qRT-PCR measured plcH transcription to determine that it is positively regulated by GbdP. Deletion mapping identified the region necessary for GbdR-dependent regulation. LacZ fusion assays were used to analyze plcH promoter activity, demonstrating that the gbdR knockout mutant did not display induction. GFP assays were used to analyze the activity of the pchP promoter, showing that induction did not occur in the absence of GbdR. EMSA confirmed GbdR binding to the plcH and pchP promoters. Alignment of the plcH and pchP promoters identified the sequences necessary for GbdR regulation and binding. Site-directed mutagenesis was then used to further test the putative binding regions, demonstrating that the site was valid. Incubation of P. aeruginosa in mice lungs demonstrated GbdR regulation in-vivo and elucidating the subject of nosocomial P. aeruginosa infection in cystic fibrosis patients.
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|