Figure 3. BpiB09 affects the QS-dependent pyocyanin production, C. elegans paralysis in PAO1 and decreases AHL response in A. tumefaciens AHL reporter strain.

A) Decreased pyocyanin production. OD₅₂₀ of pyocyanin extracts from culture supernatants of P. aeruginosa carrying pBBR1MCS-5::bpiB09 and empty pBBR1MCS-5. Data represent mean values of at least five independent experiments. Bars indicate the standard deviations. B) C. elegans paralysis induced by PAO1 on BHI medium. Data represent mean values of eight independent assays per treatment (+/−) standard error. Incubation was carried out on BHI agar plates at room temperature. Per PAO1 strain eight replicate plates were assayed, each with 30 one-day-old adult C. elegans. GLM analysis revealed a significant PA strain effect between the PAO1 wildtype and pBBR1MCS-5::bpiB09 (Likelihood ratio test, χ² = 132.04, df = 1, P<0.001), whereas there was no significant difference between the PAO1 wildtype and the empty vector control (Likelihood ratio test, χ² = 1.7 * 10⁻⁷, df = 1, P>0.99). PAO1, wild-type P. aeruginosa strain PAO1; PAO1 pBBR1MCS-5, control strain carrying an empty vector pBBR1MCS-5; PAO1 pBBR1MCS-5::bpiB09, expressing the bpiB09 gene constitutively. C) Reduced ß-galactosidase activity (i.e. traI-lacZ activity) caused by spent medium of P. aeruginosa cultures after 16 hours of growth using the A. tumefaciens NTL4 reporter strain. Data represent mean values of at least five independent experiments. Bars indicate the standard deviations. D) Inhibition of quorum sensing dependent violacein production in Chromobacterium violaceum strain DSM # 30191. 200 µl C. violaceum culture incubated (16 h, 30°C) with 50 µl (10 µg/ml) of E. coli cell extract expressing an empty vector (pBBR1MCS-5) - upper row; or with cell extract of E. coli expressing the bpiB09 gene in pBBR1MCS-5 - lower row.