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. Author manuscript; available in PMC: 2015 May 1.
Published in final edited form as: Mol Microbiol. 2014 Apr 9;92(3):488–506. doi: 10.1111/mmi.12587

Figure 3. Multicopy expression of PA4843 encoding a predicted diguanylate cyclase restores BrlR production and recalcitrance of ΔsagS biofilm cells to killing by bactericidal antimicrobial agents.

Figure 3

(A) Indicated P. aeruginosa strains were grown in tube reactors for 3 days and subsequently treated with tobramycin (150 μg/ml) or norfloxacin (450 μg/ml) for 1 hr under flowing conditions. P. aeruginosa harboring the empty vector pJN105 was used as a vector control. (B) Multicopy expression of PA4843 restores recalcitrance of ΔsagS biofilm cells to killing by antimicrobial agents as determined using biofilm-MBC assays. P. aeruginosa PAO1, ΔsagS and ΔsagS cells overexpressing PA4843 and PA4929 were grown for 3 days as biofilms and subsequently treated for 24 hr under continuous flowing conditions before recovering and enumerating surviving cells. Biofilm susceptibility to tobramycin (75 and 300 μg/ml) and norfloxacin (100 and 300 μg/ml) was determined by viable counts (CFU). The concentrations were chosen based on findings by Gupta et al. (Gupta et al., 2013) indicating complete killing of ΔsagS mutant biofilm cells following treatment with tobramycin concentrations exceeding 100 μg/ml and norfloxacin concentrations exceeding 200 μg/ml. (C) Total cell extracts obtained from P. aeruginosa wild-type and ΔsagS biofilms expressing a chromosomally located His6/V5-tagged brlR construct under the control of its own promoter (PbrlR-brlR-V5/His6) in the absence of presence of plasmid-borne PA4843 and PA4929 were probed for the presence of BrlR by immunoblot analysis using anti-V5 antibodies (anti-V5, see “BrlR” and top panel). Lower panel shows Coomassie-stained gel after immunoblotting demonstrating similar loading of all four total cell extract (TCE) used for immunoblotting and probing for BrlR using anti-V5 antibodies. A total of 15μg total cell extract was loaded. All experiments were done in triplicate and a representative image is shown. Error bars denote standard deviation. **, Significantly different from PAO1, p < 0.01 as determined by ANOVA and SigmaStat.