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Infection and Immunity, September 1998, p. 4123-4129, Vol. 66, No. 9
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

SirR, a Novel Iron-Dependent Repressor in Staphylococcus epidermidis

Philip J. Hill,1,2,* Alan Cockayne,1,2,3 Patrick Landers,1,2 Julie A. Morrissey,1,2 Catriona M. Sims,1,2 and Paul Williams1,2,3

Institute of Infections and Immunity,1 School of Pharmaceutical Sciences,2 and School of Clinical Laboratory Sciences,3 University of Nottingham, Nottingham NG7 2UH, United Kingdom

Received 6 March 1998/Returned for modification 28 April 1998/Accepted 10 June 1998

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.

* Corresponding author. Present address: School of Biological Sciences, Sutton Bonington Campus, University of Nottingham, Sutton Bonington, Leicestershire LE12 5RD, United Kingdom. Phone: 115 9516169. Fax: 115 9516162. E-mail: Phil.Hill{at}nottingham.ac.uk.

Infection and Immunity, September 1998, p. 4123-4129, Vol. 66, No. 9
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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