Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming, and twitching motilities

Abstract

Pseudomonas aeruginosa is a ubiquitous environmental bacterium capable of forming biofilms on surfaces as a survival strategy. It exhibits a large variety of competition/virulence factors, such as three types of motilities: flagellum-mediated swimming, flagellum-mediated swarming, and type IV pilus-mediated twitching. A strategy frequently used by bacteria to survive changing environmental conditions is to create a phenotypically heterogeneous population by a mechanism called phase variation. In this report, we describe the characterization of phenotypic variants forming small, rough colonies that spontaneously emerged when P. aeruginosa 57RP was cultivated as a biofilm or in static liquid cultures. These small-colony (S) variants produced abundant type IV fimbriae, displayed defective swimming, swarming, and twitching motilities, and were impaired in chemotaxis. They also autoaggregated in liquid cultures and rapidly initiated the formation of strongly adherent biofilms. In contrast, the large-colony variant (parent form) was poorly adherent, homogeneously dispersed in liquid cultures, and produced scant polar fimbriae. Further analysis of the S variants demonstrated differences in a variety of other phenotypic traits, including increased production of pyocyanin and pyoverdine and reduced elastase activity. Under appropriate growth conditions, cells of each phenotype switched to the other phenotype at a fairly high frequency. We conclude that these S variants resulted from phase variation and were selectively enriched when P. aeruginosa 57RP was grown as a biofilm or in static liquid cultures. We propose that phase variation ensures the prior presence of phenotypic forms well adapted to initiate the formation of a biofilm as soon as environmental conditions are favorable.

FIG. 1

Visual differences between growth phenotypes. (A to C) Colonies of L variant (A), S1 variant (B; the arrow indicates the emergence of a L-type revertant sector emerging from the side of a colony), and S2 variant (C) on LB agar plates incubated at 30°C. (D) Overnight growth in broth medium with shaking. The L and S1rev variants grew homogeneously dispersed in the medium, whereas the S1 and S2 variants preferred the interface and the glass surface. S1rev is an L variant resulting from the reversion of an S1 variant.

FIG. 2

Kinetics of biofilm formation. L (●), S1 (□), and S2 (▴) variants were cultivated in polystyrene tubes at 32°C without agitation. At the indicated time intervals, triplicate tubes were rinsed and stained with crystal violet. The amount of stained cells was then quantified by spectrophotometry (A₆₀₀) after solubilization of the dye in ethanol.

FIG. 3

Evaluation of cell surface hydrophobicity and adhesion potential by the MATH (A) and MATS (B) tests, respectively, in L (●) and S1 (□) variants. Various amounts of hexadecane (MATH) or silica sand (MATS) were mixed with a washed cell suspension in PBS, and the optical densities at 600 nm before and after were compared. Values for the MATH test are means ± standard deviations of duplicates.

FIG. 4

Differences in motility phenotypes of L and S variants. (A) Swimming motility on a tryptone swim plate (0.3% agar). (B) Swarming motility on a 0.5% agar plate. (C) Twitching motility on a thin (3-mm) LB plate containing 1% agar. Twitching is observed as a hazy zone of interstitial growth surrounding the surface colony. (D) Staining with crystal violet of cells in twitching zones that remained attached to the polystyrene surface after removing the agar layer and washing with water. (E to H) Light microscopy of the outside of the twitching zone stained with crystal violet. (E and F) S1 and S2 variant cells at a magnification of ×90; (G and H) rafts of S1 and S2 variant cells oriented toward the expending direction of the twitching area at a magnification of ×900.

FIG. 5

Comparison of chemotactic responses of L and S variants. Capillary apparatus with or without 0.1% tryptone as a chemoattractant was prepared as described in Materials and Methods and incubated at 37°C for 45 min. The content of the syringe was then plated onto TSA plates for cell enumeration. Error bars represent the standard deviations of duplicates.

FIG. 6

Transmission electron micrographs of L (A) and S2 (B) variants. Cells were grown overnight on LB agar plates, transferred onto Formvar-coated copper grids, and stained with phosphotungstic acid. Arrows indicate type IV pili. Bars = 0.2 μm.