Fig. 5.

YhaJ promotes activation of the fimS element by binding exclusively in the OFF orientation. (A) Schematic depiction of the phase-variable fimS element, containing an invertible promoter, in the ON orientation. EMSA analysis showing that YhaJ does not bind UPEC fimS in the ON orientation. (B) Schematic depiction of fimS in the OFF orientation. EMSAs demonstrating YhaJ binding over increasing concentrations to both UPEC and EHEC exclusively in the OFF orientation (Bottom). Excess unlabeled probe was added as a competitor to demonstrate binding specificity. Positions of primers used to amplify EMSA probes are indicated in blue. (C) DNaseI footprinting analysis of YhaJ bound to fimS. The protected region is indicated in red with the corresponding YhaJ/LTTR sequence motif indicated. The asterisks denote canonical hypersensitive sites flanking the protected region. (D) T1F phase-switching assay used to demonstrate YhaJ-activated inversion of fimS. The fimS locus contains a single HinfI restriction site, and DNA from cultures of WT UPEC, ΔyhaJ, and +pyhaJ was digested with HinfI and analyzed on 2% agarose. The banding pattern indicates the abundance of the population expressing T1F ON (red) or OFF (gray). WT EHEC which harbors a locked-OFF fimS was used as a control. (E) Quantification of T1F phase-switching assays illustrates the relative percentage of the population expressing fimS ON or OFF. Data represent the mean of 4 biological replicates ±SD.