Flagellin diversity in Clostridium botulinum groups I and II: a new strategy for strain identification: Appl.Environ.Microbiol.

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TypeArticle
Journal titleAppl.Environ.Microbiol.
Volume73
Issue9
Pages29632975; # of pages: 13
SubjectAmino Acid Sequence; analysis; BAND; Base Sequence; Canada; Chromatography,Liquid; Clostridium; Clostridium botulinum; Cluster Analysis; Computational Biology; DNA; DNA Primers; Electrophoresis; Electrophoresis,Polyacrylamide Gel; Flagellin; FRAME; GENE; Genes; genetics; Genome; IDENTIFICATION; ISOLATION; mass spectrometry; MASS-SPECTROMETRY; Microscopy,Electron,Transmission; MOLECULAR; Molecular Sequence Data; Multiple; Open Reading Frames; peptide; Peptides; Phylogeny; protein; Proteins; REGION; SEQUENCE; Sequence Analysis,DNA; Sodium; Species Specificity; STRAIN; STRAINS; STRUCTURAL; Tandem Mass Spectrometry; TARGET; ultrastructure; Variation (Genetics)
AbstractStrains 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
Publication date
LanguageEnglish
AffiliationNRC Institute for Biological Sciences; National Research Council Canada
Peer reviewedNo
NRC numberPAUL2007A
NPARC number9382893
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Record identifierf3a7dd58-e1b9-4ad7-b678-1eee38c24a49
Record created2009-07-10
Record modified2016-05-09
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