From the Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, Michigan 48201
Received for publication, November 22, 2000, and in revised form, January 29, 2001
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ABSTRACT |
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The cadCA operon of
Staphylococcus aureus plasmid pI258 confers resistance to
salts of the soft metals lead, cadmium, and zinc. The operon is
regulated by CadC, a member of the ArsR family of metal-responsive
transcriptional repressors. In this study the role of the five cysteine
residues of CadC in soft metal ion sensing was investigated. Cys-7,
Cys-11, Cys-52, Cys-58, and Cys-60 were changed individually to glycine
or serine residues. The effect of the cadC mutations was
examined in Escherichia coli using a green fluorescent
protein reporter system. None of the mutations affected the ability of
CadC to repress gfp expression. Neither Cys-11 nor Cys-52
was required for in vivo response to Pb(II), Zn(II), or
Cd(II). Cys-7, Cys-58, or Cys-60 mutations each reduced or eliminated
soft metal sensing. Wild-type and mutant CadC proteins were purified,
and the effect of the substitutions on DNA binding was determined using
a restriction enzyme protection assay. Binding of wild-type CadC
protected cad operator DNA from digestion at the single
SspI site, and the addition of Pb(II), Zn(II), or Cd(II) resulted in deprotection. Chemical modification of the cysteine residues in CadC had no effect on protection but eliminated
deprotection. C11G and C52G proteins exhibited wild-type properties
in vitro. C7G, C58S, and C60G proteins were able to be
protected from SspI digestion but had reduced responses to
soft metal ions. The results indicate that Cys-7, Cys-58, and Cys-60
are involved in sensing those soft metals and suggest that they are
ligands to Pb(II), Zn(II), and Cd(II).
Because the first cells could have evolved in hydrothermal vents
rich in toxic soft metals such as cadmium and lead (1), the development
of resistance mechanisms to such metals was essential for their
survival. In bacteria soft metal resistance often is catalyzed by
P-type ATPases (2, 3). The first to be identified was the cadmium
resistance (cadCA) operon of Staphylococcus
aureus plasmid pI258 (4). The cadA gene encodes a
P-type ATPase that has been shown to transport Cd(II), Zn(II), and
Pb(II) (5). The cadC gene encodes a transcriptional
regulator that is a member of the ArsR family of metalloregulatory
proteins (6). When expressed in S. aureus, CadC responds to
metals (7, 8). In contrast, induction of the cad operon
in vivo by cadmium or zinc was difficult to observe in
Escherichia coli, which is intrinsically resistant to those
metals. We have shown that resistance in E. coli is caused
by expression of the chromosomally encoded zntA gene, and a
zntA-disrupted strain exhibits increased sensitivity to
zinc, cadmium, and lead (5). The disruption has been used as a
background strain for examining cadC function in E. coli. In that study CadC was shown to respond to soft metals with
the order of effectiveness Pb(II) > Cd(II) > Zn(II).
Here we investigate the role of cysteine residues in metal ion
selectivity by CadC. CadC contains five cysteines at residues 7, 11, 52, 58, and 60. Each was altered by site-directed mutagenesis. The
effect of the substitutions on activity in E. coli and
in vitro was examined. None of the cysteine residues was
required for repression in vivo or for binding to the
cad promoter in vitro. Neither Cys-11 nor Cys-52
seems to be required for soft metal recognition by CadC. Substitution
of Cys-7, Cys-58, or Cys-60 resulted in loss of soft
metal-dependent regulation by CadC both in vivo
and in vitro. The results suggest that these three residues are involved in sensing Pb(II), Zn(II), or Cd(II).
Growth of Cells--
Cells of E. coli were grown in
LB medium (9) at 37 °C. Ampicillin (50 µg/ml), kanamycin (40 µg/ml), chloramphenicol (80 µg/ml),
isopropyl- Cloning of cadC--
A 406-nucleotide base pair
(bp)1 fragment from plasmid
pYPK11 (11) containing the plasmid pI258 cadC gene was
amplified by polymerase chain reaction (12). The polymerase chain
reaction product was cloned into plasmid pGEM-T (Promega), generating
plasmid pMW0. The entire cadC gene was sequenced using a
Pharmacia Cy5-labeled autosequence kit (Amersham Pharmacia Biotech) and
an ALFexpress apparatus using the method of Sanger et
al. (13) to confirm that mutations had not been introduced during
amplification. A 393-bp NdeI-PstI fragment then
was isolated from pMW0 and ligated into the plasmid pT7-7 that had
been digested with NdeI and PstI. A 448-bp
XbaI-HindIII fragment, which includes both the
ribosome-binding site from pT7-7 and cadC, was isolated
from pMW0 and ligated into the plasmid pET-28a (Novagen) that was
digested with XbaI and HindIII, generating
plasmid pMW1. Plasmid pMW1 was transformed into E. coli
HMS174(DE3) (14) for protein expression. In this plasmid,
cadC is under the control of the T7lac promoter,
which is repressed by a lacI gene, and induced with
isopropyl DNA Manipulation and Oligonucleotide-directed
Mutagenesis--
Plasmid DNA was prepared with a Wizard DNA
purification kit (Promega). Endo- and exonuclease digestions, DNA
isolation, ligation, transformation, and Klenow fragment fill-in
reactions were performed according to standard procedures (9) unless
otherwise noted. Oligonucleotide-directed mutations in cadC
were introduced by site-directed mutagenesis using the Altered
SitesTM in vitro mutagenesis system (Promega).
The 448-bp XbaI-HindIII fragment from pMW1 was
subcloned into the pALTERTM-1 vector (Promega), generating plasmid
pMW2, and mutants were constructed using single-stranded DNA from pMW2.
All mutations were confirmed by sequencing the entire cadC
gene. Multiple sequence alignments were performed with ClustalW (16)
using the ClustalW World Wide Web Service at the European
Bioinformatics Institute.
Measurement of Regulation in Vivo--
The gene for red-shifted
green fluorescent protein (GFP) (17) was used as a reporter for
monitoring the regulatory properties of the cadC gene
product. A 108-bp fragment from plasmid pYPK11 containing the pI258
cad operator/promoter was amplified by polymerase chain
reaction. The fragment was engineered with EcoRI at the 5'
end and with a BamHI site at the 3' end. The fragment was
ligated into the plasmid pQF50/red-shifted GFP (18) that had been
digested with EcoRI and BamHI, generating plasmid
pYSG1, in which gfp is controlled by the cad
operator/promoter. A BglII-HindIII fragment from
pMW1 was ligated into the vector plasmid pACYC184 that had been
digested with BamHI and HindIII, generating
plasmid pYSC1 (5). Similarly, BglII-HindIII
fragments from the pMW1 series plasmids containing mutant
cadC genes were ligated into the pACYC184 (19) that had been
digested with BamHI and HindIII, generating the
pYSCM series plasmids. All cadC genes were under the control of the T7 promoter.
Cultures of E. coli strain BL21(DE3)
zntA::km (20) harboring compatible
plasmids pYSG1 and pYSC1 or the pYSCM series were grown in LB medium
and diluted 10-fold into a low phosphate minimal medium (10) containing
1% glucose plus the appropriate antibiotics. After 2 h at
37 °C, salts of soft metal ions were added to the indicated amounts
followed by an additional 3 h of incubation at 37 °C. The
samples then were diluted to A600 0.5 with the same medium. Expression from the cad promoter was
quantified from the fluorescence of red-shifted GFP with an emission
wavelength of 510 nm and excitation wavelength of 470 nm in an SLM8000
spectrofluorometer. The fluorescence intensity of GFP-containing cells
was normalized to the fluorescence of cells carrying plasmids pYSG1 and
pACYC184, which do not produce CadC.
CadC Expression and Purification--
Wild-type CadC was
produced in E. coli, either BL21(DE3) (Novagen) or
HMS174(DE3), bearing plasmid pMW1. Cells were grown overnight in LB
medium containing kanamycin at 37 °C. The culture was diluted
50-fold with fresh, prewarmed LB medium at 37 °C. At
A600 0.6-0.8, CadC expression was induced by
the addition of 0.3 mM
isopropyl-
The mass of CadC was determined by high pressure liquid chromatography
(HPLC) gel filtration. Wild-type CadC (2 µg) was applied to a
Synchropak HPLC column (SynChrom, Inc.) equipped with a precolumn using
a Waters HPLC. Samples were eluted with a degassed buffer consisting of
50 mM Tris acetate, pH 7.5, 0.5 M potassium
acetate, and 14.4 mM Assay of CadC Binding to the cad Promoter in Vitro--
Binding
of regulatory proteins to promoters can be assayed by protection of the
DNA from digestion by restriction enzymes (22). The ability of CadC to
protect the single SspI site in the cad promoter
from SspI digestion was assayed in a total volume of 20 µl. The assay mixture contained 5 µM purified CadC, 8 units of SspI, 0.5 µg of purified plasmid pYSG1 in a
buffer consisting of 50 mM Tris-HCl, pH 7.4, 6 mM MgCl2, 0.1 M NaCl, and 50 mM KCl. Deprotection was examined by the addition of the
indicated amounts of salts of soft metals. At the concentrations used,
the metals did not inhibit SspI activity. Samples were
incubated at 37 °C for 20 min, after which they were mixed with 4 µl of 6× sample solution (0.25% bromphenol blue, 0.25% xylene
cyanol FF, and 40% (w/v) sucrose in H2O) and
electrophoresed on 1.4% agarose gels containing 0.5 µg/ml ethidium
bromide at 100 V for 60 min at 23 °C. After electrophoresis the gels
were soaked in 1 mM MgSO4 for 20 min at
23 °C to remove excess ethidium bromide and photographed on a
transilluminator using a Kodak DC120 scientific digital system. The
intensity of the bands was quantified by densitometry using UN-SCAN-ITTM gel (Silk Scientific, Inc.).
Methyl Methanethiosulfonate Modification--
Methyl
methanethiosulfonate (MMTS) is a chemical modifying reagent that reacts
specifically with cysteine residues in proteins (23). It forms the
smallest possible disulfide derivative and thus is considered to
perturb protein structure minimally. CadC (50 µM) was
reacted with 2 mM MMTS in a buffer consisting of 10 mM MOPS, pH 7.0, 0.5 M NaCl, and 0.25 mM DTT for 5 min at 23 °C. MMTS modification was
reversed by addition of 5 mM DTT for 5 min at 23 °C. The
effect of MMTS was determined using the SspI restriction site protection assay as described above.
In Vivo Soft Metal Regulation of the cad Promoter by CadC--
We
have demonstrated previously that CadC repression of the plasmid pI258
cad operator/promoter can be relieved by the addition of
Pb(II), Cd(II), or Zn(II) (in order of effectiveness) (5). Derepression
required expression in a zntA-disrupted strain, which is
unable to pump those soft metals out of the cytosol. In that study a
lacZ gene was used as a reporter. Measurement of
In vivo soft metal responsiveness was estimated from the
amount of GFP fluorescence after growth in the presence of varying concentrations of Pb(II), Zn(II), or Cd(II) (Fig. 1A). Each
value represents the mean of three separate experiments, with the
standard deviation shown by the error bars. Pb(II) was the
most effective with half-maximal derepression at 0.1 µM
Pb(OAc)2. About 10-fold more Zn(II) was required. Although
Cd(II) derepressed, the response to Cd(II) was difficult to quantify
because growth of the zntA-disrupted strain was much slower
in the presence of Cd(II) than the other two metals. Addition of other
soft metals including Ni(II), Mn(II), Cu(II), and As(III) did not
produce an increase in gfp expression (data not shown). It
should be emphasized that the added metal ion concentrations may not be
the same as the cytosolic concentrations. The free intracellular
concentration may be influenced by the rates of uptake, binding, and
efflux so that quantitative comparisons among the metals should be made
with caution.
Requirement for Cysteine Residues for Repression and
Derepression--
CadC has five cysteines at residues 7, 11, 52, 58, and 60 (Fig. 2). In the homologous
plasmid R773 ArsR repressor, Cys-32 and Cys-34 have been shown
to be required for derepression by As(III) or Sb(III), where the
thiolates form part of the three-coordinate metalloid binding site
(24). ArsR residues Cys-32 and Cys-34 correspond to Cys-58 and Cys-60
in CadC. To investigate the possibility that cysteine thiols are
ligands to Pb(II), Zn(II), or Cd(II) in CadC, each of the cysteine
codons was mutated to glycine. CadCs with glycine substitutions were
able to repress expression of the gfp reporter from the
cad promoter, except for the C58G CadC. However, a C58S CadC
was able to repress and was used for subsequent studies.
Because the C7G, C11G, C52G, C58S, and C60G mutants repressed
expression, their ability to respond to the addition of Pb(II) (Fig.
1B) or Zn(II) (Fig. 1C) to the growth medium was
examined. Although each curve in Fig. 1, B and C,
represents the results of a single experiment, each experiment was
repeated 2-6 times with equivalent results. The Cys-11 mutation had no
effect on metal responsiveness, and there was only a small effect of
the Cys-52 mutation. In contrast, the Cys-7 and Cys-58 mutations
exhibited a significant decrease in the ability to respond to either
Pb(II) or Zn(II). The C60G mutant was unresponsive to either soft
metal. Although it is possible that the mutations could have produced conformational changes in CadC that are only indirectly related to
metal binding, these results suggest that Cys-7, Cys-58, and Cys-60,
but not Cys-11 or Cys-52, are involved in metal sensing.
In Vitro Soft Metal Regulation of the cad Promoter by CadC--
To
investigate the DNA- and metal-binding properties of CadC, the
repressor was expressed and purified from E. coli as
described under "Materials and Methods." CadC purified as a
homodimer, and no evidence of monomer was observed upon molecular sieve
chromatography (data not shown). This is consistent with the reported
dimerization of the homologous ArsR (25) and SmtB (26). Upon
SDS-polyacrylamide gel electrophoresis, CadC was present as both
monomer and dimer (data not shown). If the amount of
The ability of purified CadC to bind to DNA containing the
cad operator was measured by an enzyme protection assay
(22). Plasmid pYSG1 has only a single HindIII site, so
digestion with that enzyme generates a single restriction fragment of
4.6 kbp (Fig. 3A, lane
1). The plasmid has two SspI sites, one of which is
located within the 108-bp cad operator/promoter fragment and the other in the vector. Digestion with SspI generates two
restriction fragments of 3.6 and 1 kbp (Fig. 3A, lanes
2 and 7). In the presence of purified CadC, pYSG1 was
cut only once by SspI (Fig. 3A, lanes 3 and 8). Protection from SspI digestion was
specific for CadC; neither ArsR nor bovine serum albumin protected the
cad DNA from SspI (data not shown). When Pb(II)
(Fig. 3A, lanes 4 and 9), Cd(II) (Fig.
3A, lane 5), or Zn(II) (Fig. 3A,
lane 6) was added, SspI digestion was again
observed. This deprotection is consistent with the ability of these
metals to alter the binding of CadC to the promoter. Hg(II), which we
previously reported to act as a weak inducer of the cad
operon (5), also reduced CadC protection (Fig. 3A,
lane 14). Ni(II), Co(II), As(III), or Sb(III) had little or
no effect on CadC protection (Fig. 3A, lanes
10-13, respectively). Because other members of the ArsR family
bind these metals, it was important to demonstrate that CadC does not
respond to them in vitro. The effect of Pb(II), Zn(II), or
Cd(II) was quantified (Fig. 3B). In vitro, each
of the three was nearly equally effective, varying only about 2-fold in
contrast to the in vivo results, in which Pb(II) was 10-fold
more effective than Zn(II). Each value represents the mean of three
separate experiments, with the standard deviation shown by the
error bars. As mentioned above, one possible reason for the
differences between in vivo and in vitro results may be that the metals are accumulated differentially or bound in
vivo.
Requirement for Cysteine Residues for CadC Binding and Release from
the cad Operator DNA--
The effect of the cysteine-modifying reagent
MMTS on CadC protection of cad DNA and metal-induced
deprotection was determined. MMTS converts cysteine residues to
Cys-S-S-CH3 (23). Addition of the small thiomethyl group
should not greatly perturb protein structure. After reaction with MMTS,
CadC exhibited the same protection from SspI digestion as
the untreated protein (Fig.
4A), demonstrating that this
chemical modification of CadC did not prevent DNA binding. However, the
ability of Pb(II) to deprotect was lost. The disulfide formed with MMTS
can be reduced quantitatively with DTT to regenerate the free cysteine
thiolate. When MMTS-treated CadC was reduced with DTT, the repressor
exhibited the same deprotection with Pb(II) as the untreated protein.
These results are consistent with a role for cysteine thiolates in soft
metal binding.
Each of the five cysteine-substituted CadC proteins, C7G, C11G, C52G,
C58S, and C60G, was purified, and their DNA-binding properties were
examined (Fig. 4, B-D). Each was produced in
normal amounts in the cytosol, suggesting that the mutations did not alter the structure appreciably. None of the mutations affected the
ability of the purified CadCs to bind to cad DNA as
determined by protection from SspI digestion. The C11G and
C52G proteins responded nearly the same as the wild type to the
addition of Pb(II) (Fig. 4B), Cd(II) (Fig. 4C),
or Zn(II) (Fig. 4D). The C7G and C58S proteins required
considerably more soft metal for deprotection, and the C60G protein was
nearly unresponsive to metals. Although each curve in Fig. 4
represents the results of a single experiment, each experiment was
repeated 2-6 times with equivalent results. Thus, the in
vitro DNA-binding properties of the proteins were consistent with
the in vivo effects of the mutations, suggesting that Cys-7,
Cys-58, and Cys-60, but not Cys-11 or Cys-52, participate in soft metal binding.
The ArsR family is a rapidly growing set of repressor proteins
involved in transcriptional regulation of an assortment of unrelated
types of soft metal resistances (6), including P-type ATPases (4),
ArsAB pumps (27), and metallothionines (28). At last count, 24 homologues in bacteria and 18 in archaea have been identified. These
repressors fall into several groups: ArsR repressors are
As(III)/Sb(III)-responsive (29); CadC repressors are
Pb(II)/Cd(II)/Zn(II)-responsive (5, 7, 8); and SmtB/ZiaR repressors
are Zn(II)-responsive (30, 31).
What is the molecular basis of metal ion sensing and selectivity in
members of the ArsR family? The ions that derepress are all ions of
soft Lewis acids, so it is likely ligands would be soft Lewis bases,
which include the thiolates of cysteine residues and the imidazole
nitrogens of histidines. An alignment of nine putative CadC proteins,
ArsR, ZiaR, and SmtB identifies a number of conserved residues in some
or all proteins (Fig. 2). Cys-7, Cys-58, and Cys-60 of the pI258 CadC
are conserved in all CadCs. In the present study, mutagenesis of the
three conserved cysteine residues is shown to result in loss of sensing
soft metal ions in vivo and for deprotection of CadC from
cad operator DNA in vitro. Cys-11 and Cys-52,
which are not conserved in all CadC proteins, are not required. These
results are consistent with coordination of the three cysteine
thiolates with Pb(II), Cd(II), or Zn(II). Two of the three are
equivalent to thiols involved in As(III) and Sb(III) sensing by the
plasmid R773 ArsR. Cys-58 and Cys-60 correspond to Cys-32 and
Cys-34 of ArsR, both of which are required for As(III) and Sb(III)
derepression and binding by ArsR (24). ZiaR, which is related more
closely to SmtB than to CadC, has two cysteines at residues 71 and 73 that correspond to the CXC motifs of ArsR and CadC. A
C71S/C73S double mutant is nonresponsive to Zn(II), indicating that one
or both cysteine residues are involved in Zn(II) sensing by ZiaR (31).
On the other hand, SmtB does not have a CXC motif; the SmtB
residue corresponding to Cys-60 of CadC is Gly-63. Furthermore, the
SmtB residue corresponding to Cys-60 of CadC is Cys-61, and a C61S
mutant is not defective in zinc sensing (30). Thus, the
CXC motif seems widely but not universally utilized in metal sensing.
How could metal binding at a CXC site of CadC or ArsR result
in transcription? We modeled the structure of the ArsR homodimer (32)
on the crystal structure of the SmtB aporepressor (33). In the model,
the DNA-binding domain would be composed of helix 1 (residues
33-40)-turn (residues 41-43)-helix 2 (residues 44-55). In metallated
ArsR, Cys-32, Cys-34, and Cys-37 form a three-coordinate site for
As(III) or Sb(III), where the three sulfur atoms must be within
3.2-3.5 Å of each other (24). However, from the model of the
ArsR aporepressor, this would not be possible without distortion of
helix 1. If binding of metals to ArsR or CadC disrupts helix 1, the
repressor would no longer be able to bind to DNA and would dissociate,
allowing RNA polymerase to bind and transcription to proceed.
However, the CXC motif cannot account for selectivity if it
is utilized for both As(III)/Sb(III)- and
Pb(II)/Zn(II)/Cd(II)-responsive repressors. In ArsR, the third ligand
to As(III), Cys-37, is not present in CadC. From the present results,
it seems that Cys-7 of CadC is involved in Pb(II)/Zn(II)/Cd(II)
sensing, and this residue has no counterpart in ArsR. Cys-14 in SmtB
and Cys-20 in ZiaR may correspond to Cys-7 of CadC (Fig. 2), and Cys-14
and Cys-20 are required for zinc sensing in the respective repressors (30, 31). It seems likely that this conserved cysteine residue contributes to the selectivity of CadC, ZiaR, and SmtB. Our data are
consistent with a model in which 1) Cys-7, Cys-58, and Cys-60 are metal
ligands in CadC, and 2) Pb(II), Zn(II), and Cd(II) share a common
binding site. However Pb(II), Zn(II), and Cd(II) can be four-coordinate
or higher, which implies that there could be additional protein
residues involved in metal binding. Studies with SmtB suggest that
oxygen and/or nitrogen ligands may be involved in Zn(II) binding
(33, 34). A reasonable candidate for a fourth ligand in CadC is Asp-61,
which is conserved as either an aspartate or glutamate in all
identified members of the ArsR family. Asp-61 corresponds to Asp-64 of
SmtB, which is a Hg(II) ligand in the crystal structure (33). The quest
for additional residues involved in metal sensing forms the basis of
future studies on CadC.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside (0.1 mM),
and 5-bromo-4-chloro-3-indolyl-
-D-galactosidase (80 µg/ml) were added as required. For determination of metal ion
responsiveness, a basal salts medium was used (10) with the omission of
zinc salts. Growth was monitored from the absorbance at 600 nm.
-D-thiogalactopyranoside. Production of CadC
was examined by SDS-polyacrylamide gel electrophoresis on 18%
acrylamide gels (15). The amount of CadC was estimated by Western blot
analysis. Purified CadC was used to produce a polyclonal rabbit
antiserum by Cocalico Biologicals, Inc. (Reamstown, PA). The proteins
were transferred overnight onto a nitrocellulose membrane at 25 V and
probed with a polyclonal antibody to CadC using anti-rabbit IgG (Sigma)
as the secondary antibody. Immunoblotting was performed using an
enhanced chemiluminescence assay (PerkinElmer Life Sciences) and
exposed on x-ray film at room temperature.
-D-thiogalactopyranoside for 3 h at
37 °C. E. coli HMS174(DE3) was used for production of
CadC mutant proteins using the same procedure as for the wild type.
Cells were harvested by centrifugation and washed once with buffer A
(50 mM MOPS, pH 7.0, 5 mM DTT). The cells were
suspended in 20 ml of buffer A and broken by a single pass through a
French Press cell at 20,000 p.s.i. Membrane and unbroken cells were
removed by centrifugation at 150,000 × g for 1 h.
The soluble fraction was fractionated by S-Sepharose chromatography
(Amersham Pharmacia Biotech) pre-equilibrated with buffer A. The column
was eluted with a linear gradient of 0.2-1 M NaCl in
buffer A. CadC-containing fractions were pooled, and DTT was added to a
final concentration of 10 mM. Proteins were stored at
80 °C until use. From the intensity of staining with Coomassie
Blue on SDS-polyacrylamide gel electrophoresis, CadC was estimated to
be ~90% homogeneous. Protein concentrations were determined from the
absorbance at 280 nm according to the methods of Gill and von Hippel
(21) using an extinction coefficient of 5,720 for wild-type CadC or
5,600 for single cysteine derivatives.
-mercaptoethanol at 1 ml/min.
Bovine serum albumin (66 kDa), ArsR-H6 (33 kDa), carbonic anhydrase (29 kDa), and trypsin inhibitor (17 kDa) were used as standards (10 µg of each).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity requires permeabilization of the cells. In
contrast, GFP fluorescence is noninvasive and can be assayed in
vivo. For these assays a two-plasmid system was used. One plasmid
contained a gfp gene under control of the cad
promoter. The second plasmid had a wild-type or mutant cadC
gene behind the T7 promoter. Although transcription of the T7
polymerase is under lac promoter control, there was
sufficient basal level CadC expression to repress GFP in the absence of
isopropyl-
-D-thiogalactopyranoside (Fig.
1A).
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Fig. 1.
Soft metal ion regulation of gfp
expression from the cad promoter and the effect
of mutagenesis of conserved cysteine residues of the CadC repressor on
the in vivo response to soft metal ions. A
two-plasmid system was used to measure the ability of CadC to repress
expression from the cad promoter and the response to metals
as described under "Materials and Methods." A wild-type
cadC gene from plasmid pI258 was expressed constitutively
from plasmid pYSC1 in E. coli BL21(DE3)
zntA::km. The same cells contained
plasmid pYSG1, in which the gene for red-shifted GFP was under the
control of the cad promoter. Cells were excited at 470 nm,
and GFP fluorescence was measured at 510 nm. A, soft metal
ion derepression was assayed after the addition of the indicated
concentrations of Pb(OAc)2 (circles),
Cd(OAc)2 (inverted triangles), or
ZnSO4 (squares). Each value represents the mean
of three separate experiments, with the standard deviation shown by the
error bars. B and C, the ability of
CadC cysteine mutants to derepress in the presence of soft metal ions
was assayed after the addition of the indicated concentrations of
Pb(OAc)2 (B) or ZnSO4
(C). CadC mutants were C7G (circles), C11G
(inverted triangles), C52G (squares), C58S
(diamonds), and C60G (triangles).
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Fig. 2.
Multiple alignment of CadC homologues.
Representative CadC homologues are shown (with GenBankTM accession
numbers in parentheses) from: CadCs from plasmid pI258 (B32561),
Stenotrophomonas maltophilia (CAB96928), Bacillus
firmus (C42707), Listeria monocytogenes (AAA25276),
Staphylococcus lugdunensis (AAB18271), S. aureus
(AAA26609), Bacillus haldurans (BAB07756),
Pyrococcus abyssi (CAB49845), Methanobacterium
thermoautotrophicum (AAB86261), ArsR from plasmid R773
(CAA34168), ZiaR from Synechocystis sp. strain PCC 6803 (Q55940), and SmtB from Synechococcus sp. strain PCC 7942 (BAA10706). The positions of Cys-7, Cys-11, Cys-52, Cys-58, and Cys-60
in pI258 CadC and the corresponding cysteine residues in homologues are
shaded. The multiple alignment was calculated with ClustalW
(16).
-mercaptoethanol was increased to 1 M, the protein
migrated predominately as a monomer (data not shown). Thus CadC is a
noncovalent homodimer that can form disulfide bonds in
vitro.
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Fig. 3.
Effect of Pb(II), Cd(II), and Zn(II) on the
ability of CadC to protect cad promoter DNA from
digestion by the restriction enzyme SspI.
SspI restriction site protection assays were performed as
described under "Materials and Methods." A, the 4.6-kbp
plasmid pYSG1, which contains the pI258 cad promoter, was
digested with HindIII (lane 1) or with
SspI in the absence (lanes 2 and 7) or
presence (lanes 3 and 8) of 5 µM
purified CadC. There is a single SspI site in the
cad promoter and a second site in the vector. Thus complete
digestion produces two DNA fragments of 3.6 and 1 kbp. Deprotection by
soft metal ions was assayed after the addition of 20 µM
Pb(OAc)2 (lanes 4 and 9),
Cd(OAc)2 (lane 5), ZnSO4 (lane
6), NaAsO2 (lane 10), potassium antimonial
tartrate (lane 11), CoSO4 (lane 12),
NiCl2 (lane 13), or
Hg(NO3)2 (lane 14). B,
the effect of soft metal ions on protection of cad promoter
DNA from SspI digestion by CadC was assayed after the
addition of the indicated concentrations of Pb(OAc)2
(circles), Cd(OAc)2 (inverted
triangles), or ZnSO4 (squares). Each value
represents the mean of three separate experiments, with the standard
deviation shown by the error bars.
View larger version (14K):
[in a new window]
Fig. 4.
Effect of chemical modification and
mutagenesis of CadC cysteine residues on the ability to bind to the
cad promoter DNA in vitro and to
respond to soft metal ions. A, methyl
methanethiosulfonate (MMTS) is a chemical modifying reagent
that reacts specifically with cysteine residues in proteins (23).
Inhibition by MMTS frequently is considered to reflect a requirement
for the cysteine thiolates. Purified wild-type CadC (5 µM) was reacted with 2 mM MMTS or with 2 mM MMTS followed by 5 mM DTT, after which its
ability to protect the plasmid pI258 cad operator DNA was
assayed with or without the indicated concentrations of
Pb(OAc)2 as described under "Materials and Methods."
Treatment of CadC: none (circles), MMTS (inverted
triangles), and MMTS followed by DTT (squares).
B-D, the ability of mutant CadC proteins to bind
to the cad promoter and soft metal ion deprotection assayed
after the addition of the indicated concentrations of
Pb(OAc)2 (B), Cd(OAc)2
(C), and ZnSO4 (D). CadC mutants were
C7G (circles), C11G (inverted triangles), C52G
(squares), C58S (diamonds), and C60G
(triangles).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Sylvia Daunert, University of Kentucky, for plasmid pQF50/red-shifted GFP and Dr. Simon Silver, University of Illinois School of Medicine, for plasmid pYPK11.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant AI45428.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.
Both authors contributed equally to this study.
§ To whom correspondence 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@med.wayne.edu.
Published, JBC Papers in Press, February 14, 2001, DOI 10.1074/jbc.M010595200
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ABBREVIATIONS |
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The abbreviations used are: bp, base pair; kbp, kilobase pair; GFP, green fluorescent protein; MOPS, 4-morpholinepropanesulfonic acid; DTT, dithiothreitol; HPLC, high pressure liquid chromatography; MMTS, methyl methanethiosulfonate.
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