Western blot assay showed that the smcR mutant exhibited a 10-fold decrease in vvpE expression compared to its parental wild type strain during stationary growth. Reintroduction of recombinant smcR restored vvpE expression to the wild type levels. Comparison of the expression levels in the wild type and the rpoS mutant confirmed that vvpE activation by SmcR is RpoS-dependent. Primer extension analysis confirmed that vvpE expression is regulated by SmcR. Additionally, primer extension showed that CRP and SmcR coactivate vvpE expression. Deletion analysis of the vvpE promoter in conjunction with the luxAB reporter assay identified that the CRP and SmcR binding regions extended from -221 to -114 bp. EMSAs confirmed that SmcR and CRP bound specifically to a 200-bp DNA fragment encompassing residues –221 and –114. The precise location of CRP and SmcR binding sites were determined by DNase I footprinting.
Regulated genes for each binding site are displayed below. Gene regulation diagrams show binding sites, positively-regulated genes, negatively-regulated genes, both positively and negatively regulated genes, genes with unspecified type of regulation. For each indvidual site, experimental techniques used to determine the site are also given.
|Site sequence||Regulated genes||Gene diagram||Experimental techniques||TF function||TF type|