title: Flagellin glycosylation in Paenibacillus alvei CCM 2051T.
abstract: Flagellin glycosylation impacts, in several documented cases, the functionality of bacterial flagella. The basis of flagellin glycosylation has been studied for various Gram-negative bacteria, but less is known about flagellin glycans of Gram-positive bacteria including Paenibacillus alvei, a secondary invader of honeybee colonies diseased with European foulbrood. Paenibacillus alvei CCM 2051(T) swarms vigorously on solidified culture medium, with swarming relying on functional flagella as evidenced by abolished biofilm formation of a non-motile P. alvei mutant defective in the flagellin protein Hag. Here, the glycobiology of the polar P. alvei flagella was investigated. Analysis on purified flagellin demonstrated that the 30-kDa Hag protein (PAV_2c01710) is modified with an O-linked trisaccharide comprised of one hexose and two N-acetyl-hexosamine residues, at three sites of glycosylation. Downstream of the hag gene on the bacterial chromosome, two open reading frames (PAV_2c01630, PAV_2c01640) encoding putative glycosyltransferases were shown to constitute a flagellin glycosylation island. Mutants defective in these genes exhibited altered migration in sodium dodecyl sulfate polyacrylamide gel electrophoresis as well as loss of extracellular flagella production and bacterial motility. This study reveals that flagellin glycosylation in P. alvei is pivotal to flagella formation and bacterial motility in vivo, and simultaneously identifies flagella glycosylation as a second protein O-glycosylation system in this bacterium, in addition to the well-investigated S-layer tyrosine O-glycosylation pathway.
authors.affiliation: Department of NanoBiotechnology, NanoGlycobiology Unit, Universität für Bodenkultur Wien, Muthgasse 11, Vienna A-1190, Austria.
authors.forename: Bettina
authors.initials: B
authors.lastname: Janesch
authors.affiliation: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Potsdam 14424, Germany Institute of Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 22, Berlin 14195, Germany.
authors.forename: Falko
authors.initials: F
authors.lastname: Schirmeister
authors.affiliation: Department of Chemistry, Division of Biochemistry, Universität für Bodenkultur Wien, Muthgasse 18, Vienna A-1190, Austria.
authors.forename: Daniel
authors.initials: D
authors.lastname: Maresch
authors.affiliation: Department of Chemistry, Division of Biochemistry, Universität für Bodenkultur Wien, Muthgasse 18, Vienna A-1190, Austria.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.affiliation: Department of NanoBiotechnology, NanoGlycobiology Unit, Universität für Bodenkultur Wien, Muthgasse 11, Vienna A-1190, Austria.
authors.forename: Paul
authors.initials: P
authors.lastname: Messner
authors.affiliation: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Potsdam 14424, Germany christina.schaeffer@boku.ac.at daniel.kolarich@mpikg.mpg.de.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kolarich
authors.affiliation: Department of NanoBiotechnology, NanoGlycobiology Unit, Universität für Bodenkultur Wien, Muthgasse 11, Vienna A-1190, Austria christina.schaeffer@boku.ac.at daniel.kolarich@mpikg.mpg.de.
authors.forename: Christina
authors.initials: C
authors.lastname: Schäffer
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D005408:Flagellin / Q000378:metabolism*; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D006601:Hexoses / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D056507:Paenibacillus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
chemical_list: D001426:Bacterial Proteins; D006601:Hexoses; D005408:Flagellin; D016695:Glycosyltransferases
keywords: Paenibacillus alvei; flagellum; glycoproteomics; glycosylation island; protein O-glycosylation; self-assembly; trisaccharide