DNA affinity purification using the exoA and lctO intergenic region was used to pull down a binding protein, identified by MALDI-TOF mass spectrometry as ccpA. A consensus search for catabolite responsive elements (CRE’s) identified two putative binding sites on the lctO probe (narrowed to one by further probe affinity analysis) and one each on the cfa and speB promoters. qRT-PCR was performed on growth phase- and ccpA-variable cultures; lctO and cfa shared similar expression patterns, repressed in exponential phase and derepressed in stationary phase in the wild type, while ccpA mutants demonstrated loss of growth-regulated repression. SpeB transcription was upregulated in stationary phase for both mutant and wild type, though mutant strains showed decreased transcription in both phases, suggesting that – though ccpA is a speB activator – it is not a growth phase regulator for that gene. DNA affinity confirmed specific binding of ccpA to the speB promoter. qRT-PCR of mouse lesions from ccpA-variable strains confirmed earlier expression data.
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|