FIGURE 1.

ToxS and ompU operator binding sites are key players in ToxR PPIs in E. coli. PPIs of the indicated ToxR and ToxS‐FLAG variants were tested in E. coli W3110 ∆cyaA using a bacterial cAMP‐based two‐hybrid system (BACTH), which is based on the functional complementation of the adenylate cyclase CyaA (Karimova et al., 1998). Strains are N‐terminal ToxR, ToxR^W76R and ToxS‐FLAG translational fusions linked to the C‐termini of the B. pertussis CyaA T18 or T25 domains with or without V. cholerae ompU operator fragments (op^ompU), and co‐expressed ToxS (a, b). The leucine zipper of the yeast GCN4 protein (zip) was used as a positive complementation control (+), while the empty plasmids pKT25 and pUT18C served as negative controls (‐) (e, f). For the drop test (c, d, e), strains were grown in LB overnight and subsequently transferred to a single MacConkey maltose indicator plate to reveal the CyaA⁺ phenotype (red colonies indicate the utilization of maltose as a C‐source). PPIs are shown between the indicated ToxR or ToxR^W76R translational fusions (designated as X) in the presence or absence of co‐expressed ToxS and ompU operator binding sites (op^ompU) (c). Panel (d) displays PPIs between ToxR and ToxS‐FLAG (designated as Y). Panel (f) shows quantifications of functional complementation between the indicated ToxR, ToxR^W76R and ToxS‐FLAG hybrid proteins (white bars) in dependence of ompU operator binding sites (op^ompU, lined white bars) by measuring β‐galactosidase activities. The cells were grown in LB supplemented with 0.05 mM IPTG to the stationary phase. Strains in which ToxR and ToxR^W76R PPIs were measured in the presence of co‐expressed ToxS were labeled with ToxRS or ToxR^W76RS. Strains in which interactions between ToxR and ToxS‐FLAG were analyzed were labeled with ToxR + ToxS‐FLAG. The positive and negative controls are represented by black bars. The values are means of three biological replicates, each with technical triplicates with error bars, which represent the standard deviation. Interactions are reported as Miller Units. The asterisks indicate significantly different means with p < .05 for the respective columns, each tested against E. coli W3110 ∆cyaA pKT25‐ToxR pUT18C‐ToxR or pKT25‐ToxR^W76R put18C‐ToxR^W76R using one‐way ANOVA test with Bonferroni post hoc analysis [Colour figure can be viewed at wileyonlinelibrary.com]