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Inactivations of rsbU and sarA by IS256 represent novel mechanisms of biofilm phenotypic variation in Staphylococcus epidermidis - PubMed

Inactivations of rsbU and sarA by IS256 represent novel mechanisms of biofilm phenotypic variation in Staphylococcus epidermidis

Kevin M Conlon et al. J Bacteriol. 2004 Sep.

Abstract

Expression of ica operon-mediated biofilm formation in Staphylococcus epidermidis RP62A is subject to phase variable regulation. Reversible transposition of IS256 into icaADBC or downregulation of icaADBC expression are two important mechanisms of biofilm phenotypic variation. Interestingly, the presence of IS256 was generally associated with a more rapid rate of phenotypic variation, suggesting that IS256 insertions outside the ica locus may affect ica transcription. Consistent with this, we identified variants with diminished ica expression, which were associated with IS256 insertions in the sigmaB activator rsbU or sarA. Biofilm development and ica expression were activated only by ethanol and not NaCl in rsbU::IS256 insertion variants, which were present in approximately 11% of all variants. sigmaB activity was impaired in rsbU::IS256 variants, as evidenced by reduced expression of the sigmaB-regulated genes asp23, csb9, and rsbV. Moreover, expression of sarA, which is sigmaB regulated, and SarA-regulated RNAIII were also suppressed. A biofilm-forming phenotype was restored to rsbU::IS256 variants only after repeated passage and was not associated with IS256 excision from rsbU. Only one sarA::IS256 insertion mutant was identified among 43 biofilm-negative variants. Both NaCl and ethanol-activated ica expression in this sarA::IS256 variant, but only ethanol increased biofilm development. Unlike rsbU::IS256 variants, reversion of the sarA::IS256 variant to a biofilm-positive phenotype was accompanied by precise excision of IS256 from sarA and restoration of normal ica expression. These data identify new roles for IS256 in ica and biofilm phenotypic variation and demonstrate the capacity of this element to influence the global regulation of transcription in S. epidermidis.

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Figures

FIG. 1.
FIG. 1.

Characterization of S. epidermidis RP62A, 33A, 1457, and M15 genotypes and phenotypes. (A) Comparative measurement of icaA, icaR, and gyrB (control) transcription in RP62A and 33A. RT-PCR analysis was performed on RNA prepared from cultures grown at 37°C to an OD600 of 4.0 in BHI medium or BHI medium supplemented with 4% NaCl, 10% NaCl, or 4% ethanol. (B) Biofilm formation in tissue culture treated 96-well plates by RP62A and 33A in BHI medium or BHI medium supplemented with 4% NaCl, 10% NaCl, or 4% ethanol. (C) Comparative measurement of icaA, icaR, and gyrB (control) transcription in 1457 and M15. RT-PCR analysis was performed on RNA prepared from cultures grown at 37°C to an OD600 of 4.0 in BHI medium or BHI medium supplemented with 4% NaCl, 10% NaCl, or 4% ethanol. (D) Biofilm formation in tissue culture treated 96-well plates by RP62A and 33A in BHI medium or BHI medium supplemented with 4% NaCl, 10% NaCl, or 4% ethanol. Biofilm values represent OD490 readings after staining with crystal violet and are the means of at least three independent assays. Standard deviations are indicated where applicable. EtOH, ethanol.

FIG. 2.
FIG. 2.

Comparative measurement of rsbV, asp23, csb9, and gyrB (control) transcription in RP62A and 33A (A) and 1457 and M15 (B). RT-PCR analysis was performed on RNA prepared from cultures grown at 37°C to an OD600 of 4.0 in BHI medium.

FIG. 3.
FIG. 3.

Location of IS256 insertions in the rsbU gene. (A) Diagrammatic representation of the rsbU, rsbV, rsbW, sigB operon structure in S. epidermidis. (B) Exact locations of IS256 insertions detected in the rsbU gene of RP62A variants. The nucleotide binding sites for the PCR primers rsbUFor and rsbU2, the rsbU start codon, and the location of a unique HindIII site are all indicated. The duplicated 8-bp IS256 target sequences—TTAAAGAA for Red 1; AGCATTCA for 33A; CAAATGAA for Red 2, Red 3, and Red 4; and AATTATTT for Red 5—are underlined, and arrows indicate the exact insertion sites.

FIG. 4.
FIG. 4.

Analysis of 17 phenotypic variants produced by S. epidermidis RP62A. (A and B) Long-range PCR analysis of the sigB operon (A) and ica operon (B) in the wild-type RP62A and in 17 phenotypic variants. (C) Comparative measurement of asp23, icaA, and gyrB (control) transcription in wild-type RP62A and the 17 variants. RT-PCR analysis was performed on RNA prepared from cultures grown at 37°C to an OD600 of 4.0 in BHI medium.

FIG. 5.
FIG. 5.

Comparative measurement of asp23, icaA, and gyrB (control) transcription in wild-type RP62A, biofilm-negative variant 33A and three revertants from 33A. RT-PCR analysis was performed on RNA prepared from cultures grown at 37°C to an OD600 of 4.0 in BHI medium.

FIG. 6.
FIG. 6.

Comparative measurement of RNAIII, sarA, and gyrB (control) transcription in 1457 and M15 (A) and RP62A and 33A (B). RT-PCR analysis was performed on RNA prepared from cultures grown at 37°C to an OD600 of 4.0 in BHI medium.

FIG. 7.
FIG. 7.

Location of IS256 insertion in the sarA gene. (A) Diagrammatic representation of the sarA open reading frame and triple promoter regulatory region in S. epidermidis. The P1 promoter has homology to σB-dependent promoters, whereas P2 appears to be σA dependent (19). (B) Exact location of the IS256 insertion detected in the sarA gene of RP62A variant Red S1. The nucleotide binding sites for the PCR primers SEsarA1 and SEsarA2 and the sarA start and stop codons are indicated. The duplicated 8-bp IS256 target sequence, ATAAAAAA, for Red S1 is underlined, and an arrow indicates the exact insertion site.

FIG. 8.
FIG. 8.

Analysis of ica operon, icaR, sarA, and RNAIII expression in a sarA::IS256 insertion mutant. (A) Comparative measurement of icaA, icaR, and gyrB (control) transcription in RP62A, Red S1 (sarA::IS256), and Red S1/R1 (revertant 1). RT-PCR analysis was performed on RNA prepared from cultures grown at 37°C to an OD600 of 2.0 in BHI medium. (B) Comparative measurement of sarA and gyrB (control) transcription in RP62A and Red S1 (sarA::IS256) and Red S1/R1 (revertant 1). RT-PCR analysis was performed on RNA prepared from cultures grown to an OD600 of 2.0 at 37°C in BHI medium. Primers SEsarA3 and SEsarA4, located between the sarA P1 promoter and the IS256 insertion site, were used to measure sarA transcription in the sarA mutant. (C) Comparative measurement of RNAIII and gyrB (control) transcription in RP62A, Red S1 (sarA::IS256), and Red S1/R1 (revertant 1). RT-PCR analysis was performed on RNA prepared from cultures grown to an OD600 of 8.0 at 37°C in BHI medium.

FIG. 9.
FIG. 9.

Characterization of biofilm and ica operon environmental regulation in S. epidermidis RP62A, Red S1 (sarA::IS256) and Red S1/R1 (revertant 1). (A) Comparative measurement of icaA, icaR, and gyrB (control) transcription in Red S1 (sarA::IS256) and Red S1/R1 (revertant 1). RT-PCR analysis was performed on RNA prepared from cultures grown at 37°C to an OD600 of 4.0 in BHI medium or in BHI medium supplemented with 4% NaCl or 4% ethanol. (B) Biofilm formation in tissue culture-treated 96-well plates by RP62A, Red S1 (sarA::IS256), and Red S1/R1 (revertant 1) in BHI medium or BHI medium supplemented with 4% NaCl or 4% ethanol. EtOH, ethanol.

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