Figure 2

Bacterial two-hybrid analysis of protein-protein interactions involving VipA and VipB. (A) Contact between VipB and wild-type or mutant VipA, fused to Zif and to the ω subunit of E. coli RNAP respectively, induces transcription from the lacZ promoter of the E. coli reporter strain KDZif1ΔZ, resulting in β-galactosidase activity. As a positive control, MglA-Zif and SspA-ω was used while the negative control corresponds to empty vectors. Shown is the mean β-galactosidase activity ± standard deviation in Miller units produced from 3 independent experiments where two independent transformants were tested on each occasion. Data was subjected to a student’s 2-sided t-test to determine whether the β-galactosidase activity produced by a VipA mutant was significantly different from that of wild-type VipA (*, P < 0.05; ***, P < 0.001). (B) To determine levels of VipA mutants (upper panel) or VipB (lower panel), B2H cultures were pelleted and equivalent amounts were separated by SDS-PAGE and analyzed by Western blot using polyclonal antibodies recognizing VipA or VipB. The experiment was repeated twice.