title: Characterization of the Pseudomonas aeruginosa Glycoside Hydrolase PslG Reveals That Its Levels Are Critical for Psl Polysaccharide Biosynthesis and Biofilm Formation.
abstract: A key component of colonization, biofilm formation, and protection of the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharide Psl. Composed of a pentameric repeating unit of mannose, glucose, and rhamnose, the biosynthesis of Psl is proposed to occur via a Wzx/Wzy-dependent mechanism. Previous genetic studies have shown that the putative glycoside hydrolase PslG is essential for Psl biosynthesis. To understand the function of this protein, the apo-structure of the periplasmic domain of PslG (PslG(31-442)) and its complex with mannose were determined to 2.0 and 1.9 Å resolution, respectively. Despite a domain architecture and positioning of catalytic residues similar to those of other family 39 glycoside hydrolases, PslG(31-442) exhibits a unique 32-Å-long active site groove that is distinct from other structurally characterized family members. PslG formed a complex with two mannose monosaccharides in this groove, consistent with binding data obtained from intrinsic tryptophan fluorescence. PslG was able to catalyze the hydrolysis of surface-associated Psl, and this activity was abolished in a E165Q/E276Q double catalytic variant. Surprisingly, P. aeruginosa variants with these chromosomal mutations as well as a pslG deletion mutant were still capable of forming Psl biofilms. However, overexpression of PslG in a pslG deletion background impaired biofilm formation and resulted in less surface-associated Psl, suggesting that regulation of this enzyme is important during polysaccharide biosynthesis.
authors.affiliation: Program in Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
authors.forename: Perrin
authors.initials: P
authors.lastname: Baker
authors.affiliation: Program in Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada; Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Whitfield
authors.affiliation: Division of Infectious Disease, Center for Microbial Interface Biology, Ohio State University, Columbus, Ohio 43210.
authors.forename: Preston J
authors.initials: PJ
authors.lastname: Hill
authors.affiliation: Program in Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada; Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Dustin J
authors.initials: DJ
authors.lastname: Little
authors.affiliation: Division of Infectious Disease, Center for Microbial Interface Biology, Ohio State University, Columbus, Ohio 43210.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Pestrak
authors.affiliation: Photon Sciences Division, Brookhaven National Laboratory, Upton, New York 11973-5000.
authors.forename: Howard
authors.initials: H
authors.lastname: Robinson
authors.affiliation: Division of Infectious Disease, Center for Microbial Interface Biology, Ohio State University, Columbus, Ohio 43210. Electronic address: daniel.wozniak@osumc.edu.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Wozniak
authors.affiliation: Program in Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada; Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: P Lynne
authors.initials: PL
authors.lastname: Howell
mesh_terms: D018441:Biofilms; D002240:Carbohydrate Sequence; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000096:biosynthesis*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D011134:Polysaccharides; D006026:Glycoside Hydrolases
keywords: Psl; PslG; bacterial genetics; biofilm; crystal structure; exopolysaccharide biosynthesis; glycoside hydrolase; polysaccharide