From the School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
Received for publication, October 17, 2002, and in revised form, November 15, 2002
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ABSTRACT |
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The copA gene of Escherichia
coli encodes a copper transporter and its promoter is normally
regulated by Cu(I) ions and CueR, a MerR-like transcriptional
activator. We show that CueR can also be activated by gold salts and
that Cys112 and Cys120 are involved in
recognition of gold, silver, and copper salts. Gold activation is
unaffected by copper chelating agents but is affected by general metal
chelators. This is the first example of specific regulation of
transcription by gold, and we briefly speculate that the biological
effects of gold antiarthritic drugs may be through their effects on
copper management in eukaryotic systems.
There is no known natural biological function of gold or its
salts, but it has biological effects. Solutions of gold chloride are
toxic to bacteria (1), disimilatory Fe(III)-reducing bacteria and
archaea are capable of reducing Au(III) to Au(0), precipitating the
metal (2), and may be responsible in part for the formation of some
gold deposits (1). Medical uses of gold have been limited largely to
prosthetics and antiarthritic Au(I)-thiolate drugs, although the basis
of antiarthritic action has been unclear (3). In contrast, the
biological requirements for copper are quite well understood. Mammalian
systems require copper for many enzymes (4). Recently, new elements of
copper homeostasis in bacteria have been identified, including
transporters, multicopper oxidases, and two-component and MerR-like
regulators (5-9).
The Escherichia coli CopA protein is a
Cu(I)/Ag(I)-translocating P-type ATPase involved in copper export and
resistance (5). It shows similarity to copper pumps from several
prokaryotic and eukaryotic sources, including 31 and 29% identity to
the human Wilson's and Menkes' proteins, respectively. The
transcription of copA is regulated solely by CueR
(GeneBankTM AF318185), a copper- and silver-responsive (7,
8, 10) member of the MerR family of transcriptional activators (Fig. 1). MerR acts as a dimer on the promoter
of bacterial mercury resistance (mer) genes recruiting RNA
polymerase to the promoter in the process (11), and it is activated by
Hg(II) binding to 3 cysteine residues in a trigonal planar fashion
(12). CueR activates expression of the copA promoter
(PcopA) upon addition of copper or silver, but not nickel,
lead, cadmium, mercury, or zinc salts (8). Here we show that CueR
activates the PcopA promoter in the presence of gold salts
and that cysteine residues, equivalent to those in MerR, are required
for activation by copper, silver, and gold.
Bacterial Strains and Plasmids--
All experiments, other than
the construction of mutants and some DNA Manipulations--
Standard DNA manipulations were carried
out as described elsewhere (18). Oligonucleotides were synthesized by
Alta Bioscience Ltd., University of Birmingham, by
Invitrogen, or by MWG Biotech. Cloned PCR
products were sequenced using an PE Applied Biosystems Big
DyeTM sequencing kit according to the manufacturers'
instructions and analyzed on an ABI 3700 sequencing apparatus.
The direct effect of gold salts on Mutagenesis of cueR--
The cueR mutants
C112S; C120S; C129S,C130S; and H131N,H132N were generated by the gapped
duplex method (13) using 24- to 30-mer oligonucleotides containing the
1 or 2 mismatched bases. Site-directed mutagenesis by PCR overlap
extension (19) was used in later stages for creating H76N,H94N using
primer pairs containing complementary mutating nucleotides, and
flanking forward and reverse primers containing EcoRI and
BamHI sites, on pSU18cueR as template. Truncated
cueR mutants were generated by PCR using a downstream primer
containing a stop codon and a BamHI site, with a flanking
upstream primer containing an EcoRI site. PCR products were
digested with EcoRI and BamHI and cloned into
EcoRI-BamHI-cut pSU18, creating
pSUcueR-mutant. All mutated DNA fragments were fully sequenced to confirm the required mutation and screen against undesired mutations.
Generation and Assay of Cu(I)--
Solutions of cuprous ions,
Cu(I), were made freshly on a daily basis as described elsewhere
(20).
Gold Specifically Induces Activation of the PcopA
Promoter--
The dynamic response of the copA promoter was
tested at a range of copper, silver, and gold ion concentrations in
minimal medium (Fig. 2A), and
maximal responses were observed with additions of 100 µM
CuSO4, 2.5 µM AgNO3, or 25 µM AuCl3 for mid-exponential phase cells.
Gold toxicity caused a reduction in cell growth at higher metal
concentrations. Similar relative concentrations for maximal induction
were observed at different growth stages, although the absolute
concentrations differed, presumably due to the production of
metal-chelating extracellular material. AuCl3 does not
inhibit
Induction by gold of PcopA was abolished in a disruptant
strain, E. coli
TG1[cueR::kanR] (8). As
with previous results for copper and silver (8), metal induction of
PcopA could be recovered by providing the cueR gene in trans (Fig. 2B). Therefore, CueR
activates transcription of PcopA in response to gold ions.
To check that the response of CueR to gold was specific, a second
member of this class of metal regulators, MerR and its promoter
PmerTPAD, was tested in both E. coli TG1 and in
E. coli CSH26
The growth response of E. coli W3110 Cysteine Residues 112 and 120 Are Required for Activation by
Copper, Silver, and Gold--
The CueR protein contains several
cysteine and histidine residues which are candidates for recognition
and specific binding of activating metals. Cysteine residues at
positions 112, 120, 129, and 130 were mutated to serine and histidines
at positions 76, 94, 131, and 132 were altered to asparagine. These
variants of the cueR gene were expressed in pSU18 vector
(16) in E. coli TG1[cueR::kanR](pMUPcopA).
The response of the complemented E. coli
TG1[cueR::kanR] strain to
metals was more variable between experiments than when the wild-type
E. coli TG1 was used, possibly due to the variable copy
number of pSU18::cueR plasmids. However,
qualitatively clear results were obtained, showing that mutations C112S
or C120S eliminated the response to copper, silver, and gold salts
(Fig. 3) as did all double and truncated
mutants containing one of these mutations (data not shown).
Furthermore, none of these mutants responded to lead, mercury, nickel,
cadmium, or cobalt. A deletion from position 120 failed to
respond to metals, while a deletion from position 128 had an attenuated
response. The cysteines at positions C112 and C120 are analogous to
C117 and C126 of Tn501 and Tn21 MerR, which are
important for binding Hg(II) and activation of the transcription of the
mer operon (11). Alanine A78 was replaced by cysteine to
mimic the equivalent essential cysteine C82 in MerR (Fig. 1), but the
mutant protein did not activate transcription in response to Hg(II)
in vivo (data not shown).
Double mutations H131N,H132N and, separately, C129S,C130S, were
generated to investigate the importance of these distal, potentially metal-binding, amino acids and were tested in the induction assay (Fig.
3). The mutations H131N,H132N gave no significant change in the
response to copper but gave an altered response to silver (decreased)
and gold (increased) compared with the wild type. There was a slight
increase of the background response without metal. The mutant
C129S,C130S also showed relatively small changes in the response to
copper and silver but there was a slight decrease in the response to
gold. For neither mutant was the specificity of reaction with other
metals altered. The retention of a response to copper, silver, and gold
salts, albeit altered in magnitude, by these double mutations and by
the seven amino acid C-terminal deletion from Gly128
indicates that the full CCHH motif at positions 129-132 is not responsible for metal recognition by CueR, as we had originally hypothesized. A decrease of 30-40% but not abolition of the response to copper was found with the separate H76N and H94N mutations (data not
shown), indicating that these two histidines are not essential to metal recognition.
The Effect of Chelators Indicates That the Metal Responses Are
Specific--
The effect of different chelating agents on the
induction of PcopA was tested (Fig.
4). Bathocuproine disulfonate (21), cuprizone (22), and neocuproine (21, 22) were used as copper chelators;
EDTA was used as a nonspecific metal chelator. When PcopA
was induced in the presence of 100 µM bathocuproine,
activation by Cu(I) was reduced by 84-86% and the activation by
Cu(II) decreased by 35-69%; activation by silver was reduced by 70%;
and activation by gold was increased by 9-34%. Activation of the
mercury resistance promoter, PmerTPAD, in the presence of
merR and 1 µM Hg(II) showed no significant
difference in the presence and absence of bathocuproine. With 100 µM cuprizone, activation by Cu(I) was reduced 33-65% and by Cu(II) 16-46%, suggesting that cuprizone may not be specific for Cu(II), as advertised. Activation by silver increased by 29% and
activation by gold increased by 42-45%, both of which may be due to
the removal of relatively more poorly inducing copper ions by the
chelator. No difference was observed for the activation of
PmerTPAD by Hg(II).
Activation of PcopA was observed in the presence of the
membrane-permeable copper chelator neocuproine even in the absence of
metal, resulting in 8.8-fold base level increase (Fig. 4). The
increased induction by different metals was calculated by subtracting
from each sample the background level in the control with chelator but
without metal and comparing with the equivalent samples without
chelator (with the "no chelator, no metal" control subtracted),
thus enabling direct comparison of the results. The values were as
follows: 3-4-fold increase in activation by Cu(I), 1.8-3-fold
increase in activation by Cu(II), no change in activation by silver or
with gold. Again there was no change in the response of
PmerT and merR. These results suggest that
neocuproine specifically carries copper into the cell. The background
level may have increased due trace amounts of copper present in SM9 medium.
Gold (as extracellular Au(III)) is not chelated by copper-specific
chelators and activates the copA promoter in their presence to approximately the same extent as in the presence or absence of these
chelators. Au(I) salts are too insoluble to use directly in chelation
experiments. EDTA (200 µM) decreased the activation of
the copA promoter to background levels by all metals (Fig. 4). None of the copper chelators had any effect on the induction of the
mer promoter by MerR and mercury, which suggests that all of
the effects observed for copper, silver, and gold are metal-specific and not caused by the chelating agents.
Au(I) is a soft metal ion with a covalent radius of 134 pm and and
electronegativity of 1.42 (Allred-Rochow), identical to those
properties of silver, but different to those of copper, 117 pm and
1.75, respectively (23). The effective nuclear charge, the charge due
to the protons of the nucleus less a screening factor due to the outer
electrons of the atom, for gold, copper, and silver is identical at
4.20 (Slater). No other element has the same effective nuclear charge.
The activation of PcopA by CueR and gold salts was
unexpected from a biological point of view. The physical chemistry of
gold ions suggests that they are mimicking Cu(I) and Ag(I) in the metal binding site of CueR, leading to activation of PcopA. Au(I)
is a common valence for this element and may be produced in the
reducing environment of the cytoplasm or in the periplasm by a
reductase such as NDH2 (24, 25). Although Au(I) is practically
insoluble in water, which has prevented us from simply confirming our
in vivo data with in vitro experiments, the local
production of Au(I) salts is the most chemically convincing explanation
for the mimicry of Cu(I) by gold salts, given the identical effective
nuclear charge among the three elements. The exact levels of metal
required for half-maximal induction of the promoter are extracellular
values and differential uptake and reduction may account for the
differences between the metal ions; Ag(I) alone would not be involved
in redox reactions and has the lowest concentration for half-maximal
induction. Carefully designed experimental conditions for in
vitro study of the effect of the metals on CueR will need to be
performed to confirm the valence state of gold required for activation
of CueR.
The lack of effect of a The details of recognition of Cu(I) Ag(I) and Au(I) by the CueR
regulator remain to be fully dissected. The observation that cysteine
residues 112 and 120 are required for activation by all three metals
indicates that the metals bind to the same site on the regulator, and
the equivalence of these amino acids to Cys117 and
Cys126 in MerR (Fig. 1) suggest that the metal binding site
is in the same relative position on both regulators. Thus, it is not
surprising that replacing alanine 78 with cysteine to mimic the
equivalent essential cysteine in MerR (Fig. 1) has no effect on CueR
response to Hg(II), as the spacing between Cys112 and
Cys120 in CueR is different to that between
Cys117 and Cys126 in MerR, and more subtle
positioning and charge effects will undoubtedly be invovled in metal
recognition. The recently described copper-dependent
regulator, HmrR, from rhizobia (27) contains cysteines equivalent to
Cys112 and Cys120, but lacks a sequence
equivalent to the CCHH motif at positions 129-132, providing indirect
confirmation of our analysis. The detail of metal binding will require
structural information on the CueR protein. The existing structures of
MerR family regulators are for proteins responding to effectors other
than metals (28, 29), and cysteines 112 and 120 lie outside the regions
of similarity between CueR and these regulators.
The molecular homeostatic mechanisms for copper in humans continue to
be identified. The toxicity of copper is due in part to its redox
chemistry. Cuprous ions can be oxidized by peroxide to generate
hydroxyl ions and radicals, which can attack and damage phospholipids
in biological membranes and inactivate membrane bound enzymes (30).
This type of damage is characteristic in people with copper toxicosis
(Wilson's disease) due to a defect in the WND copper transport
protein (31). Rheumatoid arthritis is a disease characterized by
migration of phagocytes and other leukocytes into synovial and
periarticular tissue and damage of these tissues by activated oxygen
species from triggered phagocytes (32). Increased copper levels were
found in the blood plasma of rheumathoid arthritis patients (33),
copper toxicity and arthritis were linked in a patient with Wilson's
disease (34), and a copper-supplemented diet was shown to ameliorate
Mycobacterium butyricum-induced arthritis in rats (35).
Our speculation is that by mimicking copper, gold may act as an
effective activator of the expression of copper transport proteins and
alter the copper homeostasis within the arthritic tissues, by removing
copper from locations where it might generate reactive oxygen species.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Alignment of CueR and other metal-responsive
members of the MerR family. The consensus amino acids indicate
where three of the four regulators have an identical amino acid at that
position. The sites of directed mutations tested for their effect on
the response to copper, silver, and gold salts are marked with
arrows above the sequences; Cys112 and
Cys120 are numbered.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase assays, were
carried out in E. coli strain TG1 (K-12, lac-pro supE
thi hsdD5 [F' traD36
proA+B+ lacIq
lacZ M15]). E. coli WK6
(lac-proAB)galEstrA
[F' lacIq lacZ
M15
proAB+] and its mutS215:Tn10
variant were used for gapped duplex mutagenesis (13). E. coli CSH26
(lac-proAB)thi ara
recA (14) was used in some
-galactosidase assays to show that the observed effects were
not strain specific. The following plasmids have been described elsewhere: pMU2385 (15), pSU18 (16), pMUPcopA (8),
pSUcueR (8), pMUPmerT (17), and
pSUmerR (17). Bacterial cells were grown at 37 °C in
Luria-Bertani (LB) medium or M9 medium supplemented with casamino acids
and thiamine (18). For metal induction assays, sodium chloride was
omitted to avoid AgCl precipitation (called SLB and SM9 media).
Antibiotics were used as described earlier (17).
-Galactosidase Assays--
E. coli cells
transformed with plasmid pMUPcopA (8) or pMUPmerT
(17) were assayed for
-galactosidase activity (14) as described
previously (17). E. coli TG1
[cueR::kan] cells containing pMUPcopA
(8) were transformed with pSUcueR or other pSU18 vectors
containing mutant cueR, and induced overnight with 1 mM
isopropyl-
-D-thiogalactopyranoside. Cultures for
metal induction experiments were grown overnight in the required medium
with antibiotic selection to maintain resident plasmids. The overnight
culture was diluted 100-fold into fresh medium without antibiotics and grown to mid-exponential phase. Metal induction was performed for
2 h at 37 °C. Assays were performed in triplicate and repeated several times.
-galactosidase was tested by a
standard enzyme assay using 96 units of
-galactosidase in 1 ml of
Z-buffer (14) in the presence of 0, 50, or 150 µM AuCl3. Reactions were started with 0.1 ml 4 mg
ml
1
o-nitrophenyl-
-D-galactoside and stopped with
0.5 ml 1 M Na2CO3. Z-buffer is
reducing, containing 50 mM 2-mercaptoethanol. No inhibition by gold was seen.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase in vitro (data not shown), so
reduction in induction at high gold concentrations is not due to direct
effects on the reporter enzyme activity. The values of half-maximal
induction, calculated from the induction profiles, were: for copper,
~21 µM; for gold, ~15.2 µM; and for
silver, ~1.9 µM; these values include differences in
access of the metal to the regulator (sequestration, uptake, and
reduction) as well as the induction per se. The values for
silver and copper agree with those determined previously (8). The
increases in induction with increasing metal ion concentrations are
different for the three metals.
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Fig. 2.
A, the response of the copA
promoter to copper, silver, and gold salts. -Galactosidase activity
was measured in wild-type E. coli TG1 (pMUPcopA)
in SM9 medium with added CuSO4 (filled bars),
AgNO3 (open bars), or AuCl3
(hatched bars); the micromolar metal concentrations are
shown on the abscissa. B, the response of the
copA promoter to gold salts is dependent on CueR.
-Galactosidase activity was measured in response to 50 µM copper and 12.5, 25, and 50 µM gold
salts in (i) wild-type E. coli TG1(pMUPcopA),
(ii) the cueR deletion strain E. coli
TG1[cueR::kanR](pMUPcopA),
and (iii) the same deletion strain containing the plasmid
pSUcueR. Metal concentrations (µM) are given
on the abscissa. Higher gold concentrations were tolerated
in B compared with A, probably because of
sequestration of gold at higher cell concentrations in
B.
recA (17). Induction of the
mercury resistance promoter, PmerTPAD, was observed only
when the merR gene was provided in trans and
induced with 1 µM HgCl2, but was not observed
after induction with copper, silver, and gold or when merR
was not present, even though cueR was present on the
chromosome (data not shown).
copA (5)
to increasing concentrations of AuCl3 in LB medium was
indistinguishable from that of the parental W3110 strain (data not
shown), indicating that the CopA transporter does not confer gold resistance.
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Fig. 3.
Effect of cysteine and histidine mutations on
copper, silver, and gold recognition. -Galactosidase activity
was measured in SM9 medium following induction with 50 µM
CuSO4, 2.5 µM AgNO3, or 35 µM AuCl3. pSU18 bearing wild-type or mutant
cueR was provided in trans in E. coli
TG1[cueR::kanR](pMUPcopA).
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Fig. 4.
Response of the copA
promoter to Cu(I), Cu(II), Ag(I), and Au(III) in the presence of
chelators. -Galactosidase activity in E. coli TG1
(pMUPcopA) was measured in Chelex-treated SM9 medium
following induction with 50 µM Cu(I) as CuCl, 50 µM Cu(II) as CuSO4, 2.5 µM AgCl
or 37 µM AuCl3 in the presence of 100 µM concentrations of bathocuproine (b),
cuprizone (c), neocuproine (n), or EDTA
(e). The results were normalized relative to the value
determined for Cu(II) in the absence of chelating agents, given an
arbitrary value of 100%.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
copA mutation on the gold
sensitivity of growth of E. coli cells may be due to gold
salts not being recognized by the CopA transporter, indicating that
there are different mechanisms for proteins discriminating between
copper and gold salts, or may be due to change to the valence state of gold as it is exported to the outside (oxidizing) side of the cytoplasmic membrane, or transport may not confer resistance to the
metal. Such an effect has been shown for the CopB copper exporter of
Enterococcus hirae, which transports Cu(I) and Ag(I), but
confers resistance only to Cu(I) and not to Ag(I) (26).
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ACKNOWLEDGEMENTS |
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The Medical Research Council provided infrastructure support in bioinformatics. We are grateful to Dr. J. L. Hobman, Professor T. Southwood, and R. Sukchawalit for useful discussion; to R. Godfrey for technical assistance; and to Drs. P. A. Lund, C. Constantinidou, and C. Rensing for gifts of strains and plasmids.
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FOOTNOTES |
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* This work was supported by Biotechnology and Biological Sciences Research Council Grants G14071 and JIF13209 (to N. L. B.) and by the Darwin Trust of Edinburgh Studentship (to J. V. S.).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.
Present address: Dept. of Clinical Pharmacology, University of
Berne, Murtenstrasse 35, CH-3010 Bern, Switzerland.
§ To whom correspondence should be addressed. Tel.: 44-121-414-4567; Fax: 44-121-414-5907; E-mail: n.l.brown@bham.ac.uk.
Published, JBC Papers in Press, November 22, 2002, DOI 10.1074/jbc.C200580200
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