Statistical analysis of Pseudomonas aeruginosa biofilm development: impact of mutations in genes involved in twitching motility, cell-to-cell signaling, and stationary-phase sigma factor expression - PubMed (original) (raw)

Statistical analysis of Pseudomonas aeruginosa biofilm development: impact of mutations in genes involved in twitching motility, cell-to-cell signaling, and stationary-phase sigma factor expression

Arne Heydorn et al. Appl Environ Microbiol. 2002 Apr.

Abstract

Four strains of Pseudomonas aeruginosa (wild type, Delta(pil)HIJK mutant, lasI mutant, and rpoS mutant) were genetically tagged with the green fluorescent protein, and the development of flow chamber-grown biofilms by each of them was investigated by confocal laser scanning microscopy. The structural developments of the biofilms were quantified by the computer program COMSTAT (A. Heydorn, A. T. Nielsen, M. Hentzer, C. Sternberg, M. Givskov, B. K. Ersbøll, and S. Molin, Microbiology 146:2395-2407, 2000). Two structural key variables, average thickness and roughness, formed the basis for an analysis of variance model comprising the four P. aeruginosa strains, five time points (55, 98, 146, 242, and 314 h), and three independent rounds of biofilm experiments. The results showed that the wild type, the Delta(pil)HIJK mutant, and the rpoS mutant display conspicuously different types of temporal biofilm development, whereas the lasI mutant was indistinguishable from the wild type at all time points. The wild type and the lasI mutant formed uniform, densely packed biofilms. The rpoS mutant formed densely packed biofilms that were significantly thicker than those of the wild type, whereas the Delta(pil)HIJK mutant formed distinct microcolonies that were regularly spaced and almost uniform in size. The results are discussed in relation to the current model of P. aeruginosa biofilm development.

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Figures

FIG. 1.

FIG. 1.

Construction of a P. aeruginosa Δ_pilHIJK_ mutant strain. A schematic diagram showing the pilGHIJK gene cluster on the P. aeruginosa PAO1 chromosome and relevant restriction sites for _Eco_RI and Eco_RV is presented. Also shown is the region of pJKΔ_pilHIJK that contains P. aeruginosa DNA with a Kmr cartridge replacing part of the pilGHIJK gene cluster (described in Materials and Methods).

FIG. 2.

FIG. 2.

Twitching motility assays of wild-type and Δ_pilHIJK_ mutant P. aeruginosa. Thin agar plates (1.5%) were stab inoculated with a toothpick to the bottom of the plate and incubated for 48 h at 37°C. The light haze of growth, the twitch zone, at the agar-plate interface is a measure of twitching motility. The smaller, denser zone represents surface colony growth.

FIG. 3.

FIG. 3.

Spatial structures of 98-h-old biofilms of wild-type P. aeruginosa PAO1 (A), P. aeruginosa rpoS (B), P. aeruginosa Δ_pilHIJK_ (C), and P. aeruginosa lasI (D) growing in citrate minimal medium. The bacteria are expressing the GFP. The larger central plots are simulated fluorescent projections in which a long shadow indicates a large, high microcolony. Shown in the right and lower frames are vertical sections through the biofilms collected at the positions indicated by the white triangles. The scale bars shown in the central plots are also valid for the right and lower frames.

FIG. 4.

FIG. 4.

Roughness versus average thickness of 98-h-old (A) and 146-h-old (B) biofilms of wild-type P. aeruginosa (•), P. aeruginosa rpoS (▿), P. aeruginosa Δ_pilHIJK_ (▪), and P. aeruginosa lasI (⋄). Images were acquired by CLSM in three independent biofilm experiments and quantified by the computer program COMSTAT. In each of the three rounds, 18 image stacks were acquired from two flow channels totaling 54 image stacks for each strain at each time point. Each spot represents a single stack of images. Note that there are only 36 spots for the P. aeruginosa rpoS mutant at the 98-h time point because, inadvertently, no images were acquired for this strain at the 98-h time point in the second round.

FIG. 5.

FIG. 5.

Comparison of P. aeruginosa biofilm architectures. Mean values for log average thickness and log roughness together with 90% confidence ellipses is shown for wild-type P. aeruginosa (•), P. aeruginosa rpoS (▿), P. aeruginosa Δ_pilHIJK_ (▪), and P. aeruginosa lasI (◊). Two nonoverlapping ellipses indicate that a particular pair of biofilms were different at a specific time point. Two overlapping ellipses mean that a pair of biofilms were not significantly different at a particular time point. This type of comparison corresponds to a multivariate t test on approximately a 5% level. Panels: A, 55 h; B, 98 h; C, 146 h; D, 242 h; E, 314 h.

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