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oai:figshare.com:article/33998480 2026-09-25T17:32:26Z category_1 category_4 category_915 category_8 category_12 category_13 category_14 category_24 category_61 portal_5 item_type_3 month_year_09_2026

<p>Plasmids used and generated in this study.</p> Abdelraouf O. Dapour (22687088) Moustafa Shehata (25111244) Rania Siam (141257) Nahla A. Hussein (5324402) Biophysics Biochemistry Physical Sciences not elsewhere classified Microbiology Cell Biology Genetics Molecular Biology Evolutionary Biology Developmental Biology sensor histidine kinases partial mantel r k12 mg1655 revealed integrating auxiliary proteins 9 – 2 large periplasmic domain outer membrane lipoprotein mol ), suggesting escherichia coli </ conserved phosphorelay system two transmembrane helices evolutionary coupling independent div >< p response regulator rcsb rcsd periplasmic domain cytosolic domain response regulators rcs phosphorelay rcs ), inner membrane functional coupling evolutionary benefit stable interaction provide insights phosphoryl group induced activation host speciation guided docking functional assays fold derepression fold ). findings demonstrate downstream components component systems capsule synthesis 0001 ). <div><p>Two-component systems (TCS) formed of sensor histidine kinases (HK) and response regulators (RR) are the primary prokaryotic signal transduction pathways. In Enterobacteriaceae, some TCS evolved architectural complexity, by integrating auxiliary proteins into the HK– RR dyad. The Regulator of Capsule Synthesis (Rcs), a conserved phosphorelay system in Enterobacteriaceae, recruits several auxiliary proteins, including an outer membrane lipoprotein, RcsF, which senses cell envelope threats and transduces the signal to downstream components. A second auxiliary protein located in the inner membrane, RcsD, transfers the phosphoryl group from the hybrid histidine kinase RcsC to the response regulator RcsB. RcsD is formed of a large periplasmic domain (Asn43-Asn308) sandwiched between two transmembrane helices and a cytosolic domain. For RcsD, the role and evolutionary benefit of maintaining a large periplasmic domain while the main phosphostransfer reaction occurs in the cytosol is not clear. 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> 2026-09-25T17:32:17Z Dataset Dataset 10.1371/journal.pone.0358890.s007 https://figshare.com/articles/dataset/_p_Plasmids_used_and_generated_in_this_study_p_/33998480 CC BY 4.0