FIG. 4.

Effects of Rbf on icaADBC and icaR expression in strain 8325-4. (A) Comparative measurements of icaA (gray bars) and icaR (hatched bars) transcription by real-time RT-PCR in the following S. aureus strains: 1112, wild type (pLI50); 6968, rbf(pLI50); 6974, rbf(pYL8565); 6973, wild type (pYL8565). Total RNA was prepared from cultures grown for 6 h at 37°C in TSB containing 3.5% NaCl, 0.75% glucose. Real-time RT-PCR was used to measure the relative expression of icaA and icaR compared to the gyrB gene. Transcript levels in all strains were compared to transcript levels in the wild-type strain, 1112, which was assigned a value of 1. The data presented are the averages of two separate experiments and standard errors are indicated. Plasmid pYL8565 carries the wild-type rbf gene, and pLI50 is the plasmid vector. (B) Regulation of PNAG synthesis by rbf in 8325-4 and derivatives. Bacterial extracts were prepared from overnight cultures grown at 37°C in TSB containing 3.5% NaCl, 0.75% glucose. PNAG was extracted from cells, serially diluted, and applied to a membrane. PNAG was detected by incubating the membrane, successively, with rabbit anti-PNAG serum, goat anti-rabbit-horseradish peroxidase, and a chemiluminescent substrate. Dilutions are indicated to the right of the figure. Strain numbers and genotypes are listed at the top of the figure.