Effect of bfmS deletion on the production of virulence-associated factors and the promoter activity of bfmR.

In all panels, MPAO1, ΔbfmS, and ΔbfmRS harbor plasmid PAK1900, respectively. A) Upper panel, P. aeruginosa MPAO1 and its derivatives were grown in Pyocyanin production broth (PPB) medium at 37°C for 36 h with shaking (250 rpm); the presence of the blue-green pigment indicates pyocyanin production. Lower panel, bacterial strains were inoculated onto a cetyltrimethylammonium bromide (CTAB) plate and incubated at 37°C for 24 h and then for 72 h at room temperature; the presence of a blue halo surrounding the colonies indicates the production of rhamnolipids. B) Relative amount of C4-HSL measured by the pDO100 (pKD-rhlA) system. MPAO1 and its derivatives were grown in M8-glutamate minimal medium supplemented with 0.2% glucose at 37°C for 24 h with shaking (250 rpm). Supernatants were subsequently prepared and measured for their relative C4-HSL contents. Plasmid pKD-rhlA carries the C4-HSL-responsive rhlA promoter fused to luxCDABE, so CPS (counts per second) values become an indirect measure of supernatant C4-HSL. C) Upper panel, MPAO1 and its derivatives were grown in PPB medium at 37°C for 24 h with shaking (250 rpm). Lower panel, MPAO1 and its derivatives were grown on CTAB plate and incubated at 37°C for 24 h and then for 72 h at room temperature. D) Expression of bfmR-lux in MPAO1 and its derivatives. Bacteria were grown in M8-glutamate minimal medium supplemented with 0.2% glucose at 37°C for 24 h with shaking (250 rpm) and then the bfmR-lux activity was measured. All experiments were independently repeated at least three times and the data shown represent comparable results. Values represent means ± standard error of the mean (SEM).