Bacterial adenylate cyclase two hybrid assay (BACTH):

For the bacterial adenylate cyclase two hybrid assay (BACTH), an adenylate cyclase mutant strain (BTH101) or a derivative if this strain was used. The proteins whose interactions were being examined were cloned into the T18 and T25 portions of adenylate cyclase [34, 35]. The reconstitution of adenylate cyclase upon their interaction allows production of cAMP, which interacts with CRP to activate the lac operon, assayed as beta-galactosidase activity. In the absence of interaction, T18 and T25 do not get reconstituted to form a functional adenylate cyclase and the strain does not express beta-galactosidase. The proteins used in this study were fused at their C-terminal to the Cya fragments. Plasmids expressing IgaA or RcsD as T18/T25 fusion constructs were cotransformed into BTH101, plated on LB-agar medium containing 100 μg/ml ampicillin and 50 μg/ml kanamycin and incubated at 30°C for 2 days. To measure the beta-galactosidase activity, the resulting colonies were inoculated and grown overnight in LB medium containing 100 μg/ml ampicillin, 50 μg/ml kanamycin, and 0.5 mM IPTG at 30°C. The beta-galactosidase assay was performed in 96-well plates. The activity was analysed by measuring the kinetics of ONPG degradation by monitoring the OD 420 nm at 28°C for 40 min at intervals of 1 min in a Tecan Spark microplate reader. The beta-galactosidase activity was calculated using the slope of OD 420 divided by their OD 600. Each protein fusion paired with the cognate vector produces very little activity and these were used as negative controls. Every graph is compiled from at least 3 separate sets of assays, and the beta-galactosidase activity is plotted relative to the IgaA/RcsD interaction in that particular experiment.

To test the effect of DsbA activity on the various IgaA/RcsD constructs, the BACTH assays were carried out in BTH101 and a dsbA mutant derivative of BTH101 (AP58). To test the effect of DjlA on the IgaA/RcsD interactions, djlA or mutants of djlA were cloned downstream of IgaA under the control of the same promoter. The assay was carried out in BTH101 or its derivatives EAW1, EAW2, and EAW4 as above and interactions plotted relative to the IgaA/RcsD interaction in WT, set to 1.