changed to an alanine residue. The level of expression of histagged wild type ArsQ and the S320A protein in cells of E. coli AW3110 was estimated by immunoblot analysis using anti-histag antibodies (Fig. S7). The altered protein was present in the membrane in approximately the same amount and migrated with the same mobility as wild type ArsQ, indicating that the mutation did not affect expression. Expression of arsQS320A led to a decrease in transport of R-AST-OH by about 80% (Fig. 8A). Interestingly, there was little change in R-AST transport activity (Fig. 8B). Serine and alanine differ only by the presence of a hydroxyl group, and R-AST-OH and R-AST differ only by the substitution of a methyl group for a hydroxyl group. We hypothesize that this apparent change in substrate specificity might be influenced by a change in polarity in the binding site, leading to loss of a hydrogen bond to R-AST-OH. This will be tested in future experiments by substitutions with other polar residues of similar size such as threonine or cysteine, or with larger amino acid residues. The structural model also allows identification of other residues in the predicted substrate binding site.

Figure 8. Effect of S32A substitution on transport of R-AST-OH and R-AST.

Figure 8.

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Uptake of (A) R-AST-OH or (B) R-AST was assayed in stationary phase cells expressing ArsQ (▽), S320A (□) or with vector plasmid pTrcHis2A (o). Substrates were added at 25 μM, final concentration. Data are the mean ± SD (n=3).

Discussion

The bacterium Burkholderia gladioli GSRB05 found in the rice rhizosphere produces AST, an arsenic-containing natural product (Kuramata et al., 2016). AST possesses a broad-spectrum antibiotic activity, and acts by the inhibition of bacterial glutamine synthetase (Nadar et al., 2019). Recently, the gene cluster for AST biosynthesis was shown to consist of a three-gene cluster, arsQML. The arsL and arsM gene products are sufficient to catalyze sequential steps in the biosynthesis of AST. ArsL is a non-canonical radical SAM enzyme that catalyzes synthesis of the precursor, R-AST-OH from inorganic arsenite and the ACP moiety from SAM. The second step is catalyzed by ArsM, a SAM methyltransferase that methylates the R-AST-OH to the reduced trivalent form of AST (R-AST). ArsQ is proposed to be an efflux permease responsible for the transport of the antibiotic out of the cell (Galvan et al., 2021). However, it is not clear whether the physiological substrate of ArsQ is the pentavalent or the trivalent species. In this study, we characterized the substrate specificity and transport properties of ArsQ.

Antibiotic-producing bacteria usually extrude the antibiotic from the cell, both to confer resistance to the producer and to inhibit growth of competitors (Li et al., 2015). Since ArsQ is found in the AST biosynthetic gene cluster, it is reasonable to expect that it serves as an efflux permease for substrates, intermediates or products in the AST biosynthetic pathway. Although ArsQ confers moderate resistance to a variety of substrates and products in the pathway, data from our study suggests that it is specific for transport of the trivalent species, both R-AST-OH and R-AST. We propose that trivalent R-AST, the end product of the B. gladioli biosynthetic pathway, is released into the medium by ArsQ and oxidized by air to the antibiotic AST, where it can kill competitors.

GlpF is a channel in the inner membrane of E. coli belonging to the major intrinsic protein (MIP) family of transmembrane channel proteins. It is a nonselective carrier protein that facilitates the diffusion of polyols such as glycerol in E. coli (Sweet et al., 1990). In addition to glycerol, GlpF serves as a channel for trivalent metalloids (Sanders et al., 1997, Yang et al., 2005). Our data show that GlpF is not a channel for trivalent R-AST-OH or R-AST.

However, similar to GlpF, ArsQ transports its substrate independent of energy, in contrast to Acr3, an As(OH)3/H+ exchanger. With ArsQ, R-AST-OH and R-AST flow out of cells down their concentration gradients following synthesis in Burkholderia gladioli GSRB05. R-AST is rapidly oxidized to AST, which prevents its reuptake. The extracellular R-AST-OH is oxidized more slowly and can re-enter the cells to be methylated to R-AST. Thus, by mass action, re-uptake of R-AST-OH results in conversion to AST. This is supported by the observation that AST-OH appears in the B. gladioli culture medium at early times, and then its levels decrease, while AST appears at later times and continues to increase, a clear precursor-product relationship (Kuramata et al., 2016, Galvan et al., 2021).

The situation in E. coli is different from that in the AST producer B. gladioli. We propose that E. coli takes up AST and derivatives adventitiously by one or more as-yet unidentified transport systems. Our data show that R-AST transport activity is low in stationary phase cells but substantially higher in exponentially growing cells (Fig. S4). The accumulated R-AST is released down its concentration gradient when ArsQ is expressed. Then why do stationary phase cells take up R-AST when ArsQ is expressed rather than releasing it (Fig. 2)? We assume that when stationary phase cells are exposed to 2.5-fold more R-AST, and uptake is low because the endogenous transporters have relatively poor activity, the extracellular concentration of R-AST is higher than the intracellular, so ArsQ would then facilitate uptake. In summary, ArsQ facilitates efflux of R-AST in exponential phase cells exposed to low R-AST, and facilitates uptake in stationary phase cells exposed to higher R-AST. Thus the direction of R-AST movement catalyzed by the bidirectional ArsQ uniporter depends both on the activity of the uptake system(s) and the concentration of added R-AST.This hypothesis will be tested in future experiments, and the endogenous uptake systems for AST and derivatives will be identified.

ArsQ is annotated as a GntP family member in the NCBI database, but annotations do not always reflect functional similarities. ArsQ is smaller (408 amino acid residues) than the average GntP protein of approximately 445 residues. GntP family members are predicted to have 12-14 transmembrane α-helices (Peekhaus et al., 1997), while ArsQ is predicted to have only 11. On the other hand, we identified the 11-TM succinate transporter from Vibrio cholerae VcINDY (PDB ID: 5ULD) as a structural homolog of ArsQ. VcINDY is a member of the DASS family of transporters that includes cotransporters and exchangers that import di- and tricarboxylates into cells. The crystal structure of VcINDY with bound terephthalate was used a template for construction of an ArsQ structural model. Ser377 in the terephthalate binding site of VcINDY corresponds to Ser320 in ArsQ, which led to the prediction that S320 may be involved in ArsQ catalysis. The serine to alanine substitution in ArsQ led to a significant decrease in transport of R-AST-OH. Interestingly, the S320A transported R-AST with nearly normal efficiency, suggesting that the ArsQ substrate binding site had a change in selectivity as a result of the mutation. The chemical difference between AST-OH and AST is replacement of a polar hydroxyl group in the former with a nonpolar methyl group in the latter. The hydroxyl and methyl groups are isosteric, both should fit equally well into the substrate binding site of ArsQ. We speculate that the nonpolar alanine residue is less able to interact with the hydroxyl group in R-AST-OH but retains the ability to interact with the methyl group in R-AST. Later experiments will include mutagenesis of other residues in the predicted substrate binding site of ArsQ to elucidate their contribution to ArsQ function. In summary, our data suggests that ArsQ is an energy-independent uniporter for trivalent R-AST. Future studies will focus on elucidating the structure of ArsQ and its relationship to function.

Experimental Procedures

Chemicals.

Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich. The L-enantiomer of arsinothricin (L-AST) was purified from cultures of B. gladioli GSRB05, whereas D,L-hydroxyarsinothricin (D,L-AST-OH) was chemically synthesized, as described previously (Kuramata et al., 2016, Nadar et al., 2019, Suzol et al., 2020). In the experiments described below, the concentration of L-AST-OH was calculated as half of the total added D,L-AST-OH. Pentavalent arsenicals were reduced as described (Reay & Asher, 1977). The reduced species were analyzed by high pressure liquid chromatography (NexSar HPLC, Perkin Elmer, Waltham, MA) coupled with inductively coupled mass spectroscopy (ICP-MS) (NexION 1000; PerkinElmer) (Qin et al., 2006).

Strains, media and growth conditions.

E. coli AW3110 (Δars::cam F− IN(rrn-rrnE) (Carlin et al., 1995), which is As(III) hypersensitive, was used for most studies. OSBR1 (Δars::cam F-IN(rrn-rrnE, ΔGlpF), an AW3110 derivative with a glpF disruption (DOI: 10.1128/JB.187.20.6991-6997.20), was used for transport assays. E. coli strains were grown aerobically at either 30 °C or 37 °C in either lysogeny broth (LB) medium or M9 basal salts medium (Sambrook et al., 1989), as noted, supplemented with 0.4% glycerol, 0.1 mM CaCl2 and 1 mM MgSO4.

Preparation of wild type and mutant arsQ genes.

B. gladioli wild type arsQ and mutant arsQS320A genes GSRB05 arsQ was purchased from GenScript (Piscataway, NJ, USA) and cloned in plasmid pTrcHisA (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Resistance assays.

For resistance assays, competent cells of AW3110 were transformed with constructs bearing arsQ genes or the control vector. Cells were grown overnight with shaking at 37 °C in LB medium with antibiotics supplemented with 25 μg/ml chloramphenicol (Cm) and 100 μg/ml ampicillin (Ap), final concentrations. Overnight cultures were washed with and suspended in 0.9% NaCl, and diluted 100-fold or to an A600nm=0.05 in M9 medium containing various concentrations of organoarsenicals and incubated at 37 °C with shaking for an additional 24 h. Growth was estimated from the absorbance at 600nm.

Transport assays.

Transport assays were performed with either exponential or stationary phase cells. Overnight cultures of E. coli cells were diluted 100-fold and grown to A600nm = 0.5 (exponential phase) or A600nm = 1 (stationary phase) at 37 °C with aeration in LB medium. The cells were harvested, washed with and suspended in M9 medium at A600nm = 10. To initiate the transport reaction, AST, R-AST, AST-OH or R-AST-OH was added at a final concentration of 25 μM to 2 ml of cell suspension. The uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) was added at a final concentration of 10 μM. 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 at room temperature with 5 ml of M9 medium. The filters were digested with 0.2 ml of concentrated HNO3 (70%, ≥ 99.999% trace metals basis) at 70 °C for 30 min. The dissolved filters were allowed to cool to room temperature and diluted with HPLC-grade water to produce a final HNO3 concentration of 2%. Arsenic was quantified by ICP-MS. Standard solutions were made in the range of 1 - 50 ppb in 2% nitric acid using arsenic standard (Ultra Scientific, N. Kingstown, RI).

Immunological detection of ArsQ.

Immunoblot analysis was used to detect expression of wild type and mutant ArsQ proteins. Membranes from E. coli were prepared for immunoblot analysis as described previously (Villadangos et al., 2012). E. coli cultures were grown in 1 L of LB medium with 50 μM isopropyl β-d-1-thiogalactopyranoside as inducer at 37 °C to A600nm = 0.6 for 2 h. Cells were harvested by centrifugation, and the pellet suspended in a buffer consisting of 75 mM HEPES-KOH, pH 7.5, 0.15 M KCl, 1 mM MgSO4 and 0.25 M sucrose, and lysed by passage through a French pressure cell at 4000 psi. The lysate was treated with 0.5 mM diisopropyl fluorophosphate and 10 μg/ml DNase I, final concentrations, at 37 °C for 15 min, and centrifuged at 27,000 x g for 20 min at 4 °C to remove unbroken cells and cell debris. The supernatant suspension was centrifuged at 105,000 x g for 1 h, and the membrane fraction was suspended in the same buffer. Protein content was determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.). Membrane proteins were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) on 10% acrylamide gels and transferred to a Schleicher & Schuell Protran® nitrocellulose transfer membrane (PerkinElmer). Immunoblot analysis was performed according to the manufacturer’s directions using a Western Lighting Ultra Chemiluminescence Substrate Kit and an anti-mouse IgG to the six-histidine tag (PerkinElmer).

Phylogenetic analysis

Multiple alignment of ArsQ homolog sequences was performed using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). Acquisition of ArsQ sequences was performed by searching the National Center for Biotechnology Information (NCBI) protein database using a BLASTP protein basic local alignment search (Johnson et al., 2008). Phylogenetic analysis was performed to infer the evolutionary relationship among the ArsQ proteins from various organisms. The phylogenetic trees were constructed using the Neighbor-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.

Topological analysis and construction of an ArsQ structural homology model

The prediction of the transmembrane helices (TMs) in B. gladioli ArsQ 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., 2014).

The ArsQ 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). To examine the structure-function relationship of ArsQ, a homology model was generated using the SWISS-MODEL online tool (https://swissmodel.expasy.org/). The structure of VcINDY in complex with terephthalate (PDB ID: 6WTX) used as a template (Sauer et al., 2020). 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

SUPINFO

Acknowledgements

This work was supported by National Institutes of Health Grant R35 GM55425 to BPR and NSF BIO/MCB grant 1817962 to MY.

Abbreviations:

AST

Arsinothricin

AST-OH

hydroxyarsinothricin

HPLC

high pressure liquid chromatography

ICP-MS

inductively coupled plasma-mass spectroscopy

R-AST

reduced trivalent form of AST

R-AST-OH

reduced trivalent form of AST-OH

SAM

S-adenosine methionine

TM

transmembrane spanning helix

FCCP

carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone

SDS

sodium dodecyl sulfate

PAGE

polyacrylamide gel electrophoresis

Data availability statement

Data are available on request from the authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

SUPINFO
NIHMS1884169-supplement-SUPINFO.pdf (714.7KB, pdf)

Data Availability Statement

Data are available on request from the authors.

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