(Received for publication, August 26, 1996, and in revised form, October 9, 1996)
From the Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, Michigan 48201
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
73AsO21 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.
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.
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.
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--D-galactopyranoside (IPTG)1 was used at concentrations
indicated.
|
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 PlasmidPlasmid 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
T G
TC ACG TAG GGC AGC-3
; to introduce a
HindIII site: 5
-CTG TCA CAT TGT AAT
AG
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 AssaysEverted 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.
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
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
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 MethodsProtein 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).
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 LE392
uncIC
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).
73AsO2
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).
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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 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.
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.
Effect of pH and Oxyanions on 73AsO2
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.
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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).
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.
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 (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).
We thank Dr. T. H. Wilson for plasmid pKK223-3 and Dr. T. B. Gladysheva for purified ArsC.