Fig. 3. Expression of PA14 R-body genes is stochastic and controlled by RcgA and FecI2, and does not confer a competitive advantage during growth in biofilms.
a Reporter strain growth and expression of mScarlet under the control of the rebP1 promoter (PrebP1-mScarlet), no promoter (MCS-mScarlet), or a constitutive promoter (PPA1/04/03-mScarlet) in shaken 1% tryptone liquid cultures incubated at 25 °C. n = 6 biological replicates from two experiments. Data are presented as mean values ± SD. b Image of fluorescence from PA14 cells containing PrebP1 transcriptional reporter construct (PrebP1-mScarlet) and a constitutive promoter driving gfp (PPA1/04/03-gfp) in shaken 1% tryptone liquid cultures grown at 25 °C for 16 h. Image is representative of six replicates from three experiments. Scale bar is 10 µm. c Left: Schematic indicating plane of view, and legend defining fluorescence signal, in right-hand panels. Right: Bright-field and fluorescence microscopy images of 3-day-old biofilms formed by the indicated genotypes, containing the PrebP1 transcriptional reporter construct (PrebP1-mScarlet). Dotted lines demarcate the edges of biofilms. Scale bar is 2 mm. d Left: schematic indicating plane of view, and legend defining fluorescence signals, for right-hand panels. Right: representative images of thin sections prepared from 3-day-old biofilms formed by strains containing PrebP1-mScarlet and constitutively expressing GFP (via PPA1/04/03-gfp). Dotted lines denote the interfaces with air (top) and agar (bottom). Scale bar is 20 μm. e Three-day-old WT and ∆rebP1P2 biofilms grown on colony morphology medium. Images are representative of nine replicates from three experiments. Scale bar is 4 mm. f Relative percentages of colony forming units (CFUs) of each strain obtained from mixed-strain colony biofilms grown for 3 days. Each mixed biofilm contained one unlabeled strain and one constitutively expressing mScarlet. Error bars represent standard deviation of biological triplicates.