title: Motility and flagellar glycosylation in Clostridium difficile.
abstract: In this study, intact flagellin proteins were purified from strains of Clostridium difficile and analyzed using quadrupole time of flight and linear ion trap mass spectrometers. Top-down studies showed the flagellin proteins to have a mass greater than that predicted from the corresponding gene sequence. These top-down studies revealed marker ions characteristic of glycan modifications. Additionally, diversity in the observed masses of glycan modifications was seen between strains. Electron transfer dissociation mass spectrometry was used to demonstrate that the glycan was attached to the flagellin protein backbone in O linkage via a HexNAc residue in all strains examined. Bioinformatic analysis of C. difficile genomes revealed diversity with respect to glycan biosynthesis gene content within the flagellar biosynthesis locus, likely reflected by the observed flagellar glycan diversity. In C. difficile strain 630, insertional inactivation of a glycosyltransferase gene (CD0240) present in all sequenced genomes resulted in an inability to produce flagellar filaments at the cell surface and only minor amounts of unmodified flagellin protein.
authors.affiliation: Institute for Biological Sciences, National Research Council, 100 Sussex Drive, Ottawa, ON K1A 0R6, Canada.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Twine
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Reid
authors.forename: Annie
authors.initials: A
authors.lastname: Aubry
authors.forename: David R
authors.initials: DR
authors.lastname: McMullin
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Fulton
authors.forename: John
authors.initials: J
authors.lastname: Austin
authors.forename: Susan M
authors.initials: SM
authors.lastname: Logan
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002851:Chromatography, High Pressure Liquid; D016360:Clostridioides difficile / Q000235:genetics / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D005407:Flagella / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D005408:Flagellin / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000502:physiology; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D005408:Flagellin; D016695:Glycosyltransferases