Figure 7: Analysis of IgaA/RcsD interactions.

A) RcsD T411A can repress signaling by RcsD-binding defective IgaA mutants. The IgaA alleles are in the chromosome in a ΔrcsD strain as in 3F. These strains were transformed with either pBAD24 vector, rcsD (pEAW11), RcsD T411A (pEAW11T), or T411A del peri (pAP1102) and their fluorescence assayed as earlier. T411A lowers signaling by tightening cytoplasmic interactions. B) Interaction of IgaA cyt1 mutants with RcsD. Interaction of IgaA and its cyt1 mutants with RcsD, RcsD del peri, RcsD T411A del peri was tested using BACTH. C) Signaling by IgaA cytoplasmic mutant strains. The fluorescence of IgaA mutant strains was assayed as described in 3F. D) Interaction of RcsD cytoplasmic mutants with IgaA. The interaction of RcsD and its mutants was tested with IgaA using BACTH in BTH101. E) Signaling by RcsD cytoplasmic mutants with altered IgaA interaction. The ΔrcsD strain (EAW19) was transformed with either pBAD24 vector or RcsD mutants and their fluorescence assayed as in Fig. 4E. F) Updated model for Rcs phosphorelay signaling cascade. RcsF and OMPs such as BamA, sense disruptions in the outer membrane or peptidoglycan layer. The inner membrane protein IgaA receives the stress cues from RcsF, which weakens the repressive interaction between IgaA and RcsD. The signal is then transmitted to RcsC/RcsD, leading to the autophosphorylation of RcsC. RcsC then relays the phosphate to RcsD. The phosphate is then transferred to RcsB, resulting in activation of gene expression (see discussion for more details). The cytoplasmic domains of RcsD and RcsC were modeled using AlphaFold3.