Clostridium botulinum Strains Producing BoNT/F4 or BoNT/F5

Abstract

ABSTRACTBotulinum neurotoxin type F (BoNT/F) may be produced byClostridium botulinumalone or in combination with another toxin type such as BoNT/A or BoNT/B. Type F neurotoxin gene sequences have been further classified into seven toxin subtypes. Recently, the genome sequence of one strain ofC. botulinum(Af84) was shown to contain three neurotoxin genes (bont/F4,bont/F5, andbont/A2). In this study, eight strains containingbont/F4and seven strains containingbont/F5were examined. Culture supernatants produced by these strains were incubated with BoNT/F-specific peptide substrates. Cleavage products of these peptides were subjected to mass spectral analysis, allowing detection of the BoNT/F subtypes present in the culture supernatants. PCR analysis demonstrated that a plasmid-specific marker (PL-6) was observed only among strains containingbont/F5. Among these strains, Southern hybridization revealed the presence of an approximately 242-kb plasmid harboringbont/F5. Genome sequencing of four of these strains revealed that the genomic backgrounds of strains harboring eitherbont/F4orbont/F5are diverse. None of the strains analyzed in this study were shown to produce BoNT/F4 and BoNT/F5 simultaneously, suggesting that strain Af84 is unusual. Finally, these data support a role for the mobility of abont/F5-carrying plasmid among strains of diverse genomic backgrounds.