Fig. 3: Predicted and confirmed protein-protein interactions by previously uncharacterized proteins that contribute to biofilm formation or biofilm drug tolerance.

a Protein-protein interactions predicted by STRING (http://string-db.org). Proteins found to affect biofilm formation and/or susceptibility are shown in yellow. Green lines indicate interactions found for PA2171, brown lines for PA2146, and blue lines for PA2134. *, PA2184 is absent from the database in STRING version 11.0. Proteins shown to interact by STRING are predicted to be located in the cytoplasm, based on pseudomonas.com. b Detection of a physical interaction between PA2146 and PA2184 by bacterial two-hybrid analysis. The PA2146 gene was cloned into the vector pKT25, and PA2184 into the vector pUT18C. Plasmids were co-transformed into E. coli DHM1 cells. The transformants were spotted (2 µl) onto LB agar containing ampicillin, kanamycin, IPTG, and X-Gal. E. coli harboring empty vectors was used as negative control while cells producing TorR and TorS were used as positive control. Plates were incubated at 30°C for 48 h. Cleavage of X-Gal (blue) indicates a positive protein-protein interaction. Representative image is shown. Additional images and controls are shown in Supplementary Fig. 5a. c Quantitative analysis of protein-protein interactions, by E. coli DHM1 harboring pUT18C + pKT25, TorR+TorS, and PA2146 + PA2184, as determined using the Miller assay. Experiments were carried out in triplicates but only representative image is shown. Additional images and controls are shown in Supplementary Fig. 5a. Error bars indicate standard deviation. **significantly different from E. coli DHM1 harboring empty vectors (pUT18C + pKT25, p < 0.05, as determined by ANOVA followed by a Dunnett’s post-hoc test.