Fig. 3.

HicB interacts with HicA. a Two hybrid analysis of HicA and HicB interaction. HicB (black squares) and HicA (white squares) were fused to T25 (white arrow) or T18 (grey arrow) catalytic domains of Bordetella pertussis adenylate cyclase both at the N or C terminus of these domains. The whole set of combinations is presented in Fig.S1. For this purpose hicA and hicB open reading frames were amplified by PCR using ToxXba-ToxEco and DopXba-DopEco primers (Table 3). The amplicons cleaved by XbaI and EcoRI were cloned into pKT25, pKNT25, pUT18 and pUT18C, the resulting plasmids are listed in Table 2. After introduction of the different recombinant plasmids into E. coli strain BTH101, β-galactosidase activities were assayed after growth in LB medium containing 1 mM IPTG. Empty vectors pUT18 and pKNT25 were used to determine basal level of β-galactosidase activity and pKT25-Zip and pUT18-Zip as positive control of a high interaction. β-galactosidase activity (Miller units) is indicated, results are the average of three independent experiments. b. SDS-PAGE of both HicA-Strep and HicB-6His. A synthetic operon hicA-strep, hicB-6His was cloned in pET22b(+). E. coli BL21 containing this plasmid was grown in LB medium to an OD_570nm of 0.6, 1 mM IPTG was added, and the growth of cells was carried on three hours after induction. Cells were collected and broken, protein extract was applied to Ni affinity chromatography using imidazole for elution (lane 1) and Strep-Tactin affinity chromatography using desthiobiotin for elution (lane 2). The eluted proteins were resolved on a 12.5% SDS PAGE.