In Staphylococcus epidermidis and Staphylococcus aureus, a number of cell wall- and cytoplasmic membrane-associated
lipoproteins are induced in response to
iron starvation. To gain insights into the molecular basis of
iron-dependent gene regulation in the staphylococci, we sequenced the
DNA upstream of the 3-kb S. epidermidis sitABC operon, which Northern blot analysis indicates is transcriptionally regulated by the growth medium
iron content. We identified two DNA sequences which are homologous to elements of the Corynebacterium diphtheriae DtxR regulon, which controls, in response to
iron stress, for example, production of
diphtheria toxin,
siderophore, and a
heme oxygenase. Upstream of the sitABC operon and divergently transcribed lies a 645-bp open reading frame (ORF), which codes for a
polypeptide of approximately 25 kDa with homology to the DtxR family of
metal-dependent
repressor proteins. This ORF has been designated SirR (staphylococcal
iron regulator repressor). Within the sitABC promoter/operator region, we also located a region of dyad symmetry overlapping the transcriptional start of sitABC which shows high homology to the DtxR operator consensus sequence, suggesting that this region, termed the Sir box, is the SirR-binding site. The SirR
protein was overexpressed, purified, and used in
DNA mobility shift assays; SirR retarded the migration of a synthetic
oligonucleotide based on the Sir box in a
metal (Fe2+ or Mn2+)-dependent manner, providing confirmatory evidence that this motif is the SirR-binding site. Furthermore, Southern blot analysis of staphylococcal chromosomal
DNA with the synthetic Sir box as a probe confirmed that there are at least five Sir boxes in the S. epidermidis genome and at least three in the genome of S. aureus, suggesting that SirR controls the expression of multiple target genes. Using a monospecific polyclonal antibody raised against SirR to probe Western blots of whole-cell lysates of S. aureus, S. carnosus, S. epidermidis, S. hominis, S. cohnii, S. lugdunensis, and S. haemolyticus, we identified an approximately 25-kDa cross-reactive
protein in each of the staphylococcal species examined. Taken together, these data suggest that SirR functions as a divalent
metal cation-dependent transcriptional repressor which is widespread among the staphylococci.