AraC-Type Regulator Rbf Controls the Staphylococcus epidermidis Biofilm Phenotype by Negatively Regulating the icaADBC Repressor SarR

Abstract

Regulation of icaADBC-encoded polysaccharide intercellular adhesin (PIA)/poly-N-acetylglucosasmine (PNAG) production in staphylococci plays an important role in biofilm-associated medical-device-related infections. Here, we report that the AraC-type transcriptional regulator Rbf activates icaADBC operon transcription and PIA production in Staphylococcus epidermidis Purified recombinant Rbf did not bind to the ica operon promoter region in electrophoretic mobility shift assays (EMSAs), indicating that Rbf regulates ica transcription indirectly. To identify the putative transcription factor(s) involved in Rbf-mediated icaADBC regulation, the ability of recombinant Rbf to interact with the promoter sequences of known icaADBC regulators was investigated. Recombinant Rbf bound to the sarR promoter and not the sarX, sarA, sarZ, spx, and srrA promoters. Reverse transcription (RT)-PCR demonstrated that Rbf acts as a repressor of sarR transcription. PIA expression and biofilm production were restored to wild-type levels in an rbf sarR double mutant grown in brain heart infusion (BHI) medium supplemented with NaCl, which is known to activate the ica locus, but not in BHI medium alone. RT-PCR further demonstrated that although Rbf does not bind the sarX promoter, it nevertheless exerted a negative effect on sarX expression. Apparently, direct downregulation of the SarR repressor by Rbf has a dominant effect over indirect repression of the SarX activator by Rbf in the control of S. epidermidis PIA production and biofilm formation.

Importance: The importance of Staphylococcus epidermidis as an opportunistic pathogen in hospital patients with implanted medical devices derives largely from its capacity to form biofilm. Expression of the icaADBC-encoded extracellular polysaccharide is the predominant biofilm mechanism in S. epidermidis clinical isolates and is tightly regulated. Here, we report that the transcriptional regulator Rbf promotes icaADBC expression by negatively regulating expression of sarR, which encodes an ica operon repressor. Furthermore, Rbf indirectly represses the ica operon activator, SarX. The data reveal complicated interplay between Rbf and two Sar family proteins in fine-tuning regulation of the biofilm phenotype and indicate that in the hierarchy of biofilm regulators, IcaR is dominant over the Rbf-SarR-SarX axis.

FIG 1

Rbf regulates icaADBC transcription, PIA expression, and biofilm production in S. epidermidis. (A) Comparative biofilm phenotypes of CSF41498 and its isogenic rbf mutant, complemented where indicated with plasmid pEPSA5 (empty-vector control) or pEPrbf. Semiquantitative measurements of biofilm formation under static conditions were performed in tissue culture-treated 96-well plates. All the strains were grown at 37°C in BHI and BHI NaCl for 24 h. BHI and BHI NaCl were also supplemented with 0.5% xylose for strains carrying pEPrbf. Experiments were repeated at least five times, and average results are presented. Standard errors of the mean are indicated, and the asterisks denote significant differences (P < 0.05) as determined by Student's t test. (B) CSF41498 and its isogenic rbf mutant grown in BHI medium and BHI NaCl in flow cells at 0.6 dyne/cm² shear using a BioFlux 1000Z instrument. The bright-field images depicting biofilm accumulation after 18 h were captured at ×10 magnification and are representative of the results of the results of three independent experiments. (C) Comparative measurement of icaA transcription by real-time RT-PCR in CSF41498 and its isogenic rbf mutant. Total RNA was extracted from cultures grown at 37°C to an A₆₀₀ of 1.0. RelQuant software (Roche) was used to measure the relative expression of icaA compared to that of the constitutively expressed gyrB gene. Transcript levels of icaA in the rbf mutant strain were then compared to those in CSF41498 pLI50, which were assigned a value of 1. The data presented are the averages of three separate experiments, and standard deviations are indicated. Statistical significance between the rbf or rbf pEPrbf strains and CSF41498 as determined by Student's t test is indicated by the asterisks (P ≤ 0.05). (D) Comparative immunoblot analysis of PIA production in whole-cell extracts of CSF41498, its isogenic rbf mutant, and the rbf mutant complemented with pSERB6 grown overnight at 37°C in BHI. Cell extracts were diluted 1:10 and 1:100. The blot shown is representative of the results of three independent experiments.

FIG 2

Recombinant Rbf binds to the sarR promoter and regulates sarR transcription. (A) Recombinant Rbf does not bind to the ica promoter. Increasing concentrations (1.25 to 20 pmol) of recombinant Rbf protein were added to a biotinylated oligonucleotide P_ica probe. (B) Recombinant Rbf binding to the sarA, sarX, sarZ, spx, srrA, and sarR promoters. Twenty picomoles of recombinant Rbf protein was added to each biotinylated oligonucleotide probe before being separated on a 5% polyacrylamide gel. (C) Recombinant Rbf binds specifically to the sarR promoter. Increasing concentrations (1.25 to 20 pmol) of recombinant Rbf protein were added to a biotinylated oligonucleotide P_sarR probe. The protein-DNA interactions were competed with 100× specific or nonspecific competitor DNA. (D) Comparative measurement of sarR and gyrB (control) transcription in S. epidermidis strains CSF41498 pLI50, rbf pLI50, and rbf pSERB6. RNA was prepared from cultures grown overnight to stationary phase (∼16 h) at 37°C in BHI. RelQuant software (Roche, Switzerland) was used to compare the relative expression of sarR to that of the constitutively expressed gyrB gene. sarR transcript levels in all strains were compared to sarR transcript levels in CSF41498 pLI50, which was assigned a value of 1. The data presented are the averages of three separate experiments. Standard deviations are indicated, and the asterisk denotes a significant difference as determined by Student's t test (P ≤ 0.05).

FIG 3

Recombinant SarR binds to the ica operon promoter and regulates ica transcription. (A) Recombinant SarR binds to the ica promoter. Increasing concentrations (45 to 718 pmol) of recombinant SarR protein were added to a biotinylated oligonucleotide P_sarR probe. The protein-DNA interactions were competed with 100× specific or nonspecific competitor DNA. (B) Comparative measurement of icaA and gyrB (control) transcription in S. epidermidis strain 1457 and its sarR mutant. RNA was prepared from cultures grown to an A₆₀₀ of 1.0 at 37°C in BHI NaCl. RelQuant software (Roche, Switzerland) was used to measure the relative expression of icaA against the constitutively expressed gyrB gene. icaA transcript levels in all strains were compared to icaA transcript levels in CSF41498 pLI50, which were assigned a value of 1. The data presented are the averages of three separate experiments. Standard deviations are indicated, and the asterisk denotes a significant difference as determined by Student's t test (P ≤ 0.05). (C) Intergenic nucleotide sequence between SERP_RS09410 and sarR on the chromosome of S. epidermidis RP62A. The binding sites for the SEsarR_comp1 and SEsarR_comp2 oligonucleotide primers are underlined. The predicted sarR ribosome binding site (RBS) (green letters) and promoter based on canonical −10 and −35 RNA polymerase binding sites (red letters) were identified using Softberry bacterial promoter prediction software (BPROM).

FIG 4

SarR-regulated PIA production and biofilm development in S. epidermidis. (A) Comparative immunoblot analysis of PIA production in whole-cell extracts of CSF41498 and its isogenic rbf, sarR, and rbf sarR mutants grown overnight at 37°C in BHI medium and BHI NaCl. Cell extracts were diluted 1:10, 1:100, and 1:1,000. The blot shown is representative of the results of three independent experiments. (B) CSF41498 and its isogenic rbf, sarR, and rbf sarR mutants grown in BHI and BHI NaCl in flow cells at 0.4 dyne/cm² shear using a BioFlux 1000Z instrument. The bright-field images depicting biofilm accumulation after 18 h were captured at ×10 magnification and are representative of the results of three independent experiments. (C) Comparative biofilm phenotypes of CSF41498 and its isogenic rbf, sarR, and rbf sarR mutants, complemented where indicated with pLIsarR or pEPrbf. Semiquantitative measurements of biofilm formation under static conditions were performed in tissue culture-treated 96-well plates. All the strains were grown at 37°C in BHI and BHI NaCl for 24 h. Experiments were repeated at least five times, and average results are presented. Standard errors of the mean are indicated, and the asterisks denote a significant difference as determined by Student's t test (P < 0.05).

FIG 5

Rbf negatively regulates sarX transcription in S. epidermidis. Shown are comparative measurements of sarX and gyrB (control) transcription in S. epidermidis CSF41498, rbf, sarR, and rbf sarR strains. RNA was prepared from cultures grown to an A₆₀₀ of 1.0 at 37°C in BHI medium and BHI NaCl. RelQuant software (Roche, Switzerland) was used to measure the relative expression of sarX against that of the constitutively expressed gyrB gene. sarX transcript levels in all the strains were compared to sarX transcript levels in CSF41498, which were assigned a value of 1. The data presented are the averages of at least three separate experiments. Standard deviations are indicated, and the asterisks denote a significant difference as determined by Student's t test (P ≤ 0.05).

FIG 6

Model for S. epidermidis icaADBC regulation by Rbf, SarX, and SarR. Rbf indirectly regulates ica operon expression by binding to the sarR promoter and negatively regulating sarR transcription. SarR binds to the ica operon promoter to repress icaADBC transcription. SarX acts independently of both SarR and Rbf, binding to the ica operon promoter to increase icaADBC transcription (11). Under osmotic stress in medium supplemented with NaCl, SigB-dependent repression of icaR (8, 9) and the concomitant activation of the ica operon are dominant over Rbf, SarX, and SarR.