Phenazines affect biofilm formation by Pseudomonas aeruginosa in similar ways at various scales

Abstract

Some pseudomonads produce phenazines, a group of small, redox-active compounds with diverse physiological functions. In this study, we compared the phenotypes of Pseudomonas aeruginosa strain PA14 and a mutant unable to synthesize phenazines in flow cell and colony biofilms quantitatively. Although phenazine production does not impact the ability of PA14 to attach to surfaces, as has been shown for Pseudomonas chlororaphis(Maddula et al., 2006; 2008), it influences swarming motility and the surface-to-volume ratio of mature biofilms. These results indicate that phenazines affect biofilm development across a large range of scales, but in unique ways for different Pseudomonas species.

Fig. 1. Δphz1/2 has increased swarming motility

Quantification of swarming motility. Images of three swarming plates for each strain or condition were captured, exported to Adobe Photoshop and the agar surface covered by the swarms was quantified using Analysis Tools.

Fig. 2. Phenazines support development of structured biofilms in a continuous flow cell-system

Representative images of 4-day-old biofilms of the wild-type (A) and phz1/2 mutant without (B) and with addition of 25 µM pyocyanin (C) to the nutrient flow. Images of similar positions in the flow cell were taken in triplicate. Three independent experiments were performed with similar results.

Fig. 3. Phenazines modulate colony biofilm morphology

(A, B) P. aeruginosa PA14 wild-type and Δphz1/2 cultures were spotted onto 1% agar plates (containing 1% tryptone, 40 µg/ml Congo Red and 20 µg/ml Coomassie Blue) and incubated at 20°C for 7 days in the presence of 0.1 mM PCA, 0.1 mM or in the absence of supplements. Each day, colonies from three independent experiments were scanned and their surface coverage was determined. Representative images of colonies at days 2, 4 and 6 are shown (A; scale bar is 1 cm). The data in (B) show the average surface coverage and standard deviation for the three independent experiments. (C) Phenazine titrations. Colonies were grown as above, supplemented with 0, 0.1, 0.2, 0.3 or 0.4 mM PCA or pyocyanin. Surface coverage of colonies from three independent experiments was determined after incubation at 20°C for 7 days. Bars indicate the standard deviation.