Figure 2. The HisKA and Rec domains of SagS contribute to biofilm formation, but not antimicrobial tolerance.

(A) Overview of SagS domains and SagS domain constructs. Lines underneath the domains indicate the composition of the SagS domain constructs, while the names of the resulting constructs are given next to the lines. (B) Susceptibility phenotype of ΔsagS and ΔsagS mutant strains complemented with sagS or sagS domain constructs to tobramycin, norfloxacin and hydrogen peroxide. Indicated P. aeruginosa strains were allowed to form biofilms under flowing conditions for 2 days. Biofilm cells were subsequently treated with norfloxacin (450 µg/ml), tobramycin (150 µg/ml), or hydrogen peroxide (0.6%) for 1 hr under flowing conditions. P. aerugionosa PAO1 harboring the empty plasmid pJN105 was used as a control. Biofilm susceptibility was determined by log reduction. (C) Representative confocal images showing the architecture of biofilms formed by P. aeruginosa PAO1, ΔsagS, and ΔsagS mutant strains complemented with sagS and truncated sagS constructs. Biofilms were grown for 6 days in 20-fold diluted LB medium after which time confocal images were acquired. Biofilms were stained with the LIVE/DEAD BacLight viability stain (Life Technologies). White bars = 100 µm. (D) Biofilm biomass of P. aeruginosa wild-type and mutant biofilms grown for 6 days in flow cells, as determined using confocal images and subsequent COMSTAT analysis. Error bars indicate standard deviation. *, significantly different from the values for P. aeruginosa PAO1 (p ≤ 0.01).