icaR encodes a transcriptional repressor involved in environmental regulation of ica operon expression and biofilm formation in Staphylococcus epidermidis

Abstract

Biofilm formation in Staphylococcus epidermidis is dependent upon the ica operon-encoded polysaccharide intercellular adhesin, which is subject to phase-variable and environmental regulation. The icaR gene, located adjacent to the ica operon, appears to be a member of the tetR family of transcriptional regulators. In the reference strain RP62A, reversible inactivation of the ica operon by IS256 accounts for 25 to 33% of phase variants. In this study, icaA and icaR regulation were compared in RP62A and a biofilm-forming clinical isolate, CSF41498, in which IS256 is absent. Predictably, ica operon expression was detected only in wild-type CSF41498 and RP62A but not in non-IS256-generated phase variants. In contrast, the icaR gene was not expressed in RP62A phase variants but was expressed in CSF41498 variants. An icaR::Em(r) insertion mutation in CSF41498 resulted in an at least a 5.8-fold increase in ica operon expression but did not significantly alter regulation of the icaR gene itself. Activation of ica operon transcription by ethanol in CSF41498 was icaR dependent. In contrast, a small but significant induction of ica by NaCl and glucose (NaCl-glucose) was observed in the icaR::Em(r) mutant. In addition, transcription of the icaR gene itself was not significantly affected by NaCl-glucose but was repressed by ethanol. Expression of the ica operon was induced by ethanol or NaCl-glucose in phase variants of CSF41498 (icaR+) but not in RP62A variants (icaR deficient). These data indicate that icaR encodes a repressor of ica operon transcription required for ethanol but not NaCl-glucose activation of ica operon expression and biofilm formation.

FIG. 1.

Comparative measurement of icaA, icaR, and gyrB (control) transcription in wild-type and phase variants of S. epidermidis CSF41498 and RP62A. RNA was prepared from cultures grown in BHI at 37°C to the early exponential phase of the growth curve (OD₆₀₀ = 2). Expression of gyrB is constitutive (14) and was used as an internal control in this experiment.

FIG. 2.

(A) Physical and restriction maps and primer binding sites of the S. epidermidis icaR-icaA region. (B) Plasmids used to construct the icaR chromosomal mutation. The position of the icaR::ermB insertion in pSER3 is indicated

FIG. 3.

Comparative measurement of icaA, icaR (KCR1 and KCR2 [see Fig. 4A]), and gyrB (control) transcription in S. epidermidis CSF41498 and three independent icaR insertion mutants, ICAR1, ICAR2, and ICAR3. RNA was prepared from cultures grown in BHI at 37°C to the early exponential phase of the growth curve (OD₆₀₀ = 2).

FIG. 4.

(A) Physical maps and icaR primer binding sites used for RT-PCR analysis in CSF41498 and the icaR::Em^r mutants. Primers KCR2 and KCR3 were designed to measure icaR transcription in ICAR mutants. (B) Comparative measurement of icaR (primers KCR 2 and KCR3 [panel A]) and gyrB (control) transcription in S. epidermidis CSF41498 and three independent icaR insertion mutants, ICAR1, ICAR2, and ICAR3. RNA was prepared from cultures grown in BHI at 37°C to the early exponential phase of the growth curve (OD₆₀₀ = 2).

FIG. 5.

Comparative measurement of icaA, icaR, and gyrB (control) transcription in S. epidermidis RP62A and CSF41498. RNA was prepared from cultures of each strain grown simultaneously to the mid-exponential phase of the growth curve (OD₆₀₀ = 4.5) in BHI, NaCl-glucose (BHI supplemented with 4% NaCl and 0.5% glucose) and EtOH (BHI supplemented with 4% ethanol).

FIG. 6.

Comparative measurement of icaA, icaR, and gyrB (control) transcription in phase variants of S. epidermidis RP62A and CSF41498 and the icaR insertion mutant ICAR1. RNA was prepared from cultures grown to the mid-exponential phase of the growth curve (OD₆₀₀ = 4.5) in BHI, NaCl-glucose (BHI supplemented with 4% NaCl and 0.5% glucose), and EtOH (BHI supplemented with 4% ethanol).

FIG. 7.

Cell cluster formation in overnight cultures of S. epidermidis CSF41498 (left) and ICAR1 (right) bearing plasmids pBT2 (control) (lanes 1 and 3) and pSER4 (icaR gene) (lanes 2 and 4) grown at 30°C in BHI medium supplemented with chloramphenicol (10 μg/ml).

FIG. 8.

Comparative measurement of icaA, icaR, and gyrB (control) transcription in S. epidermidis CSF41498 and ICAR1 strains complemented with plasmids pBT2 (control) and pSER4 (icaR). RNA was prepared from cultures grown to an OD₆₀₀ of 1 at 30°C in BHI and BHI EtOH (4% ethanol), with chloramphenicol (10 μg/ml) selection. Lane 1, CSF41498(pBT2) in BHI; lane 2, CSF41498(pSER4) in BHI; lane 3, CSF41498(pBT2) in BHI-EtOH; lane 4, CSF41498(pSER4) in BHI-EtOH; lane 5, ICAR1(pBT2) in BHI; lane 6, ICAR1(pSER4) in BHI; lane 7, ICAR1(pBT2) in BHI-EtOH; lane 8, ICAR1(pSER4) in BHI-EtOH.