Figure 2
Regions of CpxR interacting with CpxA as monitored by BACTH analysis. Full-length CpxA1-458 was translationally fused to the N-terminus of CyaA225-399 (T18 – magenta shade) giving rise to a ‘bait’ CpxA1-458-T18 hybrid. Full-length CpxR1-232 was translationally fused to the C-terminus of CyaA1-224 (T25 – dark green shade) giving rise to a T25-CpxR1-232 ‘prey’ hybrid. CpxR was also divided into N-terminal (pistachio green), internal linker (orange) and C-terminal (sky blue) domains. Based on these divisions, additional ‘prey’ T25 hybrids were similarly constructed that consisted of only the N-terminus without linker (T25-CpxR1-117) or with linker (T25-CpxR1-132) and the C-terminus without linker (T25-CpxR132-232) or with linker (T25-CpxR117-232). CpxR1-132 was also fused to the N-terminus of T25 giving the CpxR1-132-T25 hybrid. Full-length or near full-length CpxR ‘prey’ T25 constructs were also generated consisting of T25-CpxRD51A or 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. 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 1603.6 ± 146.4 units of β-galactosidase activity/mg dry weight of bacteria. This was equivalent to ~20.2 fold more enzymatic activity produced compared to bacteria co-expressing only T18 and T25 (79.2 ± 6.2 units of β-galactosidase activity). The fold change in enzymatic activity caused by CpxA-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