Fig. 4: PA2146 encodes a conserved, small protein that is expressed under surface-associated growth conditions in P. aeruginosa PAO1 and clinical isolates.

a Sequence alignment of the P. aeruginosa PA2146 and homologs from various ɣ-proteobacteria and the gram-positive Streptococcus pneumoniae. “*“ denotes identical amino acids. For comparison, the E. coli YciG protein sequence harboring the KGG motif followed by the Walker nucleotide binding motif (highlighted in bold) is shown below the alignment in blue. b Detection of V5-tagged PA2146 in P. aeruginosa PAO1 via immunoprecipitation and immunoblotting using anti-V5 antibodies. PAO1 producing untagged PA2146 was used as a negative control. IgG_heavy/light refer to antibody bands. Experiments were carried out in triplicates but only representative image is shown. The image of the uncropped blot is shown in the supplementary material. c–e Assessment of PA2146 promoter reporter activity as determined by measuring luminescence. The putative promoter region of PA2146 (−513 to −1 relative to the translational start site) was cloned into the mini-CTX-lux vector to create CTX-PA2146-lux, which was introduced into the wild-type P. aeruginosa PAO1 strain. A promoterless lux strain was used as control. Background levels of luciferase activity observed for the control PAO1 bearing the promoterless lux operon were subtracted from the corresponding readings for PAO1::P_PA2146513-lux and normalized to total cells (A600 nm). All experiments were carried out in triplicate. Error bars indicate standard deviation. c Luminescence of the resulting strains was measured in planktonic cells grown to exponential (Expo) and stationary phase (Stat), and cells attached to the surface for 6 h (6 h), 3 days (3d), or 6 days (6d). *significantly different from exponential stage planktonic cells, p < 0.05, as determined by ANOVA followed by a Dunnett’s post-hoc test. d Relative fluorescence units of the reporter strain grown planktonically in the absence and presence of sperm DNA (0.1–1 mg/ml), 0.1–0.5 M glucose, and 0.3–0.5 M sodium chloride. No significant different was noted based on ANOVA followed by a Dunnett’s post-hoc test. e Fold-increase in transcript abundance of PA2146 in biofilms formed by P. aeruginosa PAO1 and clinical isolates relative to the same strains grown planktonically. All strains were grown planktonically to mid-log stage and as biofilms for 3 days. Experiments were performed using biological triplicates. Error bars indicate standard deviation.