Pseudomonas aeruginosa exhibits sliding motility in the absence of type IV pili and flagella

Abstract

Pseudomonas aeruginosa exhibits swarming motility on 0.5 to 1% agar plates in the presence of specific carbon and nitrogen sources. We have found that PAO1 double mutants expressing neither flagella nor type IV pili (fliC pilA) display sliding motility under the same conditions. Sliding motility was inhibited when type IV pilus expression was restored; like swarming motility, it also decreased in the absence of rhamnolipid surfactant production. Transposon insertions in gacA and gacS increased sliding motility and restored tendril formation to spreading colonies, while transposon insertions in retS abolished motility. These changes in motility were not accompanied by detectable changes in rhamnolipid surfactant production or by the appearance of bacterial surface structures that might power sliding motility. We propose that P. aeruginosa requires flagella during swarming to overcome adhesive interactions mediated by type IV pili. The apparent dependence of sliding motility on environmental cues and regulatory pathways that also affect swarming motility suggests that both forms of motility are influenced by similar cohesive factors that restrict translocation, as well as by dispersive factors that facilitate spreading. Studies of sliding motility may be particularly well-suited for identifying factors other than pili and flagella that affect community behaviors of P. aeruginosa.

FIG. 1.

PAO1 motility in strains lacking type IV pili and/or flagella. A) Flagellum-dependent swimming motility on 0.3% LB agar plates. B) TFP-dependent twitching motility at the plastic-1.5% LB agar interface. C) Motility on 0.5% M8 agar plates supplemented with 0.05% glutamate and 0.2% glucose. D) Restoration of pilin expression inhibits motility of fliC pilA bacteria. wt, wild type.

FIG. 2.

Motility organelles are not present on sliding fliC pilA bacteria. TEM of fliC pilA bacteria (A) and isogenic wild-type (wt) bacteria (B) harvested directly from swarming plates and stained with 1.5% uranyl acetate. Bar, 2 μm.

FIG. 3.

rhlA positively influences sliding motility. (A) Dispersion of fliC pilA and fliC pilA rhlA bacteria was compared on 0.5% agar M8 plates after overnight incubation at 30°C. The extent of spread is dependent upon RhlA expression. (B) Undiluted supernatants prepared from overnight LB cultures of fliC pilA rhlA and the complemented strain fliC pilA rhlA pRhlA were assayed for drop collapse activity.

FIG. 4.

GacA and RetS influence sliding motility. (A) Transposon insertion mutants of fliC pilA were screened on 0.5% M8 swarming plates for changes in motility. Mutants exhibiting both diminished (a) and increased (b) spreading were obtained; the parental fliC pilA strain was included on each plate as a control (c). Note the appearance of tendril formation in the strain marked b. (B) Sliding motility of fliC pilA gacA and fliC pilA retS mutants was assayed on 0.5% M8 plates after overnight incubation at 30°C. (C) fliC pilA gacA bacteria grown on swarming plates were harvested from the end of a tendril and visualized by TEM. No bacteria had recognizable surface structures; a representative image is shown. (D) Expression of GacA from a plasmid complements the fliC pilA gacA phenotype on 0.5% M8 plates supplemented with carbenicillin (200 μg/ml). Note that tendril formation is eliminated when GacA is reintroduced. The isogenic control strains all carry the empty vector pUCP-KS. wt, wild type.

FIG. 5.

BifA and SadC reciprocally regulate sliding motility. Dispersion of fliC pilA bacteria expressing BifA or SadC under control of the inducible P_BAD promoter was assayed on 0.5% M8 agar plates supplemented with 0.2% glucose and 0.05% glutamate plates that contained gentamicin and arabinose (0.5%). Plates were incubated overnight at 30°C and then left at room temperature for an additional 48 h. The control strain carries the empty vector pMQ80.