Biofilm formation by hyperpiliated mutants of Pseudomonas aeruginosa

Abstract

Under static growth conditions, hyperpiliated, nontwitching pilT and pilU mutants of Pseudomonas aeruginosa formed dense biofilms, showing that adhesion, not twitching motility, is necessary for biofilm initiation. Under flow conditions, the pilT mutant formed mushroom-like structures larger than those of the wild type but the pilU mutant was defective in biofilm formation. Therefore, twitching motility affects the development of biofilm structure, possibly through modulation of detachment.

FIG. 1.

Complementation of twitching motility in pilT and pilU mutants. Thin (3-mm) Luria-Bertani 1% agar plates were stab inoculated with a toothpick and incubated at 37°C for 48 h (15). Twitching motility is visualized as a halo between the agar and plastic plates, surrounding the point of inoculation. Both twitching and nontwitching strains form colonies on the agar surface. Scale bar, 10 mm. NP, mutant PAK-NP.

FIG. 2.

Transmission electron micrographs of negatively stained (phosphotungstic acid) PAK and the pilT and complemented pilT mutants. (A) Wild-type PAK strain; (B) pilT mutant R364; (C) complemented pilT mutant R364+. Images were acquired with a JEM 1230 electron microscope (JEOL, Peabody, Mass.) equipped with a charge-coupled device camera (Amount, Denver, Mass.). The arrows indicate pili. Size bars are shown on the bottom of each panel.

FIG. 3.

Biofilm formation assay under static conditions. Standardized cultures were inoculated into DMM+ in microtiter plates and incubated at RT for 24 h. The OD₆₀₀ of the planktonic culture in each well was measured to control for growth inconsistencies. The level of biofilm formation was quantitated indirectly by crystal violet staining (12). The results shown are the averages of results from five separate experiments.

FIG.4.

Confocal scanning laser micrographs of P. aeruginosa biofilms grown under flow conditions. Biofilms were stained with BacLight Live/Dead stain. Sagittal (X-Z) sections through the biofilms are presented at the top and right of each image, with arrows showing the image planes. Biofilm development at 24 h is shown for the wild type (A), R364 (C), R364+ (E), and S34 (G). Biofilm morphology at 96 h is shown for PAK (B), R364 (D), R364+ (F), and S34 (H). S34+ biofilms (not shown) resembled those of R364+. PAK-NP did not adhere to the glass (data not shown). The results shown are representative of results from three separate experiments. Scale bar, 100 μm.