FIG 4.

FimV TPR3 is critical for FimV function. fimV and fimV cyaB-R456L strains were transformed with arabinose-inducible expression constructs encoding full-length FimV (FimV) or a truncation mutant lacking TPR3 (FimV₆₈₉). As controls, strains were also transformed with empty vector (pB). (A) The levels of PilU were determined by preparing cell lysates as described in Materials and Methods of cells grown overnight on LB medium–1.5% agar supplemented with 0.1% (wt/vol) arabinose. Samples were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. PilU was visualized by immunoblotting with anti-PilU antiserum. Densitometric analysis (average, n = 3) was carried out using ImageJ, normalizing band intensity of the PilU band to a nonspecific control band, and the results are shown as percentages of the normalized band intensity of the wild type ± the standard error. Samples were considered significantly different if a pairwise Student t test gave P values of <0.05 (*). (B) The levels of surface pilins were assessed by sheared surface protein preparation of strains grown on LB medium–1.5% agar supplemented with 0.1% (wt/vol) arabinose. The surface protein fractions were prepared as described in Materials and Methods and were resolved by SDS-PAGE and visualized by Coomassie blue staining. (C) Twitching motility was determined by stab inoculation of the indicated strains into LB medium–1% agar supplemented with 0.1% (wt/vol) arabinose. Strains were incubated for 16 h, and twitching zones were visualized by removing the agar layer and staining the bacteria with 1% (wt/vol) crystal violet. Motility was measured as the diameter of the stained twitching zone. Scale bar, 1 cm. A “−” represents the negative control for each experiment, pilU strains for the anti-PilU immunoblots or a nonpiliated pilA strain for surface piliation and twitching motility. The images are representative of three experiments.