Figure 6
Regions of CpxR interacting with N-terminal CpxR as monitored by BACTH analysis. The N-terminal domain of CpxR either with linker was translationally fused to the N-terminus of CyaA225-399 (T18 – magenta shade) creating the ‘bait’ CpxR1-132-T18 hybrid. As already used in Figure2, a selection of full-length or near full-length CpxR variants translationally fused to the C-terminus of CyaA1-224 (T25 – dark green shade) were used as ‘prey’ hybrids; that is T25-CpxRD51A and T25-CpxRD51E lacking the phosphorylated aspartate residue as well as T25-CpxRΔ11–24, T25-CpxRΔ117–132 and T25-CpxRΔ188–209 lacking the putative N-terminal coiled-coil, internal linker and C-terminal winged helix-turn-helix regions respectively. We also employed the T25-CpxR1-232 hybrid containing wild type CpxR sequence. BACTH interaction analysis of ‘bait’ and ‘prey’ hybrids was quantified via measurement of β-galactosidase activity and is represented as units/mg dry weight of host E. coli BTH101 bacteria (left column; black font). The internal positive control based upon constructs expressing T18-Zip and T25-Zip yielded 1495.1 ± 237.3 units of β-galactosidase activity/mg dry weight of bacteria. This was on average ~25.6 fold more enzymatic activity produced compared to bacteria co-expressing only T18 and T25 (58.3 ± 14.1 units of β-galactosidase activity). The fold change in enzymatic activity caused by CpxR-CpxR interactions relative to this negative control is indicated in parentheses to the right. The asterisks (*) indicates a positive interaction. Data is presented as the mean (± standard error of the mean) of at least four independent experiments performed in triplicate.