Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or VSports app下载. mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. V体育官网.

. 1998 Sep;66(9):4123-9.
doi: 10.1128/IAI.66.9.4123-4129.1998.

SirR, a novel iron-dependent repressor in Staphylococcus epidermidis

P J Hill  1 , A Cockayne, P Landers, J A Morrissey, C M Sims, P Williams
Affiliations

SirR, a novel iron-dependent repressor in Staphylococcus epidermidis (V体育平台登录)

P J Hill et al. Infect Immun. 1998 Sep.

V体育平台登录 - Abstract

In Staphylococcus epidermidis and Staphylococcus aureus, a number of cell wall- and cytoplasmic membrane-associated lipoproteins are induced in response to iron starvation VSports手机版. 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. .

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Comparison of the deduced amino acid sequences of SirR and DtxR. Identical amino acid residues are boxed. Solid arrows indicate residues identified from the DtxR crystal structure as metal coordination sites (M1), the open arrows indicate metal-binding site 2 (M2), and the hatched arrow indicates the cysteine residue (C102) positioned within M2 which is replaced by a glutamate residue in SirR and TroR.
FIG. 2
FIG. 2
(A) Nucleotide sequence of the S. epidermidis sitABC operon promoter region. The −10 and −35 promoter sequences are underlined; the putative ribosome-binding site (RBS) and translation start codon of sitA are shown in bold. The vertical arrow indicates the transcriptional start of the sitABC operon, and the Sir box is indicated by convergent horizontal arrows. (B) Comparison of the S. epidermidis Sir box nucleotide sequence with sequences of known DtxR- and DesR-binding sites in C. diphtheriae and S. lividans. The 19-bp consensus sequence for DtxR derived by Lee et al. (20) is also shown.
FIG. 3
FIG. 3
Northern blot analysis of S. epidermidis sirR and sitABC transcripts. Total RNA was isolated from S. epidermidis 901 grown in iron-sufficient (+) or iron-deficient (−) RPMI 1640 medium. The 2.7-kb sitABC transcript and the 0.7-kb sirR transcript are indicated by arrows.
FIG. 4
FIG. 4
Immunoblot analysis of SitC production in S. epidermidis grown in iron-restricted RPMI 1640 or supplemented with cobalt, copper, iron, magnesium, manganese, nickel, or zinc (added at 20 μM). Whole-cell proteins prepared by lysostaphin digestion of staphylococcal suspensions adjusted to the same optical density were subjected to SDS-PAGE, immunoblotted, and probed with a monoclonal antibody to SitC. Lane 1, no addition; lane 2, Co2+; lane 3, Cu2+; lane 4, Fe2+; lane 5, Mg2+; lane 6, Mn2+; lane 7, Ni2+; lane 8, Zn2+.
FIG. 5
FIG. 5
DNA gel mobility shift assay of synthetic Sir box (labeled with digoxigenin) with partially purified SirR in the presence (+) or absence (−) of Fe2+. The Sir box oligonucleotide is clearly retarded only in the presence of both SirR and Fe2+.
FIG. 6
FIG. 6
Immunoblot analysis of SirR production in coagulase-negative staphylococci grown in iron-restricted RPMI 1640 medium. Whole-cell proteins prepared by lysostaphin digestion were subjected to SDS-PAGE, immunoblotted, and probed with a monospecific polyclonal antibody to SirR. Lane 1, S. epidermidis; lane 2, S. cohnii; lane 3, S. hominis; lane 4, S. carnosus; lane 5, S. lugdunensis; lane 6, S. warneri; lane 7, S. aureus.
FIG. 7
FIG. 7
Southern blot analysis showing the presence of multiple Sir boxes in genomic DNA prepared from S. epidermidis 901 and S. aureus BB restricted with MunI (lanes 2 and 5), HindIII (lanes 3 and 6), and SalI (lane 4). Lane 1, λ-HindIII marker; lanes 2 to 4, S. epidermidis; lanes 5 and 6, S. aureus. Strongly hybridizing bands are indicated by arrowheads.
See this image and copyright information in PMC

References

    1. Arbuthnott J P, Arbuthnott E, Arbuthnott A D J, Pike W J, Cockayne A. Investigation of microbial growth in vivo: evaluation of a novel in vivo chamber implant system. FEMS Microbiol Lett. 1992;100:75–80. - V体育官网入口 - PubMed
    1. Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K, editors. Current protocols in molecular biology. New York, N.Y: Greene Publishing Associates and Wiley-Interscience; 1994.
    1. Bagg A, Neilands J B. Molecular mechanism of regulation of siderophore-mediated iron assimilation. Microbiol Rev. 1987;51:509–518. - PMC - PubMed
    1. Boyd J, Oza M N, Murphy J R. Molecular-cloning and DNA-sequence analysis of a diphtheria toxin iron-dependent regulatory element (DtxR) from Corynebacterium diphtheriae. Proc Natl Acad Sci USA. 1990;87:5968–5972. - PMC - PubMed
    1. Bsat N, Helmann J D. Abstracts of the 97th General Meeting of the American Society for Microbiology 1997. Washington, D.C: American Society for Microbiology; 1997. Regulation of Bacillus subtilis iron uptake genes by Fur protein, abstr. H-193.

Publication types

"VSports注册入口" MeSH terms

Associated data

LinkOut - more resources