FIG 3.

UPEC type I fimbria regulation. (A) Yeast agglutination assay with dilutions of the WT (1:16); Δfnr (1:1), and Δfnr/pGEN-fnr (1:16) strains. Bacteria were grown statically for 48 h, agglutination was read after 10 min at room temperature, and the strength of the agglutination was determined by measurement of the titer of serial 2-fold dilutions of the bacterial suspensions in PBS. The experiments were performed four times in quadruplicate. (B) β-Galactosidase activity assay for expression of fimA. fimA-lacZ transcriptional fusion strains were grown statically in LB medium for 48 h at 37°C. β-Galactosidase activity was measured, and the values shown are means plus standard deviations for triplicate samples from three independent experiments. Significant differences are indicated by asterisks (***, P < 0.0001 compared to the WT and mutant). (C) Nonradioactive EMSA of binding of (FnrD154A)₂-His₆ to the promoter regions. The PCR product of the fimA promoter region was used as the probe at 300 ng per reaction mixture. Purified (FnrD154A)₂-His₆ fusion protein was added to each reaction mixture at different concentrations, as indicated; ydfZ promoter region DNA probes with and without the FNR protein were used as positive controls, and fimA coding region DNA probes with and without the FNR protein were used as negative controls. DNA fragments were stained with SYBR green.