In Pseudomonas aeruginosa , the sigma factor AlgU functions as a critical stress-responsive regulator governing essential physiological processes. This study assessed algU -dependent phenotypes through targeted gene knockout, including effects on alginate secretion and host cell invasion. Furthermore, an innovative fusion protein strategy addressed complex preparation challenges, enabling the determination of a high-resolution (2.101 Å) structure of the AlgU-MucA ^cyto complex and revealing an exceptionally low backbone deviation of merely 0.01 Å relative to the reported 6IN7 complex. Structural analysis revealed an extensive mutation-resistant interaction network at the binding interface, stabilized primarily by salt bridges, hydrogen bonds, and hydrophobic interactions. Subsequent mutational validation confirmed that triple or sextuple alanine substitutions at critical interaction sites maintained complex binding integrity while significantly compromising thermal stability, indicating that although dispensable for binding, these residues are indispensable for complex stability. Crucially, pull-down assays identified SspB as the essential adaptor bridging the AlgU-MucA ^cyto complex to the ClpX/ClpP protease; AlphaFold3 modeling and quantitative BLI measurements reveal that SspB engages AlgU via its N-terminal domain and binds ClpX through its disordered C-terminus, enabling substrate delivery and proteolysis-coupled AlgU release. Collectively, these findings elucidate a dual-safeguard regulatory mechanism: inherent mutation resistance at the binding interface prevents aberrant AlgU activation by common clinical mucA mutations, while SspB-mediated proteolysis ensures stimulus-responsive AlgU liberation. This evolutionary adaptation confers resistance against detrimental AlgU overactivation in P. aeruginosa and provides novel therapeutic targets. IMPORTANCE: Deletion of the algU gene affected the production of pyoverdine, pyocyanin, and alginate in Pseudomonas aeruginosa , as well as its cell invasiveness. By fusing AlgU and MucA ^cyto , we were able to obtain an AlgU-MucA ^cyto protein complex with both high yield and purity, and successfully obtained a crystal structure of the AlgU-MucA ^cyto complex. Structural analysis revealed an extensive and robust network of salt bridges, hydrogen bonds, and charge interactions between the two proteins. Multi-site alanine substitutions (up to sextuple mutations) in interfacial charged clusters of the AlgU-MucA ^cyto complex fail to disrupt binding but severely impair thermal stability, revealing that these residues are binding-redundant yet stability-essential. Structural modeling and BLI measurements identify SspB as a dual-interface adaptor: its N-terminal domain recognizes the preassembled AlgU-MucA ^cyto complex (via the AlgU hairpin surface), whereas its flexible C-terminal tail engages ClpX, thereby bridging AlgU-MucA ^cyto to ClpXP and positioning MucA ^cyto for proteolysis-coupled AlgU release.

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