Figure 1
BACTH analysis of CpxA-CpxA interactions . Full-length CpxA1-458 was translationally fused to the N-terminus of CyaA1-224 (T25 – dark green shade) creating a CpxA1-458-T25 hybrid used as the ‘bait’. Full-length CpxA1-458 was also translationally fused to the N-terminus of CyaA225-399 (T18 – magenta shade) giving rise to a ‘prey’ CpxA1-458-T18 hybrid. Based on CpxA divisions into sensor input (CpxA1-156, cadet blue shade), HAMP – signal transmission (CpxA157-240, soft yellow shade), Dhp – dimerization and histidine phosphorylation (CpxA241-310, metallic gold shade) and histidine kinase catalysis (CpxA311-458, grey shade) domains, an additional six ‘prey’ CpxA-T18 hybrids were constructed; CpxA1-156-T18, CpxA1-240-T18, CpxA1-310-T18, CpxA157-310-T18, CpxA187-458-T18 and CpxA311-458-T18. 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). As an internal positive control, we used the provided constructs expressing T18-Zip and T25-Zip that yielded 1547.0 ± 121.2 units of β-galactosidase activity /mg dry weight of bacteria. This was equivalent to ~83.8 fold more enzymatic activity produced compared to bacteria co-expressing only T18 and T25 (18.5 ± 3.9 units of β-galactosidase activity). The fold change in enzymatic activity caused by CpxA-CpxA interactions relative to this negative control is indicated in parentheses to the right. A level of β-galactosidase activity at least 3-fold higher than the negative control was considered to indicate a positive interaction (*). Data is presented as the mean (± standard error of the mean) of at least four independent experiments performed in triplicate.