title: Flagellin diversity in Clostridium botulinum groups I and II: a new strategy for strain identification.
abstract: Strains of Clostridium botulinum are traditionally identified by botulinum neurotoxin type; however, identification of an additional target for typing would improve differentiation. Isolation of flagellar filaments and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that C. botulinum produced multiple flagellin proteins. Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis of in-gel tryptic digests identified peptides in all flagellin bands that matched two homologous tandem flagellin genes identified in the C. botulinum Hall A genome. Designated flaA1 and flaA2, these open reading frames encode the major structural flagellins of C. botulinum. Colony PCR and sequencing of flaA1/A2 variable regions classified 80 environmental and clinical strains into group I or group II and clustered isolates into 12 flagellar types. Flagellar type was distinct from neurotoxin type, and epidemiologically related isolates clustered together. Sequencing a larger PCR product, obtained during amplification of flaA1/A2 from type E strain Bennett identified a second flagellin gene, flaB. LC-MS analysis confirmed that flaB encoded a large type E-specific flagellin protein, and the predicted molecular mass for FlaB matched that observed by SDS-PAGE. In contrast, the molecular mass of FlaA was 2 to 12 kDa larger than the mass predicted by the flaA1/A2 sequence of a given strain, suggesting that FlaA is posttranslationally modified. While identification of FlaB, and the observation by SDS-PAGE of different masses of the FlaA proteins, showed the flagellin proteins of C. botulinum to be diverse, the presence of the flaA1/A2 gene in all strains examined facilitates single locus sequence typing of C. botulinum using the flagellin variable region.
authors.affiliation: Bureau of Microbial Hazards, HFPB, Health Canada, Sir Frederick G. Banting Research Centre, Ottawa, Ontario, Canada.
authors.forename: Catherine J
authors.initials: CJ
authors.lastname: Paul
authors.forename: Susan M
authors.initials: SM
authors.lastname: Twine
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Tam
authors.forename: James A
authors.initials: JA
authors.lastname: Mullen
authors.forename: John F
authors.initials: JF
authors.lastname: Kelly
authors.forename: John W
authors.initials: JW
authors.lastname: Austin
authors.forename: Susan M
authors.initials: SM
authors.lastname: Logan
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002853:Chromatography, Liquid; D003014:Clostridium botulinum / Q000235:genetics* / Q000648:ultrastructure; D016000:Cluster Analysis; D019295:Computational Biology; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005408:Flagellin / Q000235:genetics*; D014644:Genetic Variation; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D017931:DNA Primers; D005408:Flagellin