Fig. 5.
Binding of PmrA to the ssrB promoter is required for PmrA-mediated repression of the SPI-2 ssaG gene. (A) Electrophoretic mobility shift assays of wild-type or mutated ssrB promoter region DNA fragments using purified PmrA-His6 protein (0, 75, 150, 300, 600, and 1,200 pmoles) were carried out as described in Materials and Methods. The shifted bands can be competed out by the corresponding cold probe (unlabeled DNA) in the presence of the maximum amount of PmrA-His6 protein used. (B) Fluorescence from a plasmid-linked ssaG-gfp transcriptional fusion was determined in wild-type, pmrA mutant, the ssrB promoter mutant (pssrBmu), and pmrA ssrB promoter double mutant (pmrA pssrBmu) Salmonella grown in N-minimal medium at pH 4.6 with 1 mM Mg2+. (C) J774A.1 macrophages were infected with wild-type, pmrA mutant, ssrB mutant, and the ssrB promoter mutant (pssrBmu) strains. mRNA levels of the ssrB, ssaG, and pmrC genes were determined at the indicated times after infection.