(Received for publication, January 10, 1997, and in revised form, April 7, 1997)
From the Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, Michigan 48201
The plasmid-encoded arsenical resistance (ars) operon of plasmid R773 produces resistance to trivalent and pentavalent salts of the metalloids arsenic and antimony in cells of Escherichia coli. The first two genes in the operon, arsR and arsD, were previously shown to encode trans-acting repressor proteins. ArsR controls the basal level of expression of the operon, while ArsD controls maximal expression. Thus, action of the two repressors form a homeostatic regulatory circuit that maintains the level of ars expression within a narrow range. In this study, we demonstrate that ArsD binds to the same site on the ars promoter element as ArsR but with 2 orders of magnitude lower affinity. The results of gel shift assays demonstrate that ArsD is released from the ars DNA promoter by phenylarsine oxide, sodium arsenite, and potassium antimonyl tartrate (in order of effectiveness), the same inducers to which ArsR responds. Using the quenching of intrinsic tryptophan fluorescence to measure the affinity of the repressor for inducers, apparent Kd values for Sb(III) and As(III) of 2 and 60 µM, respectively, were obtained. These results demonstrate that the arsR-arsD pair provide a sensitive mechanism for sensing a wide range of environmental heavy metals.
The arsenical resistance (ars) operon of resistance plasmids R773 and R46 encodes an oxyanion extrusion pump that produces resistance to arsenite and antimonite (1). The operon has five genes, ArsR, -D, -A, -B, and -C (2-5). ArsR and ArsD are both trans-acting repressor proteins that homeostatically regulate the levels of ars transcript (4, 6). Although both are 13-kDa homodimers, they share no sequence similarity. ArsR is an As(III)/Sb(III)-responsive repressor with high affinity for its operator site that controls the basal level of expression of the operon (7). Binding of arsenite or antimonite produces dissociation of ArsR from the operator site, permitting transcription. As the levels of transcript rise, synthesis of the integral membrane ArsB protein becomes toxic, limiting growth. ArsD is a second regulator that controls the upper level of expression of the operon, preventing overexpression of ArsB (4). Together, ArsR and ArsD form a regulatory circuit that controls the basal and maximal levels of expression of the ars operon. Dual regulatory proteins in a single operon are rare; in metalloregulatory systems only the MerR/MerD pair has been reported (8).
Previously, it was not possible to measure ArsD binding to
ars promoter DNA (4). It was not clear whether ArsD
interacted with DNA or mRNA. In addition, initial observations
suggested that ArsD did not respond to arsenite or antimonite. In this
report, we demonstrate that ArsD binds to DNA just 5 to ars
promoter, the same site occupied by ArsR before induction. Moreover,
interaction of ArsD with arsenite, antimonite, or the organic arsenical
phenylarsine oxide (PAO)1 was demonstrated
by both in vivo and in vitro assays. In gel shift
assays, PAO was the most effective inducer. The intrinsic tryptophan
fluorescence of ArsD was quenched by arsenicals and antimonials.
Titration of the fluorescence decrease allowed determination of the
affinity for inducers. These results demonstrate that ArsR and ArsD
control expression of the operon by binding to the same operator site
but at different times. Low extracellular concentrations of arsenical
or antimonial oxysalts induce the operon by binding to ArsR, which then
dissociates from the operator element. When the intracellular
concentration of ArsD becomes sufficient to saturate the operator,
transcription is again repressed. At higher extracellular
concentrations of metalloid salt, the intracellular concentration of
As(III) or Sb(III) increases to a level that produces dissociation of
ArsD from the operator, producing an increase in ars
expression.
The bacterial
strains and plasmids used in this study are described in
Table I. E. coli cells were grown in
Luria-Bertani medium at 37 °C. Ampicillin (100 µg/ml), kanamycin
(80 µg/ml), tetracycline (15 µg/ml) or chloramphenicol (20 µg/ml)
were added as required. For protein expression, 0.5 mM
isopropyl--D-thiogalactopyranoside (IPTG) or 20 µM sodium arsenite was used as inducer, except where otherwise noted.
|
Preparation of plasmid DNA was performed by using a Wizard DNA purification kit (Promega). Endo- and exonuclease digestions, DNA fragments separations and isolations, ligations, transformations, and Klenow fragment fill-in were performed according to standard procedures (9) unless otherwise noted.
Expression and Purification of ArsDArsD was purified from
culture of strain BL21(DE3) bearing plasmid pT7-5-D (4). Cells were
grown at 37 °C overnight with aeration in 0.2 liters of LB medium
containing 0.1 mg/ml ampicillin. The cultures were diluted into 2 liters of prewarmed Luria-Bertani medium containing 0.1 mg/ml
ampicillin. At an A600 of 0.6-0.8, production
of ArsD was induced by the addition of 0.5 mM
isopropyl--D-thiogalactopyranoside for 4 h. Induced
cells were harvested by centrifugation and washed once with buffer A
(10 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 2 mM dithiothreitol). The pelleted cells were suspended in 5 ml of buffer A/g of wet cells and disrupted by a single passage through a French pressure cell at 20,000 p.s.i. Unbroken cells and membranes were removed by centrifugation at 150,000 × g for
1 h. The supernatant solution containing ArsD was loaded onto a
2.5-cm diameter column filled to 20 cm with Q-Sepharose (Pharmacia
Biotech Inc.) pre-equilibrated with buffer A. The column was eluted
with 512 ml of a linear gradient of 0-0.3 M NaCl in the
same buffer. Fractions of 4 ml were collected and analyzed by SDS-PAGE
(10). Fractions containing ArsD were pooled and concentrated. The
protein was applied to a 2-cm diameter column filled to 100 cm with
Sephacryl S-200 (Pharmacia) and eluted with buffer A. ArsD-containing
fractions were pooled, concentrated, and stored at 4 °C. The
concentration of ArsD in purified preparations was determined using a
modification of the method of Lowry et al. (11).
Gel
mobility shift and DNase I footprinting assays were performed as
described previously (7). A 160-base pair DNA fragment containing the
ars operator/promoter and partial arsR gene was generated by PCR. After digestion with restriction endonucleases, either end of this fragment was labeled with
[-32P]dATP using the Klenow fragment of DNA polymerase
I and purified with Wizard cleanup system (Promega).
Overnight cultures of E. coli strain HMS174 (DE3) harboring pBGD23 or pBGDR1 and pArsD
plasmids were diluted 50-fold to 2 ml of fresh Luria-Bertani medium
containing ampicillin and chloramphenicol. After a 2-h incubation at
37 °C, cells were induced with varying concentrations of sodium
arsenite and IPTG and grown for another 2 h. One ml of cells was
pelleted by centrifugation and suspended in 0.5 ml of a buffer
consisting of 60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl,
1 mM MgSO4 and 50 mM
-mercaptoethanol, pH 8.0 (Z buffer) (12). The cells were
permeabilized by adding 1 drop of 0.1% SDS and 2 drops of chloroform
with mixing. The reaction mixture contained 0.02 ml of cells, 0.1 ml of
8 mg/ml O-nitrophenyl-
-D-galactopyranoside, and 0.88 ml of Z buffer. Enzyme activity was estimated from the release
of nitrophenol at 420 nm and 37 °C and expressed in Miller units
(12)
Intrinsic tryptophan fluorescence was measured at room temperature using an SLM 8000 spectrofluorometer. The excitation wavelength was 295 nm. The slit widths for excitation and emission were 4 mm. Reagents were added in 1-2-µl volumes with a microliter syringe through a light-protected port to the cuvette containing 2-ml samples, and the solutions were continuously stirred during the measurement. Dilution effects were negligible. Fluorescence intensity changes were recorded at 336 nm.
To investigate the
in vivo repression of ars expression by ArsD, two
reporter gene constructs were used. In the first, pBGD23, the
lacZ gene was fused in frame with the 23rd codon of
arsD. From this plasmid, both ArsR and an
ArsD--galactosidase reporter are synthesized. In the second,
pBGD
R1, base pairs 27-259 of the 351-base pair arsR gene
were deleted (13). The arsD gene was present on a compatible
plasmid, pArsD. In this plasmid, arsD is under control of
the T7 promoter, and in strains with the DE3 lysogen, the T7 RNA
polymerase, which is under control of the lac promoter, can
be induced with IPTG. In the absence of IPTG, expression of the
reporter gene from plasmid pBGD23 was low due to the presence of ArsR
(Fig. 1A). Addition of 10 µM
sodium arsenite in the absence of IPTG induced reporter gene expression
by dissociation of ArsR from the operator/promoter. Simultaneous
induction of ArsD with IPTG decreased reporter gene expression,
demonstrating repression by ArsD. In the absence of the arsD
gene, there was no effect of IPTG on repression by ArsR (Fig.
1B). The higher level of reporter gene expression observed
in this experiment compared with the presence of ArsD (Fig.
1A) is most likely due to the leakiness of the
lac promoter so that some ArsD is produced even in the
absence of IPTG.
Although it was previously reported that repression by ArsD was not affected by inducer, in that experiment, ArsD was expressed at levels that were too high to respond to arsenite (4). In this study, when the concentration of sodium arsenite was increased, repression by ArsD was relieved, indicating that ArsD responds to inducer (Fig. 1C). Derepression of ArsD required up to 10-fold more sodium arsenite than derepression of ArsR, suggesting that ArsD has lower affinity for inducer than ArsR. In the absence of the arsD gene, repression by ArsR was fully relieved by 10 µM sodium arsenite (Fig. 1D).
ArsR and ArsD act independently and do not require each other for
repression. Expression of the reporter gene from plasmid pBGDR1,
which lacks the arsR gene, was constitutive, not requiring arsenite (Fig. 1E). Addition of IPTG to induce ArsD from
plasmid pArsD produced repression, demonstrating that ArsD does not
require ArsR for repression. In the absence of ArsR, repression by ArsD was relieved by high concentrations of sodium arsenite, demonstrating that ArsD alone responds to inducer (Fig. 1G). The control
experiments with vector plasmid in place of pArsD show that the effects
of IPTG and arsenite require ArsD (Fig. 1, F and
H).
ArsD was purified by a
combination of anion exchange chromatography and size exclusion
chromatography, as described under "Materials and Methods."
Approximately 35 mg of purified ArsD could be obtained from 2 liters of
cell culture. From the intensity of Coomassie Blue staining of samples
separated by SDS-PAGE, ArsD was judged to be >95% homogeneous (Fig.
2, inset). A small amount of dimer was
observed in the purified preparations, as judged by immunoblotting with
anti-ArsD serum. Considering the number of cysteines in ArsD, it is
possible that the dimer is held together by disulfide bonds despite
denaturation in the presence of -mercaptoethanol. The molecular mass
of purified ArsD was determined by gel filtration chromatography using
a Sephacryl S-200 column (Fig. 2). From the nucleotide sequence of the
arsD gene, the predicted mass of ArsD is 13,218 Da (4). From
its elution position, a mass of approximately 26 kDa was determined,
consistent with an ArsD homodimer.
ArsD Is a DNA Binding Protein
Gel mobility shift assays were
used to examine the DNA binding activity of ArsD. A 160-base pair
32P-labeled PCR fragment containing the R773 ars
promoter was used as target DNA. ArsD was able to retard the migration
of this DNA probe (Fig. 3, top). No
retardation of the labeled probe was observed when a 50-fold excess of
unlabeled target DNA was added. From the least squares fit, the
Kd of ArsD for the operator/promoter DNA was
calculated to be 65 µM (Fig. 3, bottom). This
compares with a Kd of 0.33 µM for ArsR
(13), 2 orders of magnitude less than ArsD.
The repressor could be dissociated from its promoter by addition of
inducer (Fig. 4). Either arsenite or antimonite produced dissociation although at relatively high amounts. In gel shift experiments, ArsR similarly dissociated only at high concentrations of
inducer (7). On the other hand, the organoarsenical PAO was 100-fold
more effective than either of the inorganic oxyanions. ArsR similarly
is induced most effectively with PAO (13, 14).
DNase I Footprint Analysis of the Binding Site for ArsD in the ars Regulatory Region
Using purified ArsD, the site of binding to the
R773 ars regulatory region was analyzed by DNase I
protection assays. Protected regions found on both the coding strand
from nucleotides 61 to
37 (Fig. 5A) and
the noncoding strand from nucleotides
64 to
40 (Fig.
5B). Protection was prevented by addition of arsenite (Fig.
5B). This is the same region protected by ArsR (7).
Intrinsic Tryptophan Fluorescence of ArsD
ArsD has two
tryptophan residues, Trp35 and Trp97. The intrinsic fluorescence of
ArsD tryptophans exhibited a considerable enhancement and blue shift of
the maximum emission wavelength (Fig. 6, curve
A) compared with free tryptophan (Fig. 6, curve C). The
fluorescence of ArsD following denaturation with guanidine was similar
to free tryptophan (Fig. 6, curve B). These results indicate
that one or both tryptophan residues are in less polar environment than
free tryptophan. ArsD tryptophan fluorescence reported inducer binding.
In the presence of inducer, the fluorescence was quenched. The
magnitude of fluorescence quenching was dependent on inducer
concentration (Fig. 7A). Quenching as a
function of inducer concentration allowed the determination of apparent
affinity constants of approximately 2 µM for Sb(III) and
60 µM for As(III) (Fig. 7B).
Arsenical resistance (ars) operons produce resistance to the metalloids As(III) and Sb(III) by encoding an active efflux system for their oxyanions (1). Resistance to As(V) is conferred by an additional protein, ArsC, that reduces pentavalent to trivalent arsenical, the substrate of the extrusion system. The first two genes of the ars operon of E. coli plasmid R773, arsR and arsD, encode trans-acting regulatory proteins (4, 6). Although both are small proteins (117 amino acid residues for ArsR and 120 for ArsD), they exhibit no significant sequence similarity. While ArsR had been shown to bind to the ars operator/promoter (7), binding of ArsD could not be measured (4). The role of ArsR was shown to be repression of the operon to a basal level, with induction through sensing of environmental metalloid. The role of ArsD is related to prevention of toxicity resulting from production of the membrane protein ArsB.
In this study, ArsD was shown to bind to the same operator region of
ars DNA as ArsR. Neither required the other for DNA binding. Physiologically, their binding would be temporally distinct. The affinity of ArsD for ars operator/promoter DNA was shown to
be 2 orders of magnitude less than ArsR. Small amounts of both ArsR and
ArsD would be synthesized at a basal level in the absence of inducer,
but ArsR would preferentially bind to the operator site, repressing
ars expression. In vivo repression by ArsR can be
fully relieved with 10 µM sodium arsenite (13), while
ArsD repression requires approximately 100 µM sodium
arsenite for induction (Fig. 1). These results suggest that ArsR has
higher affinity for inducer than ArsD. Therefore, a low level of
environmental metalloid would cause dissociation of ArsR, resulting in
transcription of the ars message and increasing amounts of
ArsD. As the intracellular concentration of ArsD exceeded the
Kd for ars DNA, it would fill the
ars operator site. Since its affinity for inducer is less
than that of ArsR, the relatively low level of inducer present in the
cell would not prevent its binding. On the other hand, exposure to high
levels of environmental metalloid would cause dissociation of ArsD,
effecting further expression of the ars genes and increased
synthesis of the Ars extrusion pump. Synthesis of high levels of the
pump proteins is itself toxic (4, 15) so that there must be a balance
between detoxification of the metalloid and expression of the pump
genes. Thus, action of the two repressors forms a homeostatic
regulatory circuit that maintains the level of ars
expression within a narrow range, with ArsR controlling basal level of
expression and ArsD controlling maximal expression (Fig.
8).
ArsD responds to the same range of inducers as ArsR, with higher
affinity for Sb(III) than for As(III), and essentially no response to
other heavy metals or metalloids. It is of interest that the R773
ars operon encodes three proteins that are regulated by
these soft metals; in addition to ArsR and ArsD, the ArsA ATPase is
allosterically regulated specifically by binding of Sb(III) or As(III)
(16, 17). These three proteins have no sequence similarity but have
apparently evolved independent binding sites for the two soft metals.
ArsR has been shown to bind to the R773 ars promoter at the
sequence TCATNNNNNNNTTTG, which is just upstream of the 35 site (7).
The results of the DNase I protection show that ArsD and ArsR bind to
the same sequence. Thus ArsR and ArsD have also evolved binding sites
for the same element on the DNA.