Figure 5
N-terminal CpxR dimerization in BACTH assays is enhanced by inclusion of the internal CpxR linker. The N-terminal domain of CpxR either with linker (A) or without linker (B) were translationally fused to the N-terminus of CyaA225-399 (T18 – magenta shade) creating the ‘bait’ CpxR1-132-T18 and CpxR1-117-T18 hybrids respectively. As ‘prey’ hybrids, the CpxR N-terminus without linker (CpxR1-117-T25) or with linker (CpxR1-132-T25) and the C-terminus without linker (CpxR132-232-T25) or with linker (CpxR117-232-T25) were fused to the N-terminus of CyaA1-224 (T25 – dark green). Interactions between ‘bait’ and ‘prey’ hybrids were again quantified via measurement of β-galactosidase activity (left columns; black font). Measurement of the interaction between T18-Zip and T25-Zip yielded 1449.1 ± 113.2 units of β-galactosidase activity was ~17.3 fold more than the enzymatic activity produced by negative-control bacteria co-expressing only T18 and T25 (83.8 ± 16.3 β-galactosidase activity units). 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.