Bacterial two-hybrid analyses.

Bacterial adenylate cyclase two-hybrid (BACTH) assays were performed using E. coli BTH101 cells based on the system first described by Karimova et al. (46). Briefly, for each assay, BTH101 cells were freshly cotransformed with ∼100 ng of each BACTH assay plasmid. The transformations were incubated on LB agar supplemented with 50 μg/ml kanamycin, 50 μg/ml carbenicillin, and 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 40 h at 30°C. After this incubation period, β-galactosidase activity was quantified in Miller units using the protocol described in references 54 and 55).