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 regulate d 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, an d Cys-60 were changed individually to glycine or serine residues. The effec t of the cadC mutations was examined in Escherichia coli using a green fluo rescent 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 mut ant CadC proteins were purified, and the effect of the substitutions on DNA binding was determined using a restriction enzyme protection assay. Bindin g of wild-type CadC protected end operator DNA from digestion at the single SspI site, and the addition of Pb(II), Zn(II), or Cd(II) resulted in depro tection, Chemical modification of the cysteine residues in CadC had no effe ct on protection but eliminated deprotection, C11G and C52G proteins exhibi ted wild-type properties in vitro, C7G, C58S, and C60G proteins were able t o 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 s ensing those soft metals and suggest that they are ligands to Pb(II), Zn(II ), and Cd(II).