Figure 5. Involvement of AafA and site-directed-mutants in biofilm formation and binding to fibronectin.

(A) Biofilm quantification. Bacteria were cultivated in LB for 20 h at 37°C in 24-well dishes and induced with 0.2% arabinose. Biofilms were fixed and stained with crystal violet, and then the stains were solubilized and quantitated spectrophotometrically at 450 nm. The bars represent the means of the results from triplicate wells; error bars indicate one standard deviation. Wt, wild type EAEC042, ΔaafA, EAEC042aafA mutant. All residues tested were mutated to Ala except Thr18Ile and Thr38Leu. *, P<0.005. (B) Fibronectin binding. EAEC 042 derivatives harboring site mutations in aafA were added to 96-well plates coated separately with 25 µg/ml of fibronectin, and the binding was determined by collecting the cells adhered to wells by scraping them into PBS. The number of adherent bacteria was determined by counting the resulting colonies in duplicate and normalized to the wild type adherence (as a 100% adherence). The bars represent the means for three experiments, with the error bars indicating 1 standard deviation. *, significantly different from EAEC042WT (P<0.005).(C) AafA surface expression. After induction of bacterial strains with 0.2% arabinose, cells were harvested, washed twice with PBS, and incubated with a polyclonal anti-aafA antibody (2 µg/ml) in PBS plus 1% bovine serum albumin for 1 h at room temperature with agitation. They were washed twice with PBS and incubated with a goat anti-rabbit IgG-fluorescein isothiocyanate (FITC) conjugate (10 µg/ml) for 30 min at room temperature in the dark. The washings were repeated, and the samples were resuspended in DAPI (4 mg/liter) and spread on slides for viewing by fluorescence microscopy.