Flagellin diversity in Group I and Group II Clostridium botulinum: a new strategy for strain identification

Bureau of Microbial Hazards, HFPB, Health Canada, Sir Frederick G. Banting Research Centre, PL: 2204A2, Ottawa, Ontario, Canada, K1A 0L2; Institute for Biological Sciences, National Research Council, 100 Sussex Dr. Ottawa, Ontario, Canada, K1A 0R6

^* To whom correspondence should be addressed. Email: john_austin{at}hc-sc.gc.ca.

	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 SDS-PAGE showed that C. botulinum produced multiple flagellin proteins. LC-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 weight for FlaB matched that observed by SDS-PAGE. In contrast, the molecular weight of FlaA was 2-12 kilodaltons (kDa) larger than the mass predicted by the flaA1/A2 sequence of a given strain, suggesting 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.