Rhamnolipids modulate swarming motility patterns of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is capable of twitching, swimming, and swarming motility. The latter form of translocation occurs on semisolid surfaces, requires functional flagella and biosurfactant production, and results in complex motility patterns. From the point of inoculation, bacteria migrate as defined groups, referred to as tendrils, moving in a coordinated manner capable of sensing and responding to other groups of cells. We were able to show that P. aeruginosa produces extracellular factors capable of modulating tendril movement, and genetic analysis revealed that modulation of these movements was dependent on rhamnolipid biosynthesis. An rhlB mutant (deficient in mono- and dirhamnolipid production) and an rhlC mutant (deficient in dirhamnolipid production) exhibited altered swarming patterns characterized by irregularly shaped tendrils. In addition, agar supplemented with rhamnolipid-containing spent supernatant inhibited wild-type (WT) swarming, whereas agar supplemented with spent supernatant from mutants that do not make rhamnolipids had no effect on WT P. aeruginosa swarming. Addition of purified rhamnolipids to swarming medium also inhibited swarming motility of the WT strain. We also show that a sadB mutant does not sense and/or respond to other groups of swarming cells and this mutant was capable of swarming on media supplemented with rhamnolipid-containing spent supernatant or purified rhamnolipids. The abilities to produce and respond to rhamnolipids in the context of group behavior are discussed.

FIG. 1.

Swarms of P. aeruginosa are able to sense and respond to neighboring swarms. (A) Two aliquots of WT cells were spotted on 0.5% swarm agar plates and monitored at 24 and 48 h. (B) Aliquots of WT and flgK mutant cells were spotted on 0.5% swarm agar plates and monitored at 24 and 48 h.

FIG. 2.

An extracellular signal modulates swarming in P. aeruginosa. (A) A flgK mutant was inoculated on 0.5% swarm agar plates and incubated at 37°C for 12 h to allow the formation of a rhamnolipid zone. The perimeter of the zone was visualized using methylene blue. (B) A flgK mutant was inoculated on 0.5% swarm agar plates and incubated at 37°C for 12 h to allow the formation of a rhamnolipid zone. The white circle depicts the edge of the rhamnolipid zone surrounding the flgK mutant colony at the time of WT inoculation. WT cells were then inoculated within the zone (top) or on the zone edge (bottom) and incubated for 16 h at 37°C. (C) Aliquots of WT and flgK cultures were spotted on 0.5% swarm agar plates containing 7.5% WT spent supernatant and incubated at 37°C for 16 h.

FIG. 3.

Rhamnolipids are required for modulating swarming behavior. (A) Aliquots of the WT cells and rhlA mutant cells were spotted on 0.5% swarm agar plates and monitored over the course of 16 h. The dotted line indicates the outline of the colony formed by the nonswarming rhlA mutant. (B) Aliquots of the WT and flgK mutant cultures were spotted on 0.5% swarm agar plates containing 7.5% rhlA spent supernatant and incubated at 37°C for 16 h. (C) Aliquots of the WT and the flgK mutant cultures were spotted on 0.5% swarm agar plates containing purified rhamnolipids and incubated at 30°C for 16 h. The amount of rhamnolipids added to these plates is approximately equivalent to the level of rhamnolipids found in agar supplemented with 7.5% spent supernatant.

FIG. 4.

Dissection of the rhamnolipid biosynthetic pathway and its role in modulating swarming behavior. (A) (Top) Thin-layer chromatographic analysis of P. aeruginosa rhamnolipid biosynthesis mutants grown in PPGAS medium for 24 h at 30°C. The positions of monorhamnolipids (mono) and dirhamnolipids (di) are shown to the left of the plate. (Bottom) Drop collapse analysis of P. aeruginosa rhamnolipid biosynthesis mutants grown in PPGAS medium for 24 h at 30°C. Samples were diluted in dH₂O, spotted on the lid of a microtiter plate, and assayed for bead formation. The dilutions are shown at the top of the gel. U, undiluted. (B) Swarming phenotypes of the WT and the rhl mutants grown on 0.5% agar for 16 h at 37°C. (C) Aliquots of the WT and flgK mutant were spotted on 0.5% swarm agar plates containing 7.5% rhlB (top) or rhlC (bottom) spent supernatant and incubated at 37°C for 24 h. (D) Rhamnolipid mutants were assayed by the BATH test to determine cell surface hydrophobicity as described in Materials and Methods. The percentage of bound cells was plotted versus the strain tested.

FIG. 5.

The sadB gene has a role in rhamnolipid sensing. (A) The swarming phenotypes of WT and the sadB mutant after 48 h of incubation inoculated individually on a 0.5% swarm agar plate. (B) The sadB mutant does not avoid other groups of swarming cells. The phenotype of the sadB mutant 24 h after inoculation on 0.5% swarm agar plates in the presence of the WT (left panel) or the flgK mutant (middle panel) is shown. Complementation of the swarming phenotype of the sadB mutant by expressing sadB in trans on plasmid pNC5 (right panel) is depicted. (C) The sadB mutant does not respond to extracellular rhamnolipids. The flgK mutant was inoculated on 0.5% swarm agar plates and incubated at 37°C for 12 h to allow the formation of a rhamnolipid zone (left panel). The white circle depicts the edge of the rhamnolipid zone surrounding the flgK colony at the time of WT and sadB inoculation. The WT (top) and the sadB mutant (bottom) were then inoculated on the zone edge and incubated for 24 h at 37°C. Aliquots of the WT, the flgK mutant, and the sadB mutant were spotted on 0.5% swarm agar plates containing 7.5% WT spent supernatant and incubated at 37°C for 24 h (middle panel). Aliquots of the sadB mutant and the flgK mutant cultures were spotted on 0.5% swarm agar plates containing purified rhamnolipids and incubated at 30°C for 16 h (right panel). The amount of rhamnolipids added to these plates is approximately equivalent to the level of rhamnolipids found in agar supplemented with 7.5% spent supernatant.