FIG 7.

The predicted RpoE promoter is required for RpoE-dependent regulation of rpoH. (A) The predicted −10 and −35 regions of the two tandem RpoE-dependent promoters as well as the transcriptional and translational start sites are shown. In order to demonstrate that these promoter regions were functional, we mutagenized nucleotides within each −35 region. The mutagenized nucleotides are indicated in bold. (B) Effect of site-directed mutagenesis on promoter activity measured in the GFP reporter assay. Plasmids containing the wild-type and mutagenized putative rpoH promoter regions were electroporated into 35000HP(pDG17). Whole-cell lysates were prepared at 0 (lane 1), 60 (lane 2), and 120 (lane 3) minutes after RpoE induction and probed with an anti-GFP monoclonal antibody. PAL detected with the monoclonal antibody 3B9 served as a loading control. Putative promoter 1 and putative promoter 2, reporters with nucleotide substitutions in each putative promoter; control, reporter containing the wild-type putative rpoH promoter region. (C) Densitometry analysis of the Western blots from panel B. The data represent the means ± SD from three independent experiments. *, P < 0.05; **, P < 0.01.