Figure 1.
Glucose starvation-induced biofilm dispersal of P. aeruginosa.
Dispersal of P. aeruginosa PAO1 WT biofilms grown in continuous-flow cells was assessed by confocal microscopy and image analysis (A, B), and by biofilm-opacity monitoring using a photometrical device (C). The biofilms were pre-grown under a continuous flow of glucose/M9-salts medium (100 µM CaCl2) and glucose starvation was induced at the time indicated (C, arrow). (A) Representative CLSM images of P. aeruginosa PAO1 MA67 WT biofilms stained with the LIVE/DEAD BacLight bacterial viability kit (Molecular Probes Inc., Eugene, OR, USA) after 4 days (left) and after 1 day glucose starvation (right) are shown in CSLM X-Y images (top) and X-Z images (bottom). (B) Percent surface coverage as determined by ImageJ analysis of live (grey) and dead (black) cells of a 4 day-old biofilm of P. aeruginosa before and 24 h after glucose starvation. The error bars represent standard errors (n = 3). (C) Representative data obtained by continuous photometrical biofilm-density measurement (top graph) of a biofilm during growth and starvation (black) in comparison to an unstarved control biofilm (grey). The glucose concentration in the effluent was determined as an indicator of the starvation event (circles). Dispersed cells in the effluent were determined as optical density (OD580 nm) (center graph) and CFU (bottom graph).
Figure 2.
Functional annotation of differentially expressed proteins.
Proteins that were differentially expressed in P. aeruginosa starved biofilms were compared to unstarved biofilm (A) and dispersal cells (B). The functional categories are according to Pseudomonas Community Annotation Project (www.pseudomonas.com).
Figure 3.
Effect of stringent response on biofilm dispersal.
Effect of 200 µg ml−1 SHMT on biofilms of P. aeruginosa PAO1 WT, grown under continuous flow conditions in M9 medium (100 µM CaCl2) was assessed. The biofilms were exposed to glucose starvation in the presence (light grey) and absence (black) of SHMT. The experiments were repeated twice on different days. Data shown are representative biofilm-opacity measurements and are from one experiment. Glucose starvation was induced at the time indicated by the vertical line.
Figure 4.
Effect of 100 µM CCCP and 150 mM arsenate on biofilms of P. aeruginosa PAO1 WT during glucose starvation.
Biofilms grown under continuous flow conditions in M9 medium (100 µM CaCl2) were exposed to glucose starvation in the presence (light grey) and absence (black) of (A) CCCP or arsenate (C). Glucose starvation was induced at the time indicated by the vertical line. (B) Comparison of biomass remaining after 1 day of glucose starvation with and without CCCP as determined by the biofilm-opacity monitoring system. Error bars represent standard error (n = 3).
Figure 5.
Biofilm formation and response to glucose starvation of P. aeruginosa PAO1 WT and mutant strains.
Strains mutated in (A) nirS, (B) bdlA, (C) Pf4 phage, (D) lasRrhlR, (E) rpoS and (F) vfr were grown under continuous flow conditions in M9 medium (100 µM CaCl2). Glucose starvation was induced at the time indicated by the vertical line. Data represent single experiments.
Figure 6.
Biofilm formation and response to glucose starvation.
P. aeruginosa PAO1 WT (grey) and cyaA mutant (black) biofilm formation and response to starvation was determined by biofilm-opacity measurement (A). Percentage of of cyaA biomass remaining after 1 day of glucose starvation (p<0.05) as determined by the biofilm-opacity monitoring system (B); error bars represent standard error. Biofilm formation and response to glucose starvation of P. aeruginosa PAO1 WT (grey) and complemented cyaA mutant (black) as determined by biofilm-opacity measurement (C). Glucose starvation was induced at the time shown by the vertical line.
Figure 7.
Effect of the inhibition of cAMP synthesis on the dispersal of PAO1 WT biofilms.
The effect of inhibition of cAMP synthesis on dispersal of biofilms grown under continuous flow conditions in M9 medium (100 µM CaCl2) or M9 medium containing 7 mM atropine (Ap) as assessed photometrically (A) and percentage of biomass remaining after 1 day of glucose starvation with and without 7 mM Ap (p<0.05) (B) were determined. Intracellular cAMP levels in WT, cyaA mutant and Ap-treated P. aeruginosa planktonic cells were determined (C). Effect of CCCP and arsenate treatment on intracellular levels of cAMP in P. aeruginosa grown in planktonic culture (D). Two sets of samples were prepared on different days, and were measured in triplicate (n = 6). Error bars indicate standard error.
Table 1.
Strains, plasmids and primers used in this work.