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Synthase-dependent secretion systems are a conserved mechanism for producing exopolysaccharides in Gram-negative bacteria. Although widely studied, it is not well understood how these systems are organized to coordinate polymer biosynthesis, modification, and export across both membranes and the peptidoglycan. To investigate how synthase-dependent secretion systems produce polymer at a molecular level, we determined the crystal structure of the AlgK-AlgX (AlgKX) complex involved in Pseudomonas aeruginosa alginate exopolysaccharide acetylation and export. We demonstrate that AlgKX directly binds alginate oligosaccharides and that formation of the complex is vital for polymer production and biofilm attachment. Finally, we propose a structural model for the AlgEKX outer membrane modification and secretion complex. Together, our study provides insight into how alginate biosynthesis proteins coordinate production of a key exopolysaccharide involved in establishing persistent Pseudomonas lung infections.
Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogenPseudomonas aeruginosa, acetylation of alginate reduces opsonic killing by phagocytes and improves reactive oxygen species scavenging. Although it is well-known that alginate acetylation inP. aeruginosarequires AlgI, AlgJ, AlgF, and AlgX, how these proteins coordinate polymer modification at a molecular level remains unclear. Here, we describe the structural characterization of AlgF and its protein interaction network. We characterize direct interactions between AlgF and both AlgJ and AlgXin vitro, and demonstrate an association between AlgF and AlgX, as well as AlgJ and AlgI, inP. aeruginosa. We determine that AlgF does not exhibit acetylesterase activity and is unable to bind to polymannuronatein vitro. Therefore, we propose that AlgF functions to mediate protein-protein interactions between alginate acetylation enzymes, forming the periplasmic AlgJFXK (AlgJ-AlgF-AlgX-AlgK) acetylation and export complex required for robust biofilm formation.
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