Flagellin glycosylation in Pseudomonas aeruginosa PAK requires the O-antigen biosynthesis enzyme WbpO.
Pseudomonas aeruginosa PAK (serotype O6) produces a single polar, glycosylated flagellum composed of a-type flagellin. To determine whether or not flagellin glycosylation in this serotype requires O-antigen genes, flagellin was isolated from the wild type, three O-antigen-deficient mutants wbpL, wbpO, and wbpP, and a wbpO mutant complemented with a plasmid containing a wild-type copy of wbpO. Flagellin from the wbpO mutant was smaller (42 kDa) than that of the wild type (45 kDa), or other mutants strains, and exhibited an altered isoelectric point (pI 4.8) when compared with PAK flagellin (pI 4.6). These differences were because of the truncation of the glycan moiety in the wbpO-flagellin. Thus, flagellin glycosylation in P. aeruginosa PAK apparently requires a functional WbpO but not WbpP. Because WbpP was previously proposed to catalyze a metabolic step in the biosynthesis of B-band O-antigen that precedes the action of WbpO, these results prompted us to reevaluate the two-step pathway catalyzed by WbpO and WbpP. Results from WbpO-WbpP-coupled enzymatic assays showed that either WbpO or WbpP is capable of initiating the two-step pathway; however, the kinetic parameters favored the WbpO reaction to occur first, converting UDP-N-acetyl-D-glucosamine to UDP-N-acetyl-D-glucuronic acid prior to the conversion to UDP-N-acetyl-D-galacturonic acid by WbpP. This is the first report to show that a C4 epimerase could utilize UDP-N-acetylhexuronic acid as a substrate.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1.
Wayne L
WL
Miller
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1.
Mauricia J
MJ
Matewish
Institute for Biological Sciences, National Research Council, Ottawa, Ontario K1A 0R6, Canada.
David J
DJ
McNally
Departments of Biochemistry and Microbiology and Immunology, McGill University, Montreal, Quebec H3A 1A4.
Noboru
N
Ishiyama
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1.
Erin M
EM
Anderson
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1.
Dyanne
D
Brewer
Institute for Biological Sciences, National Research Council, Ottawa, Ontario K1A 0R6, Canada.
Jean-Robert
JR
Brisson
Departments of Biochemistry and Microbiology and Immunology, McGill University, Montreal, Quebec H3A 1A4.
Albert M
AM
Berghuis
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1. Electronic address: jlam@uoguelph.ca.
Joseph S
JS
Lam
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000502:physiology*; D001426:Bacterial Proteins / Q000378:metabolism*; D002384:Catalysis; D005408:Flagellin / Q000737:chemistry* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D006031:Glycosylation; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D019081:O Antigens / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D019998:Racemases and Epimerases / Q000737:chemistry; D014535:Uridine Diphosphate Glucuronic Acid / Q000737:chemistry; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000737:chemistry
D001426:Bacterial Proteins; D019081:O Antigens; D011134:Polysaccharides; D005408:Flagellin; D014535:Uridine Diphosphate Glucuronic Acid; D014537:Uridine Diphosphate N-Acetylglucosamine; D000429:Alcohol Oxidoreductases; D019998:Racemases and Epimerases