Fig. 3: AI-2 regulates P. aeruginosa biofilm formation via PctA and TlpQ.

a Crystal violet quantification of biofilm formation by P. aeruginosa strains in the presence or absence of DPD/AI-2. 190 μl aliquots of P. aeruginosa strains with an OD₆₀₀ of 0.05 in TSB medium were placed into the wells of a 96-well plate and then 10 μl aliquots of DPD/AI-2 (2 μM) or a buffer control were added to the wells. Biofilms were stained with crystal violet and quantified using optical density measurement after incubation at 37 °C for 48 h. Data are mean ± s.e.m. of five independent experiments. b Quantification of biofilm formation by P. aeruginosa strains from confocal imaging. Confocal dishes were inoculated with 190 μl of mCherry-labeled P. aeruginosa strains diluted to an OD₆₀₀ of 0.01 in TSB medium and 10 μl of the DPD/AI-2 solution (2 μM) or a buffer control, and biofilms formed were detected by confocal laser-scanning microscopy after incubation at 37 °C for 1, 10 and 19 h, respectively. Images were reconstructed using the Imaris 9.0 software package (Bitplane, AG) (Supplementary Fig. 6) and biofilm biovolumes were quantified using COMSTAT (www.comstat.dk). Biovolumes were calculated from three biological replicates and each biological replicate was derived from an average of five confocal images. Similar results were obtained in three biological replicates and data are presented as mean ± s.e.m. a, b Statistical significance was evaluated using two-tailed unpaired Student’s t-test. P values < 0.05 were considered to indicate statistically significant differences. WT, wild-type.