FIG 7 .

SagS variants contributing to decreased susceptibility of biofilms to tobramycin also contribute to tolerance to tobramycin and BrlR abundance. (A) Biofilm MBC assays. The P. aeruginosa PAO1 wild type or ΔsagS and ΔsagS mutants expressing sagS or select sagS_mut variants were grown as biofilms for 3 days and subsequently treated for 24 h with increasing concentrations of tobramycin (75 to 300 µg/ml) under continuous flow conditions before recovering and enumerating surviving cells. Viability was determined by CFU counts (biofilm CFU obtained from biofilm tube reactors having an inner surface area of 25 cm²). #, no viable bacteria were detected. Experiments were carried out in triplicate. Error bars denote standard deviation. (B) Detection of BrlR by immunoblot analysis. Total cell extracts (TCEs) obtained from ΔsagS/pMJT-sagS and ΔsagS/pMJT-1 mutant biofilms (3 days old) expressing a chromosomally located V5/His₆-tagged BrlR under the control of its own promoter (P_brlR-brlR-V5/His₆) were probed for the presence of BrlR by immunoblot (IB) analysis using anti-V5 antibodies. A total of 15 µg total cell extract was loaded. The corresponding SDS-PAGE gel image obtained posttransfer demonstrates equal loading. Representative images are shown. (C) Relative BrlR level present in biofilms by the PAO1 wild type or ΔsagS and ΔsagS mutants expressing sagS or select sagS_mut variants based on relative intensity of protein bands detectable following probing for BrlR with anti-V5 antibodies and subsequent analysis using ImageJ (56). Experiments were carried out in triplicate. Error bars denote standard deviation. *, significantly different from the values for the ΔsagS/pMJT-sagS mutant (P ≤ 0.01), as determined by ANOVA and Prism5.