Alternate Energy Coupling of ArsB, the Membrane Subunit of the Ars Anion-translocating ATPase*

(Received for publication, August 26, 1996, and in revised form, October 9, 1996)

Masayuki Kuroda , Saibal Dey Dagger , Omar I. Sanders and Barry P. Rosen §

From the Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, Michigan 48201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The arsenical resistance (ars) operon of the conjugative R-factor R773 confers resistance to arsenical and antimonial compounds in Escherichia coli, where resistance results from active extrusion of arsenite catalyzed by the products of the arsA and arsB genes. Previous in vivo studies on the energetics of arsenite extrusion showed that expression of both genes produced an ATP-coupled arsenite extrusion system that was independent of the electrochemical proton gradient. In contrast, in cells expressing only the arsB gene, arsenite extrusion was coupled to electrochemical energy and independent of ATP, suggesting that the Ars transport system exhibits a dual mode of energy coupling depending on the subunit composition. In vitro the ArsA-ArsB complex has been shown to catalyze ATP-coupled uptake of 73AsO2-1 in everted membrane vesicles. However, transport catalyzed by ArsB alone has not previously been observed in vitro. In this study we demonstrate everted membrane vesicles prepared from cells expressing only arsB exhibit uptake of 73AsO2-1 coupled to electrochemical energy.


INTRODUCTION

Resistance to arsenical and antimonial compounds in bacterial cells is mediated by active extrusion of oxyanions of As(III) or Sb(III) from the cells (1, 2, 3). These efflux systems are encoded by ars operons. The ars operon of the conjugative R-factor R773 of Escherichia coli has been shown to encode an anion-translocating ATPase composed of two types of subunits, ArsA and ArsB. ArsA, the catalytic subunit, is a 63-kDa As(III)/Sb(III)-stimulated ATPase (4). ArsB is a 45-kDa inner membrane protein that catalyzes oxyanion translocation (5). The ArsA-ArsB complex has been shown both in vivo (6, 7, 8) and in vitro to be an obligatorily ATP-coupled primary pump (9).

However, recent results from several laboratories have shown that an arsB gene in the absence of an arsA gene is sufficient for resistance. While the ars operons of plasmids R773 (10) and R46 (11) have arsA genes, the homologous operons of the staphylococcal plasmids pI258 (12) and pSX267 (13) and the chromosomal ars operon of E. coli (14, 15) do not contain an arsA gene. Cells expressing the pI258 and E. coli chromosomal ars operons extrude arsenite (15, 16). There are at least two possible interpretations of these results. First, there could be a chromosomal arsA gene or homologue. Although this possibility cannot be ruled out, there are no data supporting it. Second, those ArsB proteins could function independently of ArsA, perhaps as a secondary carrier protein. The chromosomally encoded ArsB is 79% identical and overall 90% similar to the R773 ArsB. It seems intuitively unlikely that such close homologues would catalyze different reactions. When the R773 arsB gene was expressed in the absence of arsA, it conferred arsenical resistance and active extrusion (8). These findings suggest that the ArsB protein alone can catalyze energy-dependent efflux in the absence of a catalytic subunit.

For these reasons the energetics of ArsB-catalyzed efflux was compared with that of the ArsAB pump (8). In cells expressing arsB alone, arsenite transport was coupled only to electrochemical energy, not chemical energy, suggesting that a chromosomally encoded ArsA protein is probably not involved. Interestingly, the transmembrane structure of the R773 ArsB is topologically identical to secondary membrane carriers, with 12 membrane spanning segments (5). From the aggregate of these results, we postulated that ArsB functions as a secondary arsenite transporter in the absence of an ArsA subunit, a novel dual mechanism of energy coupling of a transport system (8).

However, all of the data suggesting a role of ArsB in secondary anion translocation were from physiological studies. In this study, we provide the first direct in vitro evidence that ArsB catalyzes electrophoretic anion transport. We have constructed an arsB expression plasmid that has enabled us to measure arsenite transport activity in everted membrane vesicles. NADH respiration provided electrochemical energy for ArsB-mediated transport in vesicles. Transport was sensitive to the addition of uncouplers and depolarizing permeant anions, indicating that in these everted vesicles anion transport is coupled to an electrochemical proton gradient, positive interior.


EXPERIMENTAL PROCEDURES

Materials

Restriction enzymes and nucleic acid modifying enzymes were purchased from Life Technologies, Inc. and Promega. Oligonucleotides were synthesized in the Macromolecular Core Facility of Wayne State University School of Medicine. Carrier-free 73AsO43- was obtained from Los Alamos National Laboratories. All other chemicals were obtained from commercial sources.

Strains, Plasmids, and Media

E. coli strains and plasmids used in this study are listed in Table I. E. coli strains harboring indicated plasmids were grown in LB medium (17) at 37 °C. Ampicillin (100 µg/ml), chloramphenicol (35 µg/ml), kanamycin (50 µg/ml), or tetracycline (15 µg/ml) was added as required. NaAsO2 or isopropyl-1-thio-beta -D-galactopyranoside (IPTG)1 was used at concentrations indicated.

Table I.

Strains and plasmids


Genotype/description Reference or source

E. coli strains
  JM109 recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi Delta (lac-proAB) 17
F'[traD36 proAB+ lacIq lacZDelta M15]
  ES1301mutS lacZ53 mutS201::Tn5 thyA36 rha-5 metB1 deoC IN(rrnD-rrnE) Promega
  AW3110  Delta ars::camF- IN(rrnD-rrnE) 15
  AW10 P1 transduction of Delta ars::cam from AW3110 to JM110, Delta ars::cam dam dcm supE44 hsdR17 thi leu rpsL lacY galK galT ara tonA thr tsx Delta (lac-proAB) This laboratory
   F'[traD36 proAB+ lacIq lacZDelta M15]
  LE392Delta uncICDelta ars P1 transduction of Delta ars::cam from AW3110 to LE392Delta uncIC, Delta ars::cam supE44 supF58 hsdR514 galK2 galT22 metB1 trpR55 lacY1 Delta uncIC 15
  BMH71-18mutS thi supE Delta (lac-proAB) [mutS::Tn10] [F' proA+B+ lacZDelta M15] Promega
Plasmids
  pALTER-1 Cloning and expression vector with T7, lac, and SP6 promoters, Tcr Promega
  pKK223-3 Cloning and expression vector with tac promoter, Apr Pharmacia
  pBC101 Removal of a 1.1-kilobase NdeI-PstI fragment containing the arsA gene from R773 ars operon in pWSU1, arsRDBC, Apr 8
  pArsA3 2.4-kilobase HindIII fragment containing the arsA gene inserted into HindIII digested pACYC184, Camr gene replaced by Kanr gene, Kmr 30
  pJHW101 Entire ars operon in plasmid pJBS633 5
  pAM-EK 2.9-kilobase EcoRI-KpnI fragment of pBC101 containing the arsR, arsD, and arsB genes inserted into EcoRI-KpnI digested pALTER-1, Tcr This study
  pAMB1 Disruption of BamHI site in the residual arsA gene of pAM-EK with Klenow fragment, Tcr This study
  pAMB10 Introduction of EcoRI site immediately after the arsA gene in pAMB1, Apr This study
  pAMB11 Introduction of HindIII site immediately after the arsB gene in pAMB10, Tcr This study
  pAMBT1 1.3-kilobase EcoRI-HindIII fragment of pAMB11 containing the arsB gene in pAMB11 re-ligated into the vector part of pAMB11, Tcr This study
  pKMB1 1.3-kilobase fragment of pAMBT1 containing the arsB gene inserted into EcoRI-HindIII digested pKK223-3, Apr This study
  pAO-BC arsBC gene fusion by deletion of intergenic region from pJHW101, Kmr This study
  pKMO-BC arsBC gene fusion by insertion of Csp45I-HindIII fragment from plasmid pAO-BC into Csp45I-HindIII digested pKMB1, Apr This study

DNA Manipulations

Plasmid DNA propagation, restriction enzyme treatment, ligation, and transformation were performed by minor modifications of published procedures (17). Plasmid DNA was isolated with QIAGEN Plasmid kit (QIAGEN) for sequence analysis by the method of Sanger et al. (18). Oligonucleotide-directed mutagenesis was performed using the Altered SitesTM II in vitro Mutagenesis System (Promega) according to the manufacturer's directions. All mutations introduced were confirmed by sequencing using a Cy5-labeled sequencing kit and ALFexpress system from Pharmacia Biotech Inc.

Construction of an arsB Expression Plasmid

Plasmid pBC101 was digested with EcoRI and KpnI, and a 2.9-kilobase fragment containing arsB was cloned into vector plasmid pALTER-1 (Promega), yielding plasmid pAM-EK. The BamHI site in the arsA gene in pAM-EK was inactivated by Klenow fragment treatment. The resulting plasmid pAMB1 was used for site-directed mutagenesis. Single-stranded DNA prepared from E. coli strain JM109 containing pAMB1 was used as a template. Multiround mutagenesis was done to introduce EcoRI and HindIII sites immediately after the arsA and arsB genes, respectively. The two mutagenic oligonucleotides used and the respective changes (underlined) introduced were as follows. To introduce an EcoRI site: 5'-GCT GGG TAA ATT A<UNL>A</UNL>T G<UNL>AA:T</UNL>TC ACG TAG GGC AGC-3'; to introduce a HindIII site: 5'-CTG TCA CAT TGT AAT <UNL>A</UNL>AG <UNL>C</UNL>TT CTG ATA TGA GCA AC-3'.

The resulting plasmid pAMB11 was digested with EcoRI and HindIII and religated to construct plasmid pAMBT1 that contains only arsB and its Shine-Dalgarno sequence. The EcoRI-HindIII fragment containing arsB was cloned into EcoRI-HindIII-digested vector plasmid pKK223-3 (Pharmacia), yielding plasmid pKMB1.

Transport Assays

Everted membrane vesicles were prepared essentially as described previously (19). Membrane vesicles were prepared fresh for transport assays; storage at -80 °C resulted in loss of activity. Transport assays were performed at room temperature. Unless otherwise noted, the reaction mixture contained 5 mM NADH, 0.1 mM sodium 73AsO2-, and 0.9 mg of membrane protein in a final volume of 0.6 ml of a buffer consisting of 75 mM HEPES-KOH, pH 7.5, containing 0.15 M K2SO4, 0.25 M sucrose, and 2.5 mM MgSO4. Unless otherwise noted, the reaction was initiated by addition of NADH. At intervals, 0.1-ml samples were withdrawn, filtered through nitrocellulose filters (0.22 µm pore size, Corning Costar), and washed with 5 ml of the same buffer. The filters were dried, and the radioactivity was quantified in a liquid scintillation counter. A blank value, obtained by filtering 0.1 ml of assay mixture without membrane vesicles, was subtracted from all points. To determine the initial rates, 30-s time points were used.

Detection of ArsB in Everted Membrane Vesicles

Because production of ArsB-specific antibodies has not been successful, an alternate approach to detect production of ArsB was used in which the arsC gene was fused in frame to the 3'-OH end of arsB. To construct the arsBC fusion, the intergenic sequence between arsB and arsC was deleted using the unique-site elimination method of Deng and Nickoloff (20) using single-stranded DNA from plasmid pJHW101 (arsRDABC) (5) as template. The termination codon of arsB and the intergenic region between arsB and arsC were deleted using a primer with the sequence: 5'-ATA AAT AGT GAT GTT GCT CAT CAA TGT GAC AGA GAG ACG TAG CGC <UNL>G</UNL>AG CGC GGC CAG 3'.

This primer also eliminated the BglI site near the 3'-OH end of arsB. A second primer, designed to delete the unique EcoRI site in plasmid pJHW101, had the sequence: 5' AAC TTG GA<UNL>G</UNL> TTC CCC TGT AAT 3'.

In both primers, the alteration eliminating the restriction site is underlined. Cells of E. coli strain BMH71-18mutS were transformed with the mutagenesis reaction mixture, and the cell suspension was inoculated into LB medium containing kanamycin and grown overnight. Plasmid DNA was prepared and digested by EcoRI, and the product was transformed into JM109. Plasmid DNA was prepared and screened for loss of the EcoRI and BglI sites. The arsBC fusion was confirmed by DNA sequencing. The resulting plasmid, pAO-BC, was used to clone the arsBC gene fusion into plasmid pKMB1. A Csp45I-HindIII fragment from plasmid pAO-BC was inserted into Csp45I-HindIII-digested pKMB1, yielding plasmid pKMO-BC.

The ArsBC chimeras were detected by immunoblotting of membrane protein separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Membrane protein was suspended in SDS sample buffer and heated at 80 °C for 10 min, following which the proteins were separated by SDS-PAGE on 15% polyacrylamide gels (21). Proteins were electrophoretically transferred to a nitrocellulose membrane (0.2 µm pore size, Schleicher & Schuell) overnight at 25 V and 4 °C. Antisera raised against purified ArsC was used to detect the ArsBC chimera. Immunoblotting was performed utilizing an enhanced chemiluminescence assay (DuPont NEN) and exposed to x-ray film at room temperature, as described previously (22).

Other Methods

Protein content was determined by a micromodification of the procedure of Lowry et al. (23) using bovine serum albumin as a standard. 73AsO2- was prepared by reduction of 73AsO43- (24).


RESULTS

Construction of an arsB Expression Plasmid

ATP-driven 73AsO2- transport has been measured in membrane vesicles from cells expressing the arsA and arsB genes from the native ars promoter. When the arsA gene was deleted from the R773 ars operon, the cells retained a low level resistance to arsenite (8), similar to that conferred by the chromosomal ars operon (15), which also lacks an arsA gene (14). However, no arsenite transport was observed in membrane vesicles from cells expressing arsB from the ars promoter regardless of the source of energy (data not shown). Previous attempts to express the R773 arsB gene at increased levels have been unsuccessful. The reasons for this are obscure, but possible explanations include instability of the polycistronic mRNA (25) and lethality of ArsB itself (26). In an attempt to increase expression, arsB was cloned behind the tac promoter of plasmid pKK223-3, producing plasmid pKMB1. This plasmid was transformed into three strains of E. coli in which the chromosomal ars operon had been disrupted: strains AW10, AW3110, and LE392Delta uncICDelta ars. However, only strain AW10 could be stably transformed, perhaps because, among the three strains, only AW10 contains a lacIq gene to control basal level of expression of arsB. Even in the absence of exogenously added IPTG, expression of arsB from pKMB1 produced low level arsenite resistance similar to that conferred by the chromosomal ars operon, and higher level resistance was observed when arsA was expressed in trans (Fig. 1). Although it is difficult to quantify the amount of ArsB due to the lack of a specific ArsB antiserum, relative amounts of ArsB produced from the ars promoter could be compared with those from the tac promoter in pKMB1. In both plasmids, the arsB gene was fused in frame to the downstream arsC gene, and the chimeras were detected with antiserum directed against ArsC (Fig. 2). The arsBC gene fusion produced arsenite resistance comparable to the wild type arsB gene (data not shown). In neither case could the ArsBC chimera be visualized by Coomassie staining of the gels. Even using very sensitive chemiluminescent methods, the ArsBC chimera produced from the ars promoter could not be detected (Fig. 2, lane 2). However, under control of the tac promoter, the ArsBC chimera could be detected immunologically (Fig. 2, lanes 3 and 4). ArsB was produced even in the absence of inducer, with a severalfold increase following IPTG induction. As shown above, phenotypic expresssion of arsenite resistance from the tac promoter likewise did not require induction (Fig. 1).


Fig. 1. Resistance to arsenite in cells expressing arsB. Cells of E. coli strain AW10 were grown in the presence of various concentrations of sodium arsenite at 37 °C for 8 h in the absence of IPTG. Cells contained the following plasmids: pKK223-3 (vector) (black-square), pKMB1 (arsB) (black-diamond ), pKK223-3 and pArsA3 (arsA) (bullet ), and pKMB1 and pArsA3 (black-triangle).
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Fig. 2. ArsB expression from the ars and tac promoters. Cultures of E. coli strain AW10 bearing the indicated plasmids expressing the chimeric arsBC gene were grown as as described under "Experimental Procedures." Cells were induced for 2 h with either 0.1 mM sodium arsenite (lane 2) or 0.5 mM IPTG (lane 4). Membrane vesicles prepared from those cells were suspended in SDS sample buffer and analyzed by SDS-PAGE on 15% acrylamide gels followed by immunoblot analysis using anti-ArsC serum. Lanes 1-4 contained 10 µg of membrane protein; lane 5 contained 1 µg of purified ArsC. Cells had the following plasmids: pKK223-3 (vector) (lane 1), pAO-BC (ars operon with arsBC fusion under control of ars promoter induced) (lane 2), pKMO-BC (arsBC fusion under control of the tac promoter uninduced) (lane 3), and pKMO-BC (induced) (lane 4).
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73AsO2- Transport in Everted Membrane Vesicles

The results of the in vivo study of energetics of transport suggested that ArsB alone could catalyze arsenite extrusion coupled to electrochemical energy (8). To test this hypothesis, 73AsO2- uptake was measured in everted membrane vesicles prepared from cells of E. coli strain AW10 bearing plasmid pKMB1. In these experiments, the cells were induced with IPTG to maximize expression of ArsB. The membrane vesicles exhibited time- and NADH-dependent 73AsO2- accumulation (Fig. 3). Membrane vesicles prepared from cells harboring vector plasmid pKK223-3 showed no transport. Transport required NADH oxidation, as shown by the complete inhibition by KCN. The uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) completely reversed NADH-dependent 73AsO2- uptake (Fig. 3), as did the combination of valinomycin plus nigericin (Table II). These results clearly show that arsenite transport catalyzed by ArsB alone is coupled to the electrochemical proton gradient established by NADH respiration. In contrast, little transport was observed with ATP (Table II).


Fig. 3. Energy-dependent accumulation of 73AsO2- in everted membrane vesicles. Arsenite accumulation into everted membrane vesicles prepared from strain AW10 harboring the indicated plasmids was measured as described under "Experimental Procedures." diamond , pKMB1 (arsB) in the absence of a source of energy. bullet , black-square, black-triangle, pKMB1 (arsB) with 5 mM NADH. At 1.5 min, 10 µM FCCP (black-square) or 10 mM KCN (black-triangle) was added to the reaction mixture (arrow). triangle , vector plasmid pKK223-3 in the absence of an energy source. square , pKK223-3 with 5 mM NADH.
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Table II.

Effect of permeant anions, weak base, and ionophores on 73AsO2- transport in everted membrane vesicles


Addition Initial velocity of arsenite uptakea Percent

nmol/mg membrane protein/min
Experiment I
  5 mM NADH + 0.05 M K2SO4 0.74 100
  5 mM NADH + 0.1 M KClO4 0.17 23
  5 mM NADH + 0.1 M KSCN 0.11 15
  5 mM NADH + 0.05 M (NH4)2SO4 0.67 91
  5 mM NADH + 0.1 M NH4Cl 0.35 47
Experiment IIb
  5 mM NADH + 10 µM valinomycin + 10 µM nigericin 0.04 5
  5 mM ATP 0.07 9
  5 mM ATP + 10 µM FCCP 0.04 5
pKK223-3 (vector) + 5 mM NADH 0.03 4

a  Arsenite uptake in everted membrane vesicles from cells expressing the arsB gene of plasmid pKMB1 was examined in a buffer consisting of 75 mM HEPES-KOH, pH 7.5, 0.25 M sucrose, and 2.5 mM MgSO4 containing the indicated additions.
b  Buffer contained 0.05 M K2SO4.

Effect of Permeant Anions and Weak Base on 73AsO2- Transport

The effect of permeant anions and a permeant weak base was examined (Table II). In these experiments, the vesicles were prepared in a sulfate-containing buffer. Under such conditions, the electrochemical gradient has been shown to be primarily in the form of a membrane potential, positive interior, with little or no pH gradient (27). NH4+, which dissipates the remaining Delta pH, had a small effect on the initial rate of 73AsO2- accumulation. NH4Cl was more inhibitory, attributable to an effect of Cl- as a permeant anion. The effect of SCN- and ClO4-, anions even more permeant than Cl-, considerably reduced transport activity.

Concentration Dependence for Arsenite

The concentration dependence for arsenite exhibited saturation kinetics, with an apparent Km of 0.14 mM (Fig. 4). This is essentially identical with the apparent Km of 0.1 mM for the ArsA-ArsB pump (9), suggesting that the mechanism of transport by ArsB is independent of the source of energy.


Fig. 4. Effect of substrate concentration on NADH-driven 73AsO2-1. Arsenite uptake was assayed in everted membrane vesicles with 5 mM NADH as an energy source. Inset, linearized transformation of the data; kinetic values were determined from a least squares fit.
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Effect of pH and Oxyanions on 73AsO2- Transport

Transport activity was maximal at pH 7 and decreased between 7 and 9 (Fig. 5). The effects of oxyanions on 73AsO2- transport via ArsB was examined. Among the oxyanions tested, the sodium or potassium salts of 73AsO43-, PO43-, NO3-, NO2-, SO32-, and SeO32- had little effect on the arsenite transport (Table III), indicating that ArsB does not catalyze nonspecific anion movement.


Fig. 5. Effect of pH on NADH-driven 73AsO2- transport in everted membrane vesicles. The initial rate of arsenite uptake was measured in a buffer consisting of 50 mM PIPES, 50 mM Bicine, containing 0.15 M K2SO4, 0.25 M sucrose, and 2.5 mM MgSO4. The pH was adjusted to the indicated values with either H2SO4 or KOH.
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Table III.

Effect of oxyanions on NADH-dependent arsenite transport via ArsB


Addition Initial velocity of arsenite uptake Percent

nmol/mg membrane protein/min
None 0.82 100
10 mM Na2HAsO4 0.79 96
10 mM K2HPO4 0.73 89
10 mM KNO2 0.74 90
10 mM KNO3 0.74 90
10 mM Na2SO3 0.81 99
10 mM K2SeO3 0.75 91
None 0.81 100
0.1 mM sodium potassium tartrate 0.75 93
0.1 mM potassium antimonyl tartrate 3.52 435
0.1 mM SbCl3 3.89 480

In contrast, potassium antimonyl tartrate was found to stimulate 73AsO2- transport 4- to 5-fold. The effect was specific for Sb(III) salts; sodium potassium tartrate had no effect on 73AsO2- transport, and the same stimulation was observed with antimony trichloride, which would be expected to hydrate to an antimonite oxyanion. This stimulatory effect of antimonite was further investigated. ArsB was required for Sb(III)-stimulated 73AsO2- accumulation (Fig. 6). Transport required NADH oxidation in the presence and absence of antimonite, and in both cases FCCP inhibited. Thus, this effect appears to be a property of the ArsB-mediated transport system and not a nonspecific effect of Sb(III). The degree of stimulation required stoichiometric amounts of antimonite and arsenite (Fig. 7). Importantly, at each concentration of arsenite examined, the maximal stimulation occurred at an equimolar concentration of Sb(III) (Fig. 7).


Fig. 6. Effect of Sb(III) on 73AsO2-1 uptake. Arsenite accumulation into everted membrane vesicles prepared from strain AW10 harboring the indicated plasmids was measured as described under "Experimental Procedures." bullet , open circle , pKMB1 (arsB) in the presence of 5 mM NADH. black-triangle, triangle , pKMB1 (arsB) in the presence of 5 mM NADH and 0.1 mM potassium antimonyl tartrate. At 1.5 min, 10 µM FCCP (triangle , open circle ) was added to the reaction mixture (arrow). black-square, pKK223-3 (vector) in the presence of 5 mM NADH and 0.1 mM potassium antimonyl tartrate.
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Fig. 7. Relationship of stoichiometry of Sb(III) and As(III) on 73AsO2-1 uptake. The initial rate of arsenite uptake was determined in the presence of the indicated concentrations of sodium arsenite. Assays were performed with the following concentrations of potassium antimonyl tartrate: none (open circle ), 0.05 mM (black-triangle), 0.1 mM (black-diamond ), 0.5 mM (bullet ), and 1 mM (black-square).
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The solution chemistry of arsenicals and antimonials is not well characterized. In solution it might be expected that the arsenite oxyanion (AsO2-) would be hydrated to As(OH)2O-, which is in equilibrium with the protonated form As(OH)3. However, other reasonable forms could be suggested, such as -O(HO)-As-O-As-(OH)O-, or, when arsenite and antimonite are added together, a mixed salt such as -O(HO)-As-O-Sb-(OH)O- might be formed. If antimonial oxyanions were better substrates for the transport system than arsenicals, such mixed salts could explain the substantial stimulation of 73As(III) uptake by Sb(III). In both transcriptional regulation by ArsR and allosteric regulation by ArsA, Sb(III) is more effective than As(III). Thus, it would not be surprising if antimonials were a better substrate for ArsB than arsenicals. Alternatively, an allosteric effect of Sb(III) on the carrier itself cannot be ruled out. Sb(III) allosterically activates the ArsA ATPase, but it does so by binding to a triad of cysteine thiolates (4), while there are no essential thiols in ArsB (28). Further arguing against an allosteric effect is the fact that maximal stimulation requires stoichiometric amounts of As(III) and Sb(III) at all concentrations of As(III). These results suggest that a mixed salt containing both As(III) and Sb(III) can be transported by the carrier.


DISCUSSION

From the results of previous in vivo and in vitro studies, we concluded that the arsA and arsB gene products of the R773 ars operon form a membrane-bound complex that functions as an obligatorily ATP-coupled arsenite pump. First, arsenical extrusion from cells of E. coli exhibited dependence on chemical energy; electrochemical energy was neither necessary nor sufficient (6, 7). Although the form of chemical energy could not be unambiguously identified from those physiological studies, there was a correlation between ATP levels and extrusion. Second, the transport system was shown to be a complex of two subunits, ArsA and ArsB (22), where ArsA is an As(III)/Sb(III)-stimulated ATPase. Third, everted membrane vesicles containing the ArsA-ArsB complex exhibited energy-dependent accumulation of 73AsO2- (9). In vitro transport had an absolute requirement for ATP. Again, electrochemical energy was neither necessary nor sufficient. In these cells, the H+-translocating F0F1 ATPase was deleted, so coupling of the ArsA-ArsB complex to ATP hydrolysis was direct.

However, as described above, several findings posed a question with respect to the energy coupling of the Ars system. Most intriguing was the observation that three of five ars operons lack an arsA gene. Only the operons from E. coli plasmids R773 (10) and R46 (11) have arsA genes. The two staphylococcal (12, 13) and the E. coli chromosomal (14) operons do not. Obviously it would be difficult to have an ATP-coupled pump without an ATPase subunit. One possibility is that the two types of extrusion systems have different biochemical mechanisms. The close similarity of the R773, R46, and E. coli chromosomal ArsB proteins (each exhibits over 90% similarity to the other two) would suggest that the proteins should have a common mechanism. Even the ArsBs from the staphylococcal plasmids, which are less than 60% similar to the proteins from the E. coli proteins, have essentially superimposable hydropathic profiles, suggesting similar membrane topology (29). Indeed, chimeras of the ArsB proteins from the Gram-positive and Gram-negative bacteria constructed by gene fusions of the arsB genes are functional (29). A reasonable deduction is that all ArsBs have the same biochemical mechanism.

Do ArsBs function as components of primary pumps, as the in vivo energetics studies indicate (6, 7)? Are they secondary carriers, as suggested by their transmembrane topology (5, 28)? These possibilities are not mutually exclusive. Indeed, results of in vivo transport studies suggested that the ArsB protein mediates the electrochemical energy-dependent arsenite efflux in the absence of the ArsA protein while the ArsA-ArsB complex catalyzes chemical energy-dependent transport (8). The pI258-encoded ArsB similarly had been proposed to utilize electrochemical energy (30). The results in this study clearly demonstrate that ArsB functions as a secondary arsenite transporter under conditions where there is only Delta psi (27), consistent with the carrier catalyzing electrophoretic anion movement coupled to the membrane potential. The fact that some ars operons have both arsA and arsB genes and thus encode pumps, while others have only the arsB gene and encode secondary systems suggests that the acquisition of a gene for a catalytic subunit might be a recent evolutionary event. Independence from electrochemical gradients would make the cell less susceptible to depolarizing environmental poisons. Therefore, the Ars system is a novel transport system that physiologically has two possible modes of energy coupling depending on its subunit composition (Fig. 8).


Fig. 8. Dual energy coupling of the Ars transporter. ArsB functions physiologically in either of two modes: as a potential driven secondary carrier or as a subunit of an obligatory ATP-coupled pump. In cells lacking an arsA gene, ArsB translocates arsenical and antimonial oxyanions, with energy derived from the proton-pumping respiratory chain. In cells with both genes, the ArsA-ArsB complex is an anion-translocating ATPase unable to utilize the membrane potential. Although the substrate is shown as the arsenite anion in this model, the structure of inorganic As(III) and Sb(III) oxysalts in dilute aqueous solution is unknown.
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FOOTNOTES

*   This work was supported by United States Public Health Service Grants AI19793 and GM08167. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Current address: Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda, MD 20892.
§   To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201. Tel.: 313-577-1512; Fax: 313-577-2765; E-mail: brosen{at}med.wayne.edu.
1    The abbreviations used are: IPTG, isopropyl-1-thio-beta -D-galactopyranoside; PAGE, polyacrylamide gel electrophoresis; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; PIPES, 1,4-piperazinediethanesulfonic acid; Bicine, N,N-bis(2-hydroxyethyl)glycine.

Acknowledgments

We thank Dr. T. H. Wilson for plasmid pKK223-3 and Dr. T. B. Gladysheva for purified ArsC.


REFERENCES

  1. Dey, S., and Rosen, B. P. (1995) in Drug Transport in Antimicrobial and Anticancer Chemotherapy (Georgopapadakou, N. H., ed), pp. 103-132, Dekker, New York
  2. Rosen, B. P. (1996) J. Biol. Inorg. Chem. 1, 273-277 [CrossRef]
  3. Rosen, B. P., Bhattacharjee, H., and Shi, W. P. (1995) J. Bioenerg. Biomemb. 27, 85-91 [Medline] [Order article via Infotrieve]
  4. Bhattacharjee, H., Li, J., Ksenzenko, M. K., and Rosen, B. P. (1995) J. Biol. Chem. 270, 11245-11250 [Abstract/Free Full Text]
  5. Wu, J., Tisa, L. S., and Rosen, B. P. (1992) J. Biol. Chem. 267, 12570-12576 [Abstract/Free Full Text]
  6. Mobley, H. L. T., and Rosen, B. P. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6119-6122 [Abstract]
  7. Rosen, B. P., and Borbolla, M. G. (1984) Biochem. Biophys. Res. Commun. 124, 760-765 [CrossRef][Medline] [Order article via Infotrieve]
  8. Dey, S., and Rosen, B. P. (1995) J. Bacteriol. 177, 385-389 [Abstract]
  9. Dey, S., Dou, D., and Rosen, B. P. (1994) J. Biol. Chem. 269, 25442-25446 [Abstract/Free Full Text]
  10. Chen, C.-M., Misra, T., Silver, S., and Rosen, B. P. (1986) J. Biol. Chem. 261, 15030-15038 [Abstract/Free Full Text]
  11. Bruhn, D. F., Li, J., Silver, S., Roberto, F., and Rosen, B. P. (1996) FEMS Microbiol. Lett. 139, 149-153 [CrossRef][Medline] [Order article via Infotrieve]
  12. Ji, G., and Silver, S. (1992) J. Bacteriol. 174, 3684-3694 [Abstract]
  13. Rosenstein, R., Peschel, P., Wieland, B., and Götz, F. (1992) J. Bacteriol. 174, 3676-3683 [Abstract]
  14. Sofia, H. J., Burland, V., Daniels, D. L., Plunkett, G., and Blattner, F. R. (1994) Nucleic Acids Res. 22, 2576-2586 [Abstract]
  15. Carlin, A., Shi, W., Dey, S., and Rosen, B. P. (1995) J. Bacteriol. 177, 981-986 [Abstract]
  16. Silver, S., and Keach, D. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6114-6118 [Abstract]
  17. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  18. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  19. Ambudkar, S. V., Zlotnick, G. W., and Rosen, B. P. (1984) J. Biol. Chem. 259, 6142-6146 [Abstract/Free Full Text]
  20. Deng, W. P., and Nickoloff, J. A. (1992) Anal. Biochem. 200, 81-88 [Medline] [Order article via Infotrieve]
  21. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  22. Dey, S., Dou, D., Tisa, L. S., and Rosen, B. P. (1994) Arch. Biochem. Biophys. 311, 418-424 [CrossRef][Medline] [Order article via Infotrieve]
  23. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 [Free Full Text]
  24. Reay, P. F., and Asher, C. J. (1977) Anal. Biochem. 78, 557-560 [Medline] [Order article via Infotrieve]
  25. Owolabi, J. B., and Rosen, B. P. (1990) J. Bacteriol. 172, 2367-2371 [Medline] [Order article via Infotrieve]
  26. Dou, D., Owolabi, J. B., Dey, S., and Rosen, B. P. (1992) J. Biol. Chem. 267, 25768-25775 [Abstract/Free Full Text]
  27. Perlin, D. S., Kasamo, K., Brooker, R. J., and Slayman, C. W. (1984) J. Biol. Chem. 259, 7884-7892 [Abstract/Free Full Text]
  28. Chen, Y., Dey, S., and Rosen, B. P. (1996) J. Bacteriol. 178, 911-913 [Abstract]
  29. Dou, D., Dey, S., and Rosen, B. P. (1994) Antonie Leeuwenhoek 65, 359-368 [Medline] [Order article via Infotrieve]
  30. Bröer, S., Ji, G., Bröer, A., and Silver, S. (1993) J. Bacteriol. 175, 3480-3485 [Abstract]

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