Role of cysteinyl residues in sensing Pb(II), Cd(II), and Zn(II) by the plasmid pI258 CadC repressor.

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) Ͼ Cd(II) Ͼ 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).
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 ␤-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 ni-trocellulose 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.
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 Sites TM in vitro mutagenesis system (Promega). The 448-bp XbaI-HindIII fragment from pMW1 was subcloned into the pALTER™-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 5Ј end and with a BamHI site at the 3Ј 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 pA-CYC184 (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 A 600 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 A 600 0.6 -0.8, CadC expression was induced by the addition of 0.3 mM isopropyl-␤-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 ϫ 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 Ϫ80°C until use. From the intensity of staining with Coomassie Blue on SDS-polyacrylamide gel electrophoresis, CadC was estimated to be ϳ90% 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.
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 l. The assay mixture contained 5 M purified CadC, 8 units of SspI, 0.5 g of purified plasmid pYSG1 in a buffer consisting of 50 mM Tris-HCl, pH 7.4, 6 mM MgCl 2 , 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 l of 6ϫ sample solution (0.25% bromphenol blue, 0.25% xylene cyanol FF, and 40% (w/v) sucrose in H 2 O) and electrophoresed on 1.4% agarose gels containing 0.5 g/ml ethidium bromide at 100 V for 60 min at 23°C. After electrophoresis the gels were soaked in 1 mM MgSO 4 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-IT TM 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 M) 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 ␤-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 wildtype 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).
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 M 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.
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 ␤-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.
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- 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 ZnSO 4 (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 ZnSO 4 (C). CadC mutants were C7G (circles), C11G (inverted triangles), C52G (squares), C58S (diamonds), and C60G (triangles). modifying reagent MMTS on CadC protection of cad DNA and metal-induced deprotection was determined. MMTS converts cysteine residues to Cys-S-S-CH 3 (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 DNAbinding 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. DISCUSSION 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 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 M) 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 ZnSO 4 (D). CadC mutants were C7G (circles), C11G (inverted triangles), C52G (squares), C58S (diamonds), and C60G (triangles). 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.