T4P isolation, biofilm formation, and twitching motility assays using P. aeruginosa strains

T4P from all the P. aeruginosa strains were purified following a previously described protocol (Castric, 1995). Briefly, 100 μl of an LB culture were plated on triplicate LB-agar plates supplemented with 20 μM IPTG and incubated overnight at 37°C. When needed, carbenecillin (30 μg/ml) was included in the LB-agar medium to maintain the pMMB67EH plasmid or any of its derivatives (Table ​(Table1).1). The cells were scraped off the plate and suspended in 1.5 ml of LB medium and 5 μl of the cell suspension was diluted in 2 ml water to measure its OD₆₀₀ and estimate the cell density. T4P fibers in the cell suspensions were sheared by passing the cells 4 times through a 23-gauge needle and the cells were then removed by centrifugation (2 times at 7500 × g for 15 min at room temperature). The T4P in the supernatant fluids were then precipitated by adding MgCl₂ to a final concentration of 100 mM and incubating on ice for 60 min. Once precipitated, the T4P were concentrated by centrifugation (12,000 × g for 15 min at 4°C), suspended in 100 μl of 0.1% SDS, and incubated overnight at 4°C to separate the pili bundles. The T4P content in the samples was quantified using the Bio-Rad protein assay Kit (Bio-Rad Laboratories, Inc.), following the manufacturer's recommendations for the standard procedure in microtiter plates, and its concentration (mg/ml) in each sample was normalized by the sample's cell density (in OD₆₀₀ units).

T4P purified in the 0.1% SDS solution were diluted appropriately to standardize for the cell density (OD₆₀₀) and then mixed with an equal volume of Tris-Tricine sample buffer. After incubation at 95°C for 5 min to depolymerize the fibers into the individual pilin subunits, the proteins in 15 μl of sample were separated electrophoretically in a 10–20% Mini-PROTEAN® Tris-Tricine Precast Gel (Bio-Rad). One lane was loaded with 4 μl of the Novex® Sharp Pre-stained Protein Standard (Invitrogen Life Technologies) as standards. The gel was stained overnight with Coomassie Brilliant Blue R-250 Staining Solution (Bio-Rad) and destained overnight with a solution of 50% methanol and 10% acetic acid.

Biofilm assays with strains of P. aeruginosa were performed under static conditions in microtiter plates, essentially as described elsewhere (Merritt et al., 2005) but using M63 minimal medium supplemented with 0.4% arginine as the sole carbon and energy source to stimulate biofilm development over a 24 h period (Caiazza and O'Toole, 2004). The medium was supplemented with carbenecillin (30 μg/ml) to maintain the pMMB67EH plasmid and with IPTG (20 μM) to heterologously express the cloned pilT genes. The biofilm biomass was then stained with 0.1% crystal violet, and allowed to dry before solubilizing the biofilm-associated dye with 95% ethanol and measuring its OD₅₈₀ (Merritt et al., 2005). To account for technical and biological variability, the biofilm assays included six technical replicates (wells) per strain tested and a minimum of three independent experiments. Outliers were excluded from the compiled biofilm assay results using the Quartile or Fourth-Spread method (Devore, 2000) and the data were plotted as a box-and-whisker graph using the Microsoft Excel software.

For the twitching motility assays, a thin layer (~1 ml) of molten LB medium with 1% agarose and 20 μM IPTG was deposited onto a sterile glass slide and allowed to solidify. The center of the agar was stab-inoculated with overnight LB-20 μM IPTG cultures of the P. aeruginosa strains incubated at 37°C with shaking (180 rpm). When needed, carbenecillin (30 μg/ml) was added to the liquid cultures to maintain the pMMB67EH plasmid. After stab-inoculation, the slide was placed inside a Petri dish surrounded by damp, sterile absorbent paper to maintain the humidity inside the chamber and the dish was then sealed with parafilm to prevent drying. After overnight incubation at 37°C, the areas of growth on the surface of the agar were gently removed with a coverslip and the slides were then stained with 1 ml Coomassie Brilliant Blue R-250 Staining Solution (Bio-Rad) for 2 h. The slides were briefly rinsed with 5 ml of a solution of 50% methanol and 10% acetic acid and then destained in 20 ml of the same solution for 4 h before replacing 15 ml of the solution with an equal volume of a solution of 5% methanol and 7.5% acetic acid and incubating overnight. After destaining, the agar layer on the slides was allowed to dry completely before imaging the stained twitching zones with a scanner.