FIG 3.
VqsA is involved in aphA expression by binding directly to the promoter of aphA. (A) Western blot analysis of AphA expression in WT, ΔvqsA mutant, and vqsA+ mutant cells cultured in LBS medium for 2 h. RNAP was used as a loading control. The numbers indicate the densitometric measurements. (B) qRT-PCR analysis of the aphA transcripts. The WT, ΔvqsA mutant, and vqsA+ mutant strains were cultured in LBS for 2 h, and mRNA transcripts were detected by qRT-PCR; 16S rRNA was selected as a control. The results are displayed as the mean ± standard deviation (SD) (n = 3), ***, P < 0.001 based on Student's t test. (C) EMSA analysis of the binding of VqsA to PaphA. The amount of VqsA protein used is indicated, and 20 ng of each Cy5-labeled probe was added to the EMSA reactions. The shifts were verified to be specific in experiments in which 25- to 50-fold excess of unlabeled specific DNA and nonspecific competitor DNA [poly(dI:dC)] were used. (D) Plot showing the affinity of the binding of VqsA to the aphA promoter. The densitometric intensities of bound DNA fragments were plotted against VqsA concentrations. (E) DNase I footprinting analysis of the binding of VqsA to a binding site in PaphA. Electropherograms show the PaphA promoter probe treated with DNase I after incubation with 0 or 2.0 μM VqsA. (F) EMSA performed with purified VqsA and the aphA promoter DNA (PaphA) or its variant lacking the specific VqsA-binding site (PaphA ΔVBS). Dashed boxes indicate the binding site revealed by DNase I footprinting assays.