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. 2007 Apr;189(7):2886-96.
doi: 10.1128/JB.01767-06. Epub 2007 Feb 2.

sigmaB regulates IS256-mediated Staphylococcus aureus biofilm phenotypic variation

Jaione Valle  1 , Marta Vergara-Irigaray, Nekane Merino, José R Penadés, Iñigo Lasa
Affiliations

sigmaB regulates IS256-mediated Staphylococcus aureus biofilm phenotypic variation

Jaione Valle et al. J Bacteriol. 2007 Apr.

Abstract

Biofilm formation in Staphylococcus aureus is subject to phase variation, and biofilm-negative derivatives emerge sporadically from a biofilm-positive bacterial population. To date, the only known mechanism for generating biofilm phenotypic variation in staphylococci is the reversible insertion/excision of IS256 in biofilm-essential genes. In this study, we present evidence suggesting that the absence of the sigma(B) transcription factor dramatically increases the rate of switching to the biofilm-negative phenotype in the clinical isolate S. aureus 15981, under both steady-state and flow conditions. The phenotypic switching correlates with a dramatic increase in the number of IS256 copies in the chromosomes of biofilm-negative variants, as well as with an augmented IS256 insertion frequency into the icaC and the sarA genes. IS256-mediated biofilm switching is reversible, and biofilm-positive variants could emerge from biofilm-negative sigma(B) mutants. Analysis of the chromosomal insertion frequency using a recombinant IS256 element tagged with an erythromycin marker showed an almost three-times-higher transposition frequency in a Deltasigma(B) strain. However, regulation of IS256 activity by sigma(B) appears to be indirect, since transposase transcription is not affected in the absence of sigma(B) and IS256 activity is inhibited to wild-type levels in a Deltasigma(B) strain under NaCl stress. Overall, our results identify a new role for sigma(B) as a negative regulator of insertion sequence transposition and support the idea that deregulation of IS256 activity abrogates biofilm formation capacity in S. aureus.

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Figures

FIG. 1.
FIG. 1.
Mutation of σB in a biofilm-positive strain causes a phenotype switch to a smooth morphology. (A) Colony morphology of biofilm-positive variants (rough morphology) and biofilm-negative variants (smooth morphology) of S. aureus 15981 after 24 h on CRA. (B) Percentages of rough colonies (black) and smooth colonies (gray) of the S. aureus 15981 strain and its isogenic ΔσB mutant on CRA after subcultivation in TSB-gluc for 7 days. Error bars represent the standard deviations from three independent experiments.
FIG. 2.
FIG. 2.
Influence of σB deletion on biofilm stability after 5 days in continuous-flow microfermentors. (A) Biofilm development of the wild-type strain (WT) and the ΔσB mutant in microfermentors after 24 h (upper panel) and 5 days (lower panel). (B) Quantification of the biomass of a 5-day biofilm adhering to a glass slide. Cells were removed from the glass slide, placed into 10 ml of TSB, and vortexed, and the optical density (OD) of the solution was measured at 600 nm. Error bars represent the standard deviations from three independent experiments.
FIG. 3.
FIG. 3.
Analysis of the IS256 element in biofilm-negative variants of the ΔσB strain. Southern hybridization of EcoRI-SalI-digested chromosomal DNA with an IS256-specific DNA probe. Lanes 1 to 4, ΔσB biofilm-negative variants obtained from overnight cultures of experiment 1; lanes 5 to 8, ΔσB biofilm-negative variants obtained from overnight cultures of experiment 2; lanes 9 to 10, ΔσB biofilm-negative variants obtained after 7 days of subcultivation of the ΔσB mutant in TSB-gluc. WT, wild type.
FIG. 4.
FIG. 4.
Analysis of the ica operon of biofilm-negative variants of the ΔσB strain. (A) Scheme of the ica operon EcoRI restriction sites. The icaA DNA probe was generated using primers icaA-1 and icaA-2. (B) Southern blot analysis of EcoRI-digested chromosomal DNA of several ΔσB biofilm-negative variants using the amplified icaA DNA probe. WT, wild type. (C) Dot blot analysis of PNAG accumulation in the 15981 wild-type strain, the ΔσB strain, and the ΔσB biofilm-negative variants that were classified into group I (no insertion of IS256 in the ica operon), group II (icaC::IS256), and group III (icaC deletion). Cell surface extracts of stationary-phase cultures were treated with proteinase K and spotted onto nitrocellulose filters. PNAG production was detected with anti-S. aureus PNAG antiserum. The transposon insertion mutation in the icaC gene was used as a negative control.
FIG. 5.
FIG. 5.
Analysis of the IS256 element, the biofilm formation capacity, and PIA/PNAG synthesis in ΔσB biofilm-positive revertants. (A) Southern hybridization of EcoRI-SalI-digested chromosomal DNA with an IS256-specific DNA probe. Lanes: 1, S. aureus 15981 ΔσB strain; 2, S. aureus 15981 ΔσB biofilm-negative variant; 3 to 5, S. aureus 15981 ΔσB biofilm-positive revertants. (B) Biofilm formation in microtiter dishes. (C) Dot blot analysis of PIA/PNAG accumulation.
FIG. 6.
FIG. 6.
Detection of phenotypic variants in coagulases, hemolysins, lipases, and proteases. After 5 days of subcultivation, a dilution of ΔσB biofilm-negative variant culture was plated on Baird Parker agar (Difco), TSA with 5% sheep blood (Difco), Spirit Blue agar (Difco), or TSA supplemented with skim milk (5%, wt/vol) to determine the number of phenotypic variants in coagulase, hemolysin, lipase, or protease production, respectively. The percentage of colonies that did not present phenotypic variation is represented in black. The percentage of colonies with phenotypic variation is shown in gray.
FIG. 7.
FIG. 7.
Transposition analysis of the recombinant element IS256r. (A) Construction of the recombinant element IS256r. The natural chromosomal icaC::IS256 insertion was amplified and fused with an erythromycin resistance gene that included the left inverted repeat (IRL) of the IS256 element. IS256r was cloned into the pBT2 vector. IRR, right inverted repeat. (B) Agarose gel electrophoresis of PCR fragments amplified with circle-specific, outward-directed primers. (C) Percentages of chloramphenicol-sensitive colonies among the erythromycin-resistant colonies of S. aureus 15981 and its isogenic ΔσB mutant. Error bars represent the standard deviations from nine independent experiments. WT, wild type. (D) Southern hybridization of EcoRI-NcoI-digested chromosomal DNA with IS256 (left)- and erythromycin (right)-specific DNA probes. The arrows indicate new DNA bands hybridizing with the IS256 and erythromycin probes. Lanes 1 to 4, erythromycin-resistant/chloramphenicol-sensitive derivatives obtained from the ΔσB strain.
FIG. 8.
FIG. 8.
Effect of culture media and temperature on IS256 activity in the ΔσB strain. (A) Percentages of rough colonies (black) and smooth colonies (gray) of wild-type and ΔσB strains on CRA at days 1 and 5 of continuous subcultivation under different conditions (at 30°C or 37°C) and in TSB, TSB-gluc (TSBg), or TSB-NaCl. (B) Percentages of protease-overproducing colonies (black) and non-protease-overproducing colonies (gray) of wild-type and ΔσB strains on TSA-milk at days 1 and 5 of subcultivation under different conditions as described for panel A. Error bars represent the standard deviations from three independent experiments.
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