CadC, the transcriptional regulatory protein of the cadmium resistance system of Staphylococcus aureus plasmid pI258

Abstract

, and Pb 2؉ caused the release of CadC from DNA in gel retardation assays. DNase I footprinting measurements showed that the CadC protein specifically associated with and protected a region of operator/promoter DNA from nucleotide positions ؊7 to ؉14 relative to the start point of mRNA synthesis. Runoff transcription assays with the operator/promoter region of DNA (plus the first 69 nucleotides of the cadC gene) and purified E. coli RNA polymerase gave an mRNA product of the predicted size. Added CadC protein inhibited transcription in vitro.The cadmium resistance system of Staphylococcus aureus plasmid pI258 was identified by Novick and Roth (9) and subsequently mapped by deletion analysis (8). The cadA system results in reduced net uptake of Cd 2ϩ because of increased energy-dependent efflux of the toxic cation (22). Cloning and DNA sequence analysis identified two genes called cadC and cadA (10). The cadA gene product is a P-type ATPase, a member of the large family of related cation transport ATPases found in plant and animal cells as well as in microbes (16, While the properties of CadA satisfactorily explain cadmium resistance, the role of cadC and the nature of the trans-acting component of the carefully regulated CadA system (3, 25) remain unclear. The cadC gene is involved in full cadmium resistance; cadC can be provided in trans and need not be present on the same plasmid as cadA (26). The inducible nature of transcription was established, and the initiation site for cad mRNA synthesis was identified by reverse transcriptase primer extension (25) to a position 28 nucleotides upstream of the ATG start codon of the cadC gene (Fig. 1). Operon-length mRNA of 2.6 kb, including both cadC and cadA, was identified by Northern blot DNA-RNA hybridization (25). Canonical Ϫ10 and Ϫ35 Escherichia coli-like RNA polymerase recognition sites (Fig. 1) were identified in the sequence (25). A 7-nucleotide inverted repeat segment, including the first nucleotides transcribed into mRNA, was proposed as the operator site for binding of a then-elusive trans-acting DNA-binding regulatory protein (25).Cation-inducible gene function was studied by measurements of Cd 2ϩ efflux by whole cells (25) and subcellular membranes (21) and through induction of the reporter genes ␤-lactamase (25) and luciferase (3). However, the presumed transacting regulatory protein was not identified (25). Because of the need for cadC in gene regulation (21) and the sequence homologies (1, 26) among the predicted CadC protein product, the ArsR regulatory protein (12,14,15,23) for the arsenic resistance system, and the SmtB regulatory protein for cyanobacterial metallothionein synthesis (6), we undertook a direct test of the hypothesis that CadC is the regulatory protein for cadA operon transcription. By in vitro assays with CadC protein and the appropriate operator/promoter DNA, CadC has been shown to be a DNA-binding, negatively acting regulatory protein. MATERIALS AND METHODSAbbreviations. DTT, dithiothreitol; IPTG, isopropyl-␤-D-thiogalactopyranosid...

Chromosome‐Determined Zinc‐Responsible Operon czr in Staphylococcus aureus Strain 912

“…Because of the similarities between CzrA and SmtB, it is probable that CzrA is a repressor of the czr operon, which is different from the arsR-arsABC or cadC in the pI258 of S. aureus (8,42).…”

Molecular characterization of the ferric-uptake regulator, Fur, from Staphylococcus aureus The GenBank accession numbers for the S. aureus fur gene and fhu operon reported in this paper are AF118839 and AF132117, respectively.

“…A Fur-like repressor in S. epidermidis has also been reported, however the biological function of that protein is yet to be determined (Heidrich et al, 1996). In S. aureus, various operons such as the cadC system (Endo & Silver, 1995), the znt operon (Xiong & Jayaswal, 1998) and the fur operon, co-exist, which regulate the levels of various metal ions and their effects are overlapping. It is of utmost significance to determine why bacteria possess multiple metalloregulatory systems and how these different repressors coordinate their function in vivo to regulate metal-ion concentration.…”

Structural elements of metal selectivity in metal sensor proteins

“…As the concentration of metal ion increases, the effector-binding sites of the regulators become occupied eliciting a conformational change that weakens the affinity for the O͞P region, allowing transcription to proceed. Members of the SmtB͞ArsR family include: As(III), Sb(III), Bi(III)-responsive ArsR (7), Zn(II)-responsive SmtB (8), Cd(II), Pb(II), Bi(III)-responsive CadC (9)(10)(11), Zn(II)-responsive ZiaR (12), Co(II), Zn(II)-responsive CzrA (13,14), and, most recently, Ni(II), Co(II)-responsive NmtR (15).…”