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. 2014 Dec 4;197(1):174–187. doi: 10.1128/JB.02244-14

FIG 5.

FIG 5

GcbA is regulated in a growth mode- and c-di-GMP-dependent manner. (A) Relative transcript abundances of gcbA in P. aeruginosa PAO1 exponential-phase planktonic cells, cells dispersed from 144-h-old biofilms in response to a changed in the glutamate concentration in the medium (Dispersed cells), and cells remaining within the biofilm following dispersion (Post-dispersion biofilm). Transcript abundance is reported relative to that of PAO1 cells grown as biofilms for 144 h. Error bars denote standard deviations. *, significantly different from expression in 144-h PAO1 biofilm cells (P < 0.05). (B) Detection with anti-V5 antibody of chromosomally encoded C-terminally V5/His6-tagged GcbA in total cell extracts of planktonic or biofilm (24- or 144-h-old) PAO1 cells. (C, D) Overexpression of genes encoding the PDEs DipA (C) and PA2133 (D) induced production of GcbA to detectable levels in P. aeruginosa cells grown as biofilms, as revealed by anti-V5 immunoblot analysis of chromosomally encoded C-terminally V5/His6-tagged GcbA. Planktonically grown cells harboring the empty pJN105 vector were used as positive controls. (E) ImageJ software densitometry analysis was used to determine the relative abundance of GcbA in PAO1/pJN-dipA or PAO1/pJN-PA2133 biofilm cells with respect to GcbA levels detected in PAO1 planktonic cells. Here, PAO1 refers to PAO1::PgcbA-gcbA-V5/His6. (F) Total cellular PDE activity of cell extracts obtained from P. aeruginosa PAO1 biofilms overexpressing dipA or PA2133 was determined using the synthetic chromogenic substrate bis(p-nitrophenyl) phosphate (bis-pNPP) and measuring the release of p-nitrophenol (pNP) at 405 nm. Error bars denote standard deviations. *, significantly different from results for PAO1 biofilms overexpressing dipA (P < 0.05). (G) Detection with anti-V5 antibody of chromosomally encoded C-terminally V5/His6-tagged GcbA in total cell extracts of planktonically grown PAO1 and isogenic ΔwspF mutant cells. (H) Increasing viscosity leads to reduced GcbA levels, as determined via anti-V5 antibody detection of chromosomally encoded C-terminally V5/His6-tagged GcbA in total cell extracts of planktonically grown PAO1 in the presence of increasing concentrations of Ficoll. All experiments were performed in triplicate. Panels below immunoblot images (B, C, D, G, H) are Coomassie-stained gels after transfer to ensure equal TCE protein loading.