FIGURE 2.

Implication of c-di-GMP in the response of P. aeruginosa PAO1 to NaClO. (A) P. aeruginosa PAO1 was incubated with NaClO (8 μg/ml, OD₆₀₀ = 1.0, BM2) for 1h followed by nucleotide extraction and quantification of intracellular c-di-GMP by LC-MS. Levels of c-di-GMP were normalized to the total protein content of the respective sample and compared to c-di-GMP levels in untreated controls (n = 6). The chromatogram shows c-di-GMP peaks of one representative measurement. (B) Attachment of P. aeruginosa in response to NaClO during overexpression of the c-di-GMP-degrading PDE PA2133 was evaluated by crystal violet staining. PAO1-pJN2133 and the vector control strain PAO1-pJN105 were incubated in 96-well microtiter plates either in the presence or in the absence of NaClO (2 μg/ml) for 2 h at 37°C followed by the quantification of biofilm biomass. BM2 was supplemented with 0.1% (w/v) arabinose to induce recombinant gene expression. Experiments were repeated four times, each with six wells per strain and condition (n = 24). Absorbance at 595nm (A₅₉₅) was determined and obtained values for NaClO-treated samples were normalized against the A₅₉₅ of respective untreated controls (=relative biomass). Statistical significance was evaluated by the Mann–Whitney test (^∗∗∗p ≤ 0.001, ^∗∗p ≤ 0.01, n.s., not significant). Boxes include median (thick horizontal line), 25th and 75th percentiles. Dots indicate extreme values considered as outliers.