<p>Plasmids used and generated in this study.</p>

Here, we show that RcsF and RcsD exhibit strong co-evolutionary coupling independent of host speciation (partial Mantel r = 0.950, p < 0.0001). Structure-guided docking and 500-ns molecular dynamics simulations predicted a stable interaction between RcsF and RcsD (MM-GBSA ΔG ≈ –95 kcal/mol), suggesting their functional coupling. Functional assays in <i>Escherichia coli</i> K12 MG1655 revealed that RcsD periplasmic domain represses basal Rcs activity in unstressed cells (~5-fold derepression) yet is essential for stress-induced activation (1.9–2.5-fold).

Overexpression of <i>rcsF</i> beyond the chromosomal level induces Rcs signal activation only when the RcsD periplasmic domain is present. These findings demonstrate how co-evolution of an auxiliary protein generates novel regulatory function in the Rcs phosphorelay and provide insights into the role of RcsD periplasmic domain in stress signal transduction.</p></div>