Nonmotile Subpopulations of Pseudomonas aeruginosa Repress Flagellar Motility in Motile Cells through a Type IV Pilus- and Pel-Dependent Mechanism

Abstract

The downregulation of Pseudomonas aeruginosa flagellar motility is a key event in biofilm formation, host colonization, and the formation of microbial communities, but the external factors that repress motility are not well understood. Here, we report that on soft agar, swarming motility can be repressed by cells that are nonmotile due to the absence of a flagellum or flagellar rotation. Mutants that lack either flagellum biosynthesis or rotation, when present at as little as 5% of the total population, suppressed swarming of wild-type cells. Non-swarming cells required functional type IV pili and the ability to produce Pel exopolysaccharide to suppress swarming by the flagellated wild type. Flagellated cells required only type IV pili, but not Pel production, for their swarming to be repressed by non-flagellated cells. We hypothesize that interactions between motile and nonmotile cells may enhance the formation of sessile communities, including those involving multiple genotypes, phenotypically diverse cells, and perhaps other species. IMPORTANCE Our study shows that, under the conditions tested, a small population of non-swarming cells can impact the motility behavior of a larger population. The interactions that lead to the suppression of swarming motility require type IV pili and a secreted polysaccharide, two factors with known roles in biofilm formation. These data suggest that interactions between motile and nonmotile cells may enhance the transition to sessile growth in populations and promote interactions between cells with different genotypes.

Keywords: Pel; Pseudomonas aeruginosa; microbe-microbe interaction; motility; swarming; type IV pili.

FIG 1

Motility heterogeneity represses swarming motility independent of competition. (A) Representative images from swarm assay of wild-type (WT) P. aeruginosa strain PA14 (flagellated) mixed with ΔflgK (non-flagellated) at the indicated ratios on M8 agar. WT and derivatives are labeled in black, flagellar mutants are labeled in red. Data are representative of three individual experiments. (B) Competition growth assay between WT tagged with a lacZ reporter (gray) and either an untagged WT (black) or ΔflgK mutant strain (red). Data show the average of three individual experiments. Error bars represent standard deviations between the replicate values. Statistical analysis using one-way analysis of variance showed no difference between input and output for each ratio analyzed. (C) Representative images from swarm assay of WT (flagellated) mixed with ΔflgK (non-flagellated), ΔfliC (non-flagellated), or ΔmotABCD (paralyzed flagellum) at the indicated ratios on M8 agar. Data are representative of three individual experiments. Scale bar, 30 mm.

FIG 2

Flagellated P. aeruginosa requires functional T4P to be repressed by the non-motile subpopulation. Representative images from swarm assays of the non-flagellated ΔflgK mutant (functional T4P) mixed with either wild-type (WT) P. aeruginosa strain PA14 (functional T4P), ΔpilA mutant (lacking T4P), ΔpilT mutant (no T4P retraction), or ΔpilU mutant (reduced T4P retraction) at the indicated ratios on M8 agar. Data are representative of three individual experiments. Scale bar, 30 mm.

FIG 3

Non-flagellated P. aeruginosa requires functional T4P to repress flagellum-mediated motility of motile strains on soft agar. (A) Representative images of swarm assays for wild-type (WT) P. aeruginosa strain PA14 (swarmer, T4P+) mixed with ΔflgK (non-swarmer, T4P+), ΔflgKΔpilA (non-swarmer, T4P–), ΔflgKΔpilU (non-swarmer, reduced T4P retraction), or ΔflgKΔpilT (non-swarmer, no T4P retraction) on M8 agar at the indicated ratios. WT is labeled in black, ΔflgK and derivatives are labeled in red. (B) Representative image showing the interaction between WT (black dot), ΔflgK (red dots), and ΔflgKΔpilA (purple dots) for flagellum-mediated swimming motility in soft agar (0.3% M63 agar) after 42 h incubation. Colored dots indicate points of inoculation for the respective strains. Data are representative of three individual experiments. Scale bar, 30 mm.

FIG 4

Pel matrix is required by the non-flagellated P. aeruginosa subpopulation alone to repress overall swarming motility. (A) Representative images from swarm assays of ΔflgK (non-flagellated, Pel+) mixed with wild-type (WT) P. aeruginosa strain PA14 (flagellated, Pel+) or ΔpelA (flagellated, Pel–), and of WT mixed with ΔflgKΔpelA (non-flagellated, Pel–), at the indicated ratios on M8 agar. Scale bar, 30 mm. (B) Representative images of WT (flagellated, Pel+, T4P+), ΔpilA (flagellated, Pel+, T4P–), ΔpelA (flagellated, Pel–, T4P+), ΔflgK (non-flagellated, Pel+, T4P+), ΔflgKΔpilA (non-flagellated, Pel+, T4P–), ΔflgKΔpelA (non-flagellated, Pel–, T4P+), and ΔflgKΔpilAΔpelA (non-flagellated, Pel–, T4P–) grown on Congo red plates to assess Pel production. WT and derivatives are labeled in black, ΔflgK and derivatives are labeled in red. Scale bar, 10 mm.

FIG 5

Summary model describing genes and gene products needed for repression of flagellum-mediated motility by non-flagellated cells in P. aeruginosa strain PA14. We show that a subpopulation of P. aeruginosa cells defective in flagellar motility (red) in co-culture with flagellated cells (black) impedes flagellum-mediated swarming motility in a retractable T4P-dependent manner (PilMNOP, PilA, PilU, PilT). We also show that Pel matrix (PelA), but only that from the non-flagellated strain, is required. PilU, PilJ, FimS, CyaAB, and Vfr were not required for repression of swarming motility.