Insertion site of the transposon in YB3558 and effect on CtrA abundance. A) Location of the insertion site relative to the P1 and P2 promoters of ctrA and sequence of the wild-type ctrA P2 promoter and the mutated ctrAP2::Mn promoter. Shaded boxes indicate CtrA recognition sequence half sites. Transcription start site is indicated . Triangle indicates site of transposon insertion. Transposon sequence is underlined. B) Expression of wild-type (pctrA290) and mutant (pctrAP2::Mn) ctrA promoters in wild-type and YB3558 strains. β-galactosidase assays were performed on exponentially growing cultures as described in the Methods (absorbance measurements for this experiment were carried out on a Nanodrop 2000 (Thermo Scientific) with a 0.1 mm light path and therefore activities are not Miller Units, and instead have been labeled β-galactosidase Activity). The mutant promoter displays a large reduction in activity compared to the wild-type promoter in both CB15 and YB3558. The wild-type promoter displays an expression level in YB3558 similar to that in CB15. C) Western Analysis of CtrA abundance in YB3558 and YB3559. Western blot analysis was conducted on an equal OD600 of each strain. Blots were probed with α-CtrA primary antibody and HRP-conjugated goat anti-rabbit secondary antibody (Biorad) and the signal detected with the Supersignal Pico substrate (Pierce). YB3558 demonstrates significantly reduced CtrA abundance, while complementation with ctrA under native control (YB3559) restores CtrA to near wild-type levels.
Curtis et al. BMC Microbiology 2013 13:166 doi:10.1186/1471-2180-13-166