title: Structural basis for alginate secretion across the bacterial outer membrane.
abstract: Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate. Secretion of newly synthesized alginate across the outer membrane is believed to occur through the outer membrane protein AlgE. Here we report the 2.3 Å crystal structure of AlgE, which reveals a monomeric 18-stranded β-barrel characterized by a highly electropositive pore constriction formed by an arginine-rich conduit that likely acts as a selectivity filter for the negatively charged alginate polymer. Interestingly, the pore constriction is occluded on either side by extracellular loop L2 and an unusually long periplasmic loop, T8. In halide efflux assays, deletion of loop T8 (ΔT8-AlgE) resulted in a threefold increase in anion flux compared to the wild-type or ΔL2-AlgE supporting the idea that AlgE forms a transport pathway through the membrane and suggesting that transport is regulated by T8. This model is further supported by in vivo experiments showing that complementation of an algE deletion mutant with ΔT8-AlgE impairs alginate production. Taken together, these studies support a mechanism for exopolysaccharide export across the outer membrane that is distinct from the Wza-mediated translocation observed in canonical capsular polysaccharide export systems.
authors.affiliation: Molecular Structure and Function, The Hospital for Sick Children, Toronto, ON, Canada M5G 1X8.
authors.forename: John C
authors.initials: JC
authors.lastname: Whitney
authors.forename: Iain D
authors.initials: ID
authors.lastname: Hay
authors.forename: Canhui
authors.initials: C
authors.lastname: Li
authors.forename: Paul D W
authors.initials: PD
authors.lastname: Eckford
authors.forename: Howard
authors.initials: H
authors.lastname: Robinson
authors.forename: Maria F
authors.initials: MF
authors.lastname: Amaya
authors.forename: Lynn F
authors.initials: LF
authors.lastname: Wood
authors.forename: Dennis E
authors.initials: DE
authors.lastname: Ohman
authors.forename: Christine E
authors.initials: CE
authors.lastname: Bear
authors.forename: Bernd H
authors.initials: BH
authors.lastname: Rehm
authors.forename: P Lynne
authors.initials: PL
authors.lastname: Howell
mesh_terms: D000464:Alginates; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D017124:Conserved Sequence; D020723:Glucuronic Acid / Q000378:metabolism; D006603:Hexuronic Acids; D008958:Models, Molecular; D019897:Periplasm / Q000378:metabolism; D018583:Pliability; D011134:Polysaccharides / Q000378:metabolism; D018272:Porins / Q000378:metabolism; D016062:Porosity; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D000464:Alginates; D001426:Bacterial Proteins; D006603:Hexuronic Acids; D011134:Polysaccharides; D018272:Porins; D020723:Glucuronic Acid