Figure 3: Mutations in IgaA/RcsF interface impact binding and signaling.

A-B) Detailed views of IgaA_Kp/RcsF_Kp interaction interface and the residues involved in binding. IgaA_Kp is shown in grey, RcsF_Kp in gold and Fab in purple. Note that in the assays in this figure and supplemental figures, experiments are all done with E. coli mutants and thus the E. coli numbering is used (1 residue less than K. pneumoniae numbering for both RcsF and IgaA). C) Interaction of RcsF mutants with IgaA. The interaction of E. coli IgaA-T18 with RcsF^MalF-T25 or RcsF^MalF point mutants was tested in the strains BTH 101 and BTH101 ΔrcsD (EAW12). The interaction of IgaA with RcsF^MalF is set to 100 and all other interactions are plotted relative to this. The amino acid residue numbers are for E. coli IgaA and RcsF and hence the numbering differs by 1 from those of K. pneumoniae above. D) Signaling by RcsF mutants in ΔrcsF. RcsF^MalF-T25 and its point mutants (used in Fig. 3C) were overexpressed in ΔrcsF (AP52) and the strain grown in MOPS glycerol minimal medium containing IPTG. The rprA-mCherry fluorescence signal at OD 0.4 is depicted here and the signal of pKNT25 vector (V) was set to 1. E) BACTH for IgaA mutants defective in RcsF interaction. The interaction of RcsF^MalF-T25 tested with IgaA-T18 and IgaA point mutants is plotted here. All interactions are plotted relative to the RcsF^MalF-IgaA interaction (set to 100). F) Signaling by RcsF-binding defective IgaA mutants. The igaA alleles are in the chromosome in a ΔrcsD strain carrying a P_rpra:mCherry reporter fusion. These strains were transformed with either pBAD24 vector (devoid of RcsD) or pEAW11 (rcsD⁺) and their fluorescence assayed during growth in MOPS minimal glucose medium (containing 100 g/ml ampicillin). PMBN (20 μg/ml) was added in the medium at the beginning for Rcs activation. The RFU at OD 0.4 for each strain relative to the uninduced igaA⁺ rcsD⁺ strain (set to 1) is plotted.