Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa

Abstract

Polyphosphate kinase (PPK), encoded by the ppk gene, is the principal enzyme in many bacteria for the synthesis of inorganic polyphosphate (poly P) from ATP. A knockout mutant in the ppk gene of Pseudomonas aeruginosa PAO1 is impaired in flagellar swimming motility on semisolid agar plates. The mutant is deficient in type IV pili-mediated twitching motility and in a "swarming motility" previously unobserved in P. aeruginosa. In swarming cultures, the polar monotrichous bacteria have differentiated into elongated and polar multitrichous cells that navigate the surface of solid media. All of the motility defects in the ppk mutant could be complemented by a plasmid harboring the ppk gene. Because bacterial motility is often crucial for their survival in a natural environment and for systemic infection inside a host, the dependence for motility on PPK reveals important roles for poly P in diverse processes such as biofilm formation, symbiosis, and virulence.

Figure 1

Swimming motility of P. aeruginosa PAO1, its ppk mutant, and a fliC control strain derived from a P. aeruginosa PAK strain on a semisolid agarose plate. Cells were inoculated with a toothpick from an overnight LB agar plate onto a swim plate (tryptone broth plus 0.3% agarose) and photographed after a 14-h incubation at 30°C. Strains: WT, PAO1; Δppk, PAOM5; and ΔfliC, MS159.

Figure 2

Swarming motility in P. aeruginosa PAO1. The ppk mutant is completely deficient in swarming motility which could be restored to the WT level by providing a plasmid bearing the ppk gene. Cells were inoculated onto swarm plates (Difco nutrient broth plus 0.5% Difco bacto-agar supplemented with 0.5% glucose) from an overnight swim plate with a toothpick and photographed after a 24-h incubation at 30°C. Strains: WT, PAO1; Δppk, PAOM5; ΔfliC, MS159; ΔfliD, PAO-D; ΔfliF, PA14-fliF; WT (CV), PAO1 (pMMB66HE); Δppk (CV), PAOM5 (pMMB66HE); and Δppk (PPK⁺⁺⁺), PAOM5 (pHEPAK11). CV represents the control vector pMMB66HE and PPK⁺⁺⁺ the PPK-overproducing plasmid pHEPAK11.

Figure 3

Morphology of P. aeruginosa swarm cells by (Upper) phase-contrast and (Lower) electron microscopy. (Upper) Images of cells suspended in PBS from swarm colonies (X50 magnification). (Lower) Electron micrographs of bacteria taken directly from swarm plates (×10,400.) Strains: WT (edge), edge cells of PAO1 colony; WT (center), center cells of PAO1 colony; Δppk, cells from PAOM5 colony; and Δppk (PPK⁺⁺⁺; edge), edge cells of PAOM5 colony containing pHEPAK11 plasmid. [Bars = 5 μm (Upper) and 0.5 μm (Lower).]

Figure 4

Macroscopic stab assay for twitching motility. Cells from an overnight LB agar (1.5%, wt/vol) plate were inoculated with a toothpick to the bottom of the LB agar (1%, wt/vol) plate and incubated for 24 h at 37°C. The diffuse interstitial zone is a measure of twitching motility. The denser and smaller zone represents surface colony growth. Strains: WT, PAO1; Δppk, PAOM5; ΔfliD, PAO-D; and ΔpilA, PAO-NP.

Figure 5

Light microscopy of zones of twitching motility by slide-culture assay. The strains were inoculated onto slide cultures and typical colony expansion zones were obtained at the interstitial surface between glass cover slip and medium after 3–5 h incubation at 37°C. Strains: WT, PAO1; Δppk, PAOM5; ΔpilA, PAO-NP; and Δppk (PPK⁺⁺⁺), PAOM5-harboring plasmid pHEPAK11. The bar is 10 μm and the arrow indicates the radial direction of colony expansion.