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RsbU-dependent regulation of Staphylococcus epidermidis biofilm formation is mediated via the alternative sigma factor sigmaB by repression of the negative regulator gene icaR - PubMed

RsbU-dependent regulation of Staphylococcus epidermidis biofilm formation is mediated via the alternative sigma factor sigmaB by repression of the negative regulator gene icaR

Johannes K-M Knobloch et al. Infect Immun. 2004 Jul.

Abstract

Transposon mutagenesis of rsbU leads to a biofilm-negative phenotype in Staphylococcus epidermidis. However, the pathway of this regulatory mechanism was unknown. To investigate the role of RsbU in the regulation of the alternative sigma factor sigma(B) and biofilm formation, we generated different mutants of the sigma(B) operon in S. epidermidis strains 1457 and 8400. The genes rsbU, rsbV, rsbW, and sigB, as well as the regulatory cascade rsbUVW and the entire sigma(B) operon, were deleted. Transcriptional analysis of sarA and the sigma(B)-dependent gene asp23 revealed the functions of RsbU and RsbV as positive regulators and of RsbW as a negative regulator of sigma(B) activity, indicating regulation of sigma(B) activity similar to that characterized for Bacillus subtilis and Staphylococcus aureus. Phenotypic characterization of the mutants revealed that the dramatic decrease of biofilm formation in rsbU mutants is mediated via sigma(B), indicating a crucial role for sigma(B) in S. epidermidis pathogenesis. However, biofilm formation in mutants defective in sigma(B) or its function could be restored in the presence of subinhibitory ethanol concentrations. Transcriptional analysis revealed that icaR is up-regulated in mutants lacking sigma(B) function but that icaA transcription is down-regulated in these mutants, indicating a sigma(B)-dependent regulatory intermediate negatively regulating IcaR. Supplementation of growth media with ethanol decreased icaR transcription, leading to increased icaA transcription and a biofilm-positive phenotype, indicating that the ethanol-dependent induction of biofilm formation is mediated by IcaR. This icaR-dependent regulation under ethanol induction is mediated in a sigma(B)-independent manner, suggesting at least one additional regulatory intermediate in the biofilm formation of S. epidermidis.

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Figures

FIG. 1.
FIG. 1.

Model of the regulatory pathway for the activity of the alternative sigma factor σB of S. aureus, which is homologous to the core regulatory pathway of B. subtilis. In these species, σB is negatively regulated by the anti-sigma factor RsbW, which additionally acts as a specific kinase for the anti-anti-sigma factor RsbV. RsbV activity depends on phosphorylation status, and inactive phosphorylated RsbV could be activated by dephosphorylation by the specific phosphatase RsbU. Transcriptional analysis of the deletion mutants generated in S. epidermidis in this study suggests that regulation of σB activity in this species is homologous to that in S. aureus and B. subtilis.

FIG. 2.
FIG. 2.

Physical map of the sigB operon of S. epidermidis (accession no. AF274004) and construction of deletion mutants. Arrows depict open reading frames and indicate their orientations and sizes. All deleted genes were replaced with the erythromycin resistance gene (erm) as indicated. The erm gene and chromosomal regions flanking the respective deletions were amplified by PCR and cloned into plasmid pBT2, yielding the integration vectors pSJrsbU (A), pSJrsbV (B), pSJrsbW (C), pSJsigB (D), pSJrsbUVW (E), and pSJrsbUVWsigB (F). The crosses indicate the sites of homologous recombination.

FIG. 3.
FIG. 3.

Influence of deletions on transcription of the sigB gene in S. epidermidis 1457 and its mutants. (A) Northern blot analysis with a sigB-specific probe. S. epidermidis 1457 displayed a 1.5-kb transcript. The σA-dependent transcript of the entire σB operon could not be detected under the conditions used. Mutants with a remaining sigB gene displayed transcripts of increased sizes corresponding to the erm insertion. In sigB-negative mutants, no transcripts were detected. (B) Northern blot analysis with an erm-specific probe. For all mutants, a weak transcript comprising only the erm gene was detected, indicating a weak terminator following the erm gene. In mutants with a remaining sigB gene, additional transcripts with sizes identical to those of transcripts for the sigB-specific hybridization (A) were observed. In mutants with an inactivated sigB gene, the major transcripts detected were only slightly larger than the erm transcript, indicating a strong terminator preceding the sigB gene closely downstream. The genetic maps for the mutants are shown in Fig. 2. wt, wild type.

FIG. 4.
FIG. 4.

Homology of the asp23 promoter regions of S. aureus and S. epidermidis. (A) Physical maps of the asp23 promoter regions of S. aureus N315 (accession no. AP003136) and S. epidermidis ATCC 12228 (accession no. AE016750) are displayed. Homologous genes are indicated by double-headed arrows. In S. epidermidis, no open reading frame homologous to the S. aureus SA1987 gene could be detected in the asp23 promoter region. SA1987 is a homologue to the S. aureus opuD gene. In S. epidermidis, only one opuD homologue (SE0259) could be detected in a distinct chromosomal region (accession no. AE016744). (B) Alignment of σB promoters within the asp23 promoter regions of S. aureus and S. epidermidis with the consensus sequence of σB-dependent promoters in B. subtilis (59). Bases fitting the consensus sequence are displayed in boldface type. The P1 and P3 promoters of both species represent perfect matches, whereas for the respective P2 promoters one identical mismatch with respect to the consensus sequence was observed.

FIG. 5.
FIG. 5.

Influence of deletions on transcription of σB-dependent genes asp23 and sarA. Shown are Northern blot analyses with asp23-specific (A) and sarA-specific (B) probes, as well as maps of the respective genes with published or putative promoter sites. In mutants defective in sigB or its function, a lack of σB-dependent transcripts of asp23 and sarA transcripts was observed. wt, wild type.

FIG. 6.
FIG. 6.

Biofilm formation of S. epidermidis 1457 (A) and S. epidermidis 8400 (B) as well as their respective deletion mutants in TSB and in TSB supplemented with 4% NaCl, 2% ethanol (EtOH), or 4% EtOH under different environmental conditions. OD570, optical density at 570 nm.

FIG. 7.
FIG. 7.

Influence of deletions on transcription of icaR and icaADBC. Shown are Northern blot analyses with icaA-specific (A) and icaR-specific (B) probes, as well as maps of the respective genes with published or putative promoter sites. In mutants defective in sigB or its function, icaADBC transcription was strongly repressed, whereas icaR, encoding a negative regulator (14), was up-regulated. wt, wild type.

FIG. 8.
FIG. 8.

Influence of ethanol on the transcription of icaR and icaADBC. Shown are Northern blot analyses with icaR- and icaA-specific probes. RNA was extracted in the mid-exponential growth phase in TSB supplemented with 3% ethanol. In mutants defective in sigB or its function, up-regulated icaR mRNA was repressed by the addition of ethanol, leading to increased transcription of icaADBC. wt, wild type.

FIG. 9.
FIG. 9.

Model of the regulation of PIA synthesis in S. epidermidis. Transcription of icaADBC is regulated by the activity of the negative regulator IcaR (14). Transcription of icaR is negatively regulated by ethanol (15) and independently by a σB-dependent negative regulator, which is yet unknown. Additionally, PIA synthesis is regulated posttranscriptionally by a yet-unknown glucose-dependent regulator (17).

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