Inverse regulation of biofilm formation and swarming motility by Pseudomonas aeruginosa PA14

Abstract

We previously reported that SadB, a protein of unknown function, is required for an early step in biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa. Here we report that a mutation in sadB also results in increased swarming compared to the wild-type strain. Our data are consistent with a model in which SadB inversely regulates biofilm formation and swarming motility via its ability both to modulate flagellar reversals in a viscosity-dependent fashion and to influence the production of the Pel exopolysaccharide. We also show that SadB is required to properly modulate flagellar reversal rates via chemotaxis cluster IV (CheIV cluster). Mutational analyses of two components of the CheIV cluster, the methyl-accepting chemotaxis protein PilJ and the PilJ demethylase ChpB, support a model wherein this chemotaxis cluster participates in the inverse regulation of biofilm formation and swarming motility. Epistasis analysis indicates that SadB functions upstream of the CheIV cluster. We propose that P. aeruginosa utilizes a SadB-dependent, chemotaxis-like regulatory pathway to inversely regulate two key surface behaviors, biofilm formation and swarming motility.

FIG. 1.

Surface-associated behaviors influenced by SadB levels. (A) Biofilm formation phenotypes under static conditions. (Top) Image of crystal violet (CV)-stained biofilms formed by the wild type carrying the vector control (WT/pUCP18) and the wild type overexpressing SadB (WT/pSadB⁺). Cells were grown in minimal medium containing (0.2%) glucose for 4 h at 37°C before staining with CV. (Bottom) Quantification of CV-stained wells. (B) Phase-contrast images of WT/pUCP18 and WT/pSadB⁺ attached to the surface of a 24-well plate after incubation at 37°C for 1 h. For each strain, images were recorded at a magnification of ×1,400 over 10 fields of view, and the average number of surface-associated cells for each strain is indicated below the image. (C) The graph shows the average plate surface coverage that results from swarms produced by the WT, sadB, and ΔpilJ strains. Below are representative images of WT, sadB, and ΔpilJ strains after 16 h of incubation at 37°C. The percent surface coverage of the WT is significantly less than that of the sadB mutant (P = 0.0029) and the ΔpilJ mutant (P = 0.000018). (D) Aliquots of the wild type carrying the vector control (WT/pUCP18) and the wild type overexpressing SadB (WT/pSadB⁺) were spotted on 0.5% and 0.55% swarm agar plates and incubated for 16 h at 37°C. (E) Reversal rates of WT and sadB mutant cells under low-viscosity (3% Ficoll) and high-viscosity (15% Ficoll) conditions.

FIG. 2.

The ΔpilJ mutant is biofilm defective and a hyperswarmer. (A) CheIV chemotaxis cluster. pil genes are shown in light gray and chp genes in dark gray. Black arrows represent open reading frames, and gene names are given below the arrows. (B) Biofilm formation by the WT, ΔpilJ mutant, and complemented strains at 8 h. Cells were grown at 37°C for 8 h in minimal medium containing glucose and CAA. (C) Aliquots of the ΔpilJ mutant carrying the vector control (ΔpilJ/pMQ90) or a construct expressing the pilJ gene in trans (pPilJ⁺) were spotted on 0.5% swarm agar plates and incubated at 37°C for 16 h. (D) Aliquots of the ΔpilJ mutant carrying the vector control (ΔpilJ/pUCP18) or a construct overexpressing SadB (pSadB⁺) were spotted on 0.5% and 0.55% swarm agar plates and incubated at 37°C for 16 h. (E) The extent of swarming, expressed as percent surface coverage, is shown on 0.5% agar for the strains indicated. *, statistically significant decrease in swarming compared to the other strains (P < 0.001).

FIG. 3.

The ΔchpB mutant is defective for swarming motility and displays increased biofilm formation and CR binding. (A) Aliquots of the ΔchpB mutant carrying the vector control (ΔchpB/pMQ90) and a construct expressing the chpB gene in trans (pChpB⁺) were spotted on 0.5% swarm agar plates and incubated at 37°C for 16 h. (B) Biofilm formation by the WT and ΔchpB mutant was assessed after 4 h of growth at 37°C in minimal medium containing glucose. Shown are representative wells (top) and quantification of the biofilm assays (bottom). (C) Reversal rates of the WT and the ΔpilJ and ΔchpB mutants under high-viscosity (15% Ficoll) conditions. (D) CR assays with the WT, the ΔchpB mutant, and the ΔchpB pelA double mutant. Plates were incubated for 24 h at 37°C and an additional 24 h at room temperature. (E) SEM of WT and the ΔchpB and ΔpilJ mutants. Images were prepared with ruthenium red to highlight polysaccharides. (F and G) Quantitative RT-PCR analysis of pelA (F) and pelG (G) gene expression in agar-grown cultures of the indicated strains. *, statistically significant difference from the WT (P < 0.05).

FIG. 4.

A sadB mutant displays decreased Pel polysaccharide production but no decrease in pel gene expression. (A) CR binding assays with the WT and the sadB mutant. Plates were incubated for 24 h at 37°C and an additional 24 h at room temperature. (B) SEM of the indicated strains. Images were prepared with ruthenium red to highlight polysaccharides. (C and D) Quantitative RT-PCR analysis of pelA (C) and pelG (D) gene expression in agar-grown cultures of the indicated strains. *, statistically significant difference from the WT (P < 0.05). (E) Swarming phenotype of the WT and the ΔpelA mutant. Shown are a representative swarm plate for each strain and quantification of plate coverage for each strain (five plates).

FIG. 5.

Model for inverse control of biofilm formation and swarming motility. Planktonic bacteria (top) initially interact with the surface, likely via reversible polar attachment, although which end of the cell interacts with the abiotic surface is unclear. A pathway that includes SadB and components of the CheIV chemotaxis cluster (PilJ and ChpB) controls the decision to initiate biofilm formation or move via swarming. Biofilm formation is associated with an increase in the production of Pel-dependent polysaccharide (Pel) and a decrease in flagellar reversal rate (FlaRev). Swarming is associated with decreased production of polysaccharide and an increased rate of flagellar reversals. The substratum is shown as a gradient of gray to represent the fact that variations in surface properties (viscosity, wetness, etc.) might also impact the biofilm-versus-swarming decision.