CopY is a copper-inducible repressor of the Enterococcus hirae copper ATPases.

The cop operon of Enterococcus hirae effects copper homeostasis in this organism. It encodes a repressor, CopY, an activator, CopZ, and two P-type copper ATPases, CopA and CopB. Expression of all four genes is regulated by the ambient copper. In this regulation, CopY apparently acts as a copper-inducible repressor. By DNase I footprinting, it was shown that purified CopY protected two discrete sites in the region encompassing nucleotides -71 to -11 relative to the translational start site and containing hyphenated inverted repeats. Transcription is initiated between these repeats at nucleotide -42, in a domain that remained accessible to DNase I in the DNA-repressor complex. Chemical cross-linking revealed that CopY exists as a dimer in solution. In DNA band-shift assays, it was apparent that the CopY-DNA interaction occurred in two discrete steps. Half-maximal binding of repressor to the two operator sites was observed at 2 x 10(-9) M and 5 x 10(-9) M CopY, respectively. Copper ions released CopY from the promoter/operator with an apparent half-binding constant for Cu(I) of 20 microM. The site-directed mutations A-61T and A-30T essentially abolished the binding of CopY to the respective binding sites, and the double mutation A-61T/A-30T inactivated both binding sites. Thus, CopY is a copper-inducible repressor of the cop operon of E. hirae, exhibiting highly specific DNA-protein interactions with two sites on the cop promoter/operator and playing a key role in copper homeostasis in E. hirae.

The two copper ATPase genes, copA and copB, are arranged in the cop operon, together with two regulatory genes, copY and copZ, in the order copY, copZ, copA, copB. These four genes appear to make up the copper homeostatic system of E. hirae. copY and copZ encode hydrophilic proteins of 145 and 69 amino acids, respectively. Expression of the ATPases was shown to be regulated by the ambient copper concentration in a biphasic fashion: expression was minimal at 10 M extracellular copper, and increased as well as decreased copper concentrations led to induction of the cop operon (4). Disruption of the copY gene caused massive overexpression of CopA and CopB ATPase, whereas null mutation of copZ suppressed expression to very low levels. Based on these findings, a model was proposed in which CopY acts as a repressor and CopZ as an activator of the cop operon (6). In partial support of this model, we here show the interaction of CopY with the operator/promoter region of the cop operon.
The transcriptional start site of the cop operon was mapped by primer extension, and the site of interaction of CopY with the operator was delineated by DNase I footprinting. Through site-directed mutagenesis, nucleotide residues that are critically involved in the CopY-operator interaction were identified. It was also shown that CopY is a homodimer that interacts with the operator in two steps in a copper-sensitive fashion.

EXPERIMENTAL PROCEDURES
Bacterial Strains and Plasmids-E. hirae (ATCC 9790, formerly called Streptococcus faecalis or S. faecium) was obtained from the American Type Culture Collection. Escherichia coli strains XLmutS and XL1-Blue were obtained from Stratagene, Inc. The expression vector pQE12 was purchased from Qiagen. The construction of pOA1 has been described previously (6).
Materials-Pico green was from Molecular Probes, Inc. The Chameleon mutagenesis kit (Stratagene, Inc.) was used for site-directed mutagenesis. [␥-32 P]ATP (3000 Ci/mmol) was obtained from Amersham Corp., phenylmethylsulfonyl fluoride from Merck, and growth media additives from BBL. Purification columns, column materials, and the MluI3ScaI switch-toggle primer were bought from Pharmacia Biotech Inc. Moloney murine leukemia virus reverse transcriptase was from Anawa Trading, Inc. (Wangen, Switzerland). Dynazyme DNA polymerase was provided by Finnzymes (Espoo, Finland), and oligonucleotide primers were synthesized by Microsynth, Inc. (Windisch, Switzerland). All other molecular biology reagents were obtained from Boehringer Mannheim. DNase I and other chemicals were from Sigma and were of the highest grade available.
Primer Extension-Total cellular RNA of wild-type E. hirae was prepared either from uninduced cells or from cells that were induced for 1 h with 1 mM CuSO 4 . RNA was isolated as described previously (7). The primer 5Ј-CCAATCCATCGAATCAGCTAATATC-3Ј was labeled with polynucleotide kinase and [␥-32 P]ATP to a specific activity of 0.1 Ci/nmol. 20 g of RNA in 30 l of hybridization solution (40 mM PIPES, 1 pH 6.4, 1 mM EDTA, 0.4 M NaCl, 80% formamide) were heated for 5 min to 85°C and annealed overnight at 30°C with 0.5 pmol of the labeled primer. The nucleic acids were then ethanol precipitated, washed, and dissolved in 100 l of 10 mM dithiothreitol, 0.5 mM dNTPs, 40 units of RNase inhibitor, and reverse transcribed with 100 units of Moloney murine leukemia virus reverse transcriptase in the buffer supplied by the manufacturer for 90 min at 42°C. Following ethanol precipitation, the products were analyzed by electrophoresis on 6% denaturing polyacrylamide sequencing gels.
CopY Overexpression-A PCR product of the copY gene was generated with the two primers 5Ј-CAAGAATGATCAAAGAATTT-3Ј and 5Ј-CCATTTTGGAATTCGTTGCC-3Ј and Dynazyme DNA polymerase. Following cutting with BamHI and HindIII, the PCR product was cloned into pQE12 and digested with the same enzymes. The resultant clone encoded a CopY protein with a five-amino acid N-terminal extension originating from the expression vector. These extra residues were deleted by site-directed mutagenesis. The final clone, pWY145, was verified by DNA sequencing.
CopY Purification-For overexpression, 500-ml cultures of XL1-Blue cells transformed with pWY145 were grown aerobically at 30°C to an A 550 of 0.5. Following induction with 1 mM isopropyl-1-thio-␤-D-galactopyranoside for 2 h, the cells were harvested by centrifugation for 10 min at 5000 ϫ g. The cell pellet was washed once with 200 ml of TGD buffer (50 mM Tris-SO 4 , pH 7.7, 5% (v/v) glycerol, 2 mM dithiothreitol) and resuspended in 100 ml of TGD buffer. The cells were lysed by incubation with 25 g of lysozyme and 1 mM phenylmethylsulfonyl fluoride for 10 min at room temperature and broken open by one passage through a French pressure cell at 40 MPa. The cell debris was collected by centrifugation for 1 h at 90,000 ϫ g and the supernatant passed through a Q-Sepharose column. CopY was eluted with a 0 -100 mM K 2 SO 4 gradient in TGD buffer. Final purification was achieved by gel filtration on a Superose-12 column in TGD buffer plus 150 mM K 2 SO 4 . Purified CopY was stored in 2-g aliquots at Ϫ70°C. The concentration of CopY was determined by amino acid analysis.
Cross-linking Experiments-100 pmol of purified CopY were crosslinked at room temperature with the glutaraldehyde concentrations indicated under "Results" in a total volume of 40 l of 10 mM Na-HEPES, pH 8.0, 5 mM magnesium acetate, 5 mM dithiothreitol, 50 mM sodium acetate, 12% (v/v) glycerol, for 10 min at room temperature, followed by quenching with 5 l of 1 M ethanolamine. The products were resolved on a 15% SDS-polyacrylamide gel and visualized by silver staining.
DNA Band-shift Assays-DNA band-shift assays were performed essentially as described (8). 2-3 fmol (6 -10 nCi) of labeled wild-type cop promoter/operator DNA were incubated with a 10 -300-fold molar excess of purified CopY for 30 min at room temperature in a total volume of 20 l of the buffer used for DNase I footprinting. The reactions were analyzed on 6% polyacrylamide gels in 6.8 mM Tris acetate, pH 8.0, 3.3 mM sodium acetate, 2.5% (v/v) glycerol. Gels were allowed to polymerize for at least 12 h prior to use and were run at 5 W for 90 min. Promoter/ operator fragments with the AϪ61T mutation were isolated by PCR amplification of the mutated plasmid with the two primers 5Ј-CCAATC-CATCGAATCAGCTAATATC-3Ј and 5Ј-CGAATCTTCCAACTGATCG-3Ј. The promoter/operator fragments with the mutations AϪ30T and TϪ23A and the double mutations AϪ30T/AϪ61T and TϪ23A/AϪ61T were similarly generated by PCR amplification of mutated plasmid DNA with the primers used for mutagenesis.
DNase I Footprinting-Footprinting assays were performed in 200 l of 20 mM Tris acetate, pH 8.0, 5 mM dithiothreitol, 5 mM magnesium acetate, 50 mM sodium acetate, 12% glycerol, 1 mM Ca(OH) 2 , 2 g of poly(dI-dC), and 5 g of bovine serum albumin. Assays contained 4 -6 fmol of cop promoter/operator fragment, that was labeled with 32 P at one end with polynucleotide kinase as described (8) to a specific activity of 3 Ci/pmol. The amounts of purified CopY specified under "Results" were added and the reaction incubated for 30 min at room temperature. The DNA was then digested with 0.012 unit of DNase I at 30°C for 2 min. Digestion was terminated by adding 700 l of 92% ethanol, 0.75 M ammonium acetate, 7 g/ml tRNA. The products were precipitated at Ϫ70°C for 15 min and centrifuged for 10 min at 10,000 ϫ g. The pelleted material was washed once with 70% ethanol and resolved by electrophoresis on an 8% polyacrylamide sequencing gel together with a Sanger sequencing reaction of the same fragment with the primers 5Ј-CGAATCTTCCAACTGATCG-3Ј for the sense strand and 5Ј-CCAATCCATCGAATCAGCTAATATC-3Ј for the antisense strand.
Miscellaneous Methods-Standard molecular biology methods were used according to published procedures (8). DNA was sequenced according to the method of Sanger et al. (9). The concentrations of the DNA fragments used for band-shift assays were determined by fluorescent measurements with pico green as described by the manufacturer.

RESULTS
To characterize the function of the CopY repressor and to study its interaction with the DNA target, CopY was overexpressed in E. coli and purified. When CopY was overexpressed as detailed under "Experimental Procedures," approximately 60% of the protein was trapped in inclusion bodies. However, CopY in the supernatant still made up 10% of the total protein in this fraction. CopY could be purified to approximately 90% purity by successive chromatography on Q-Sepharose and gel filtration. Fig. 1A shows the products of the different purification steps. Below the band of apparent molecular mass 17 kDa, corresponding to CopY, there was always a minor band of 16 kDa in the preparation. This contaminant increased with increasing purification and could be a degradation product of CopY. However, we could not observe variation in the results obtained with preparations containing differing amounts of the 16-kDa band. We assessed whether CopY was monomeric or multimeric in solution by cross-linking experiments. Fig. 1B shows that increasing concentrations of glutaraldehyde resulted in the formation of CopY homodimers with an apparent molecular mass of 34 kDa. No significant amounts of higher molecular mass species were generated even at very high concentrations of cross-linker, indicating that CopY homodimers are the principal species of CopY in solution. Inclusion of a 4-fold excess of lysozyme in the experiment did not result in significant crosslinking of this control protein to itself or to CopY (not shown). In analytical gel filtration, CopY migrated like a globular protein of apparent molecular mass 53 kDa, suggesting that the native CopY dimers are not globular but form an extended structure, as would be expected. 2 Since CopY acts as a repressor of the cop operon, it was expected to interact with a DNA target at or near the site of transcription initiation. Primer extension analysis was thus carried out to determine the transcription initiation site of the cop operon. Total RNA from copper-induced and uninduced wild-type E. hirae cells was reverse transcribed with a primer as specified under "Experimental Procedures," and the resultant DNA fragments were resolved on a sequencing gel (Fig. 2). A single product was generated from induced but not from uninduced cells. This is in line with the previous observation that in copper-induced cells expression of the cop operon was increased 12-fold compared with uninduced cells (6). Co-electrophoresis of the primer extension products with DNA sequencing reactions conducted with the same primer allowed assignment of the transcriptional start site to guanosine at position Ϫ42 with respect to the start of translation. This assignment was further verified by running gels of mixtures of sequencing reactions and primer-extension product (not shown).
DNA binding properties of the CopY repressor were studied by band-shift assays. When a radiolabeled 316-bp DNA fragment encompassing the transcription start site was incubated with purified CopY, DNA-protein complexes could be observed ( Fig. 3). Increasing concentrations of CopY changed the apparent mobility of the DNA fragment in two steps: to an intermediate retarded form I, and to a more retarded form II, indicating two binding events. Addition of a 10-fold molar excess of unlabeled cop promoter/operator DNA to the binding mixture competed with the DNA-protein complex formation, whereas added promoter DNA from the ␤-lactamase gene of E. coli did not act as a competitor. Similarly, the promoter of the NaH antiporter gene of E. hirae (10) did not compete with the binding (data not shown), indicating the specificity of the DNA-CopY interaction. By quantitative evaluation of the band-shift data, the half-association concentrations of CopY under these conditions were estimated to be 2 nM for retarded form I and 5 nM for retarded form II.
In vivo, the expression of the E. hirae cop operon is induced by Cu 2ϩ , Cu ϩ , Ag ϩ or Cd 2ϩ but not by Ni 2ϩ or Zn 2ϩ (3,6). It was thus of interest to test whether these heavy metal ions affected the in vitro interaction of CopY with the cop operator. Fig. 3B shows the effects of different heavy metal ions on the CopY-cop operator interaction as measured by band-shift assays. As little as 1 M added copper in the binding reaction released some CopY from the cop operator. At 50 M copper, formation of a CopY-DNA complex was completely inhibited. An approximately 50% displacement of CopY from the operator was observed with 20 M Cu ϩ , 50 M Ag ϩ , or 50 M Cd 2ϩ . The same concentrations of Ni 2ϩ and Zn 2ϩ ions released approximately 5% of the bound repressor from the DNA. These results demonstrate that Cu ϩ , Ag ϩ , and Cd 2ϩ are efficient inducers of the CopY repressor. This is in line with the induction properties of the cop operon observed in vivo. It is of course not possible to quantitatively compare in vitro band-shift experiments with whole cell studies, since it is not known how externally added metal ions change the cytoplasmic concentration in living cells. Also, the redox equilibrium between Cu(I) and Cu(II) makes it very difficult to determine the oxidation state of copper in vivo.
By site-directed mutagenesis of the cop promoter/operator DNA, specific DNA-CopY interactions were identified. Inversion of the adenosine at position Ϫ61 to a thymidine (AϪ61T) altered the binding of CopY to the operator.  1-7, 0, 1, 2, 3, 6 DNA. This suggests that adenosine Ϫ61 is an essential base for the interaction of CopY with one site on the operator. The mutation AϪ30T revealed another essential residue, inactivating the second site of interaction of CopY with the operator (Fig. 4B), whereas the mutation TϪ23A in the proximal half of the inverted repeat did not have an effect on the repressor-DNA interaction (not shown). In the double mutant AϪ30T/ AϪ61T, the repressor-DNA interaction was practically abolished. Clearly, single bases in the cop promoter/operator play key roles in the interaction with the CopY repressor. Halfmaximal binding of repressor to the single remaining high affinity binding sites in the mutants was estimated to occur at 7 nM CopY for the site proximal to the translation start and at 12 nM CopY for the distal binding site. These values are 2-3fold higher than those observed for the wild-type operators, which could indicate some cooperativity between the binding sites. Since it is unlikely that the relative binding affinities of the two sites are reversed in the mutants, this would suggest that the proximal site exhibits the higher affinity for CopY and interacts first with the repressor.
With DNase I footprinting experiments, the sites on the cop promoter/operator DNA that are occupied by the bound CopY repressor were mapped. Two regions were protected from DNase I digestion by CopY. They were slightly asymmetric on the two DNA strands and encompassed nucleotides Ϫ13 to Ϫ38 and Ϫ44 to Ϫ69 on the sense strand (Fig. 5A) and nucleotides Ϫ11 to Ϫ37 and Ϫ44 to Ϫ71 on the complementary strand (Fig.  5C). Between these protected sites, there is a region not protected from DNase I digestion, namely from Ϫ39 to Ϫ43 on the sense strand and from Ϫ38 to Ϫ43 on the antisense strand. The start of transcription, mapped to nucleotide Ϫ42, is located in this DNase I-sensitive domain (see Fig. 4C for a summary).
Band-shift experiments with mutant promoter/operator DNA had suggested that CopY binding by one site was impaired in some of the mutants. DNase I protection experiments with the mutated cop promoter/operator fragment AϪ61T showed indeed that only one site could bind repressor under the conditions used. The distal half of the inverted repeat that contained the AϪ61T mutation remained sensitive to DNase I digestion (Fig. 5B). Similarly, the AϪ30T mutated operator/ promoter DNA was protected from DNase I by CopY at the distal, nonmutated site but was protected only by high concentrations of CopY at the mutated site (Fig. 5D). Finally, the double mutant AϪ30T/AϪ61T remained essentially unprotected to DNase I digestion by CopY at both binding sites. Only high concentrations of CopY somewhat protected the proximal region from DNase I digestion (Fig. 5E). These observations are in agreement with the gel retardation experiments and show that mutations AϪ61T and AϪ30T both strongly interfere with the binding of the CopY repressor to its DNA target. Clearly, the nucleotides at these positions must fulfill crucial roles in the DNA-protein interaction.
Taken together these results show that the cop promoter/ operator possesses two independent binding sites for the CopY repressor. These sites exhibit somewhat different binding affinities for the repressor and appear well separated, since there are some DNase I-sensitive bases between the sites, including the site of transcriptional initiation. DISCUSSION We have characterized the interaction of the E. hirae copperresponsive repressor, CopY, with the cop operator in vitro. Three main features of this regulation system were revealed: (i) the CopY repressor interacts with the cop operator/promoter at two discrete sites at positions flanking the transcriptional start site; (ii) the repressor-DNA interaction is copper-sensitive; and (iii) single base changes can lead to half-of-the-site reactivity. Cross-linking and gel filtration experiments suggest that CopY is present as a homodimer in solution. Thus, it appears likely that the CopY homodimers interact with each of the two binding sites identified in the operator/promoter region.
The N terminus of the CopY repressor exhibits 27-32% sequence identity to the ␤-lactamase repressors MecI of Staphylococcus epidermis (11), PenI of Bacillus licheniformis (12), and BlaI of Staphylococcus aureus (13). These repressors possess two structurally and functionally different domains (14), similar to the well characterized proteins of the large Cro/LysR family of repressors (15). The general concept of the function of these proteins is that the N-terminal part of the protein forms a helix-turn-helix DNA binding motif, whereas the C-terminal part interacts with the effector. This concept is supported by x-ray crystallographic studies of the bacteriophage Cro and 434 repressors (16,17), which revealed that the Q28-Q29 pair in the helix-turn-helix motif of these proteins tightly interacts with the base pairs of an ACA triplet of the operator DNA (18) and that the mutation Q28A abolishes the interaction of the repressor with wild-type operator DNA (19). An analogous Q30-Q31 pair is present in CopY, and the cop promoter/operator contains four ACA triplets that would allow a similar interaction. We have shown that mutation of two of these ACA triplets to TCA almost completely abolish CopY-DNA interaction at the corresponding sites. These findings are essentially complementary to those obtained with the 434 repressors and suggest that the CopY-operator interaction is very similar in nature.
CopY concentrations required for half-maximal interaction with the two sites on the wild-type operator were determined in vitro as 2 ϫ 10 Ϫ9 M and 5 ϫ 10 Ϫ9 M, and thus are in the range of 10 Ϫ9 to 10 Ϫ10 M observed for other repressor-operator interactions (20,21). Although most repressors are homodimeric proteins, only a few of them interact with two discrete binding sites on the operator, such as NodD, IlvY, or CadC (22,23). In fact, the CadC regulatory system resembles the E. hirae CopY system in other ways. It also controls expression of heavy metal ATPases: the cad operons of cadmium-resistant bacteria encode a regulatory protein, CadC, and a cadmium efflux ATPase, CadA. The expression of CadA is strongly induced by Cd 2ϩ but also by Pb 2ϩ , Bi 3ϩ , and some other heavy metal ions (24). By gel retardation assays and DNase I footprinting, it was recently shown that CadC binds to an inverted repeat in the promoter/ operator region of the cad operon and is released by Cd 2ϩ , Pb 2ϩ , and, more efficiently, Bi 3ϩ and Hg 2ϩ (25). Similar to the CopY-DNA interaction, the binding of CadC to DNA took place in two discrete steps. However, CadC does not exhibit significant sequence identity to CopY but is related to ArsR from E. coli, which regulates resistance to arsenic ions (26), and to SmtB, which controls cyanobacterial metallothionein synthesis (27). So these proteins apparently represent another class of metalloregulators.
The promoter of the cop operon features the consensus sequences TGTAAT and TTGACA at the Ϫ10 and Ϫ35 positions relative to the transcriptional start site. These sequences are found in many bacterial promoters (28). The positioning of the two CopY binding sites relative to these transcriptional signals lies within the range of consensus positions for repressors and outside the range for activators (29). The biological significance of two CopY binding sites with similar affinities remains unclear at this time.
The control of the cop operon of E. hirae apparently also requires the function of CopZ (6). CopZ is a protein of 69 amino acids containing the presumed copper-binding motif GMX-CXXC. In copZ-disrupted strains, the transcription of the cop operon was repressed and copZ function could be complemented in trans (6). A model for the observed biphasic regulation was proposed in which derepression of the cop operon can be achieved in two ways: by copper releasing CopY from the operator under conditions of excessive copper, or through activation of transcription by CopZ under copper-limiting conditions. Although our data support the activation of the cop operon by copper-induced release of the repressor, we could not obtain evidence of an involvement of CopZ in vitro, and the elucidation of the role of this protein requires further study. FIG. 5. DNase I footprinting of the CopY-cop promoter/operator complex. 0.02 nM promoter/operator DNA, 32 P-labeled at the 3Ј-end, was incubated with (lanes 1-9) 0, 0.1, 0.2, 0.3, 0.6, 0.9, 1.5, 4.5, and 10 nM CopY, respectively, and subjected to DNase I digestion as described under "Experimental Procedures." The reaction products were resolved on 8% denaturing polyacrylamide gels and visualized by phosphorimaging. A, sense strand of wild type. B, sense strand of mutant AϪ61T. C, antisense strand of wild type. D, antisense strand of mutant AϪ30T. E, antisense strand of double mutant AϪ61T/AϪ30T. The numbers at the left and right indicate nucleotide positions relative to the translational start site.