FIGURE 3.

Characterization of the DGC PA3177. (A) E. coli BL21-pET3177 and the vector control strain BL21-pET23a were grown in LB broth supplemented with 0.4 mM IPTG for 5 h at 37°C 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 empty vector control strain BL21-pET23a (n = 6). The chromatogram shows c-di-GMP peaks of one representative measurement. (B) Attachment of P. aeruginosa during PA3177 overexpression was evaluated by crystal violet staining. PAO1-pJN3177 and empty vector control strain PAO1-pJN105 were incubated in 96-well microtiter plates for 2 h at 37°C followed by biomass quantification. For recombinant gene expression, BM2 was supplemented with 0.1% (w/v) arabinose (+Ara). Cultures without arabinose (–Ara) served as negative controls. Experiments were carried out in triplicate, each with six wells per strain and condition (n = 18). (C) Planktonic growth during 5 h at 37°C. (D) Overnight cultures of PA3177-overexpressing strain P. aeruginosa PA01-pJN3177 and vector control PAO1-pJN105 were diluted in LB broth supplemented with 0.1% (w/v) arabinose and grown for 5 h at 37°C followed by evaluation of swimming, swarming and twitching motility. Assays were carried out with three independent bacterial cultures and at least four agar plates per experiment (n ≥ 12). In all experiments 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.