Figure 2.

Results of bacterial two-hybrid analysis to assess the interactions of VirB10b with variants of VirB6b(1–168). A, bacterial two-hybrid assay measuring β-gal activity in the E. coli indicator strain BTH101 resulting from the interactions of CyaA fusion proteins to VirB10b and VirB6b(1–168) carrying substitutions of the indicated amino acids by alanine or deletions of blocks of six amino acids. Positive control (+), fusions to VirB10b and VirB6b(1–168) WT and negative control; (−), fusion to VirB10b and a strain not expressing an interaction partner. B, analysis of protein levels after SDS-PAGE and Western blotting using anti-CyaA and anti-VirB10b antibodies. Molecular masses of reference proteins are shown on the left (in kilodaltons). Values and standard deviations were calculated from five independent experiments. The p values were obtained by t test, and significant differences were observed between several variants and the WT (****, p < 0.0001).