Sequence diversity of genes encoding botulinum neurotoxin type F

Abstract

Botulism due to type F botulinum neurotoxin (BoNT/F) is rare (<1% of cases), and only a limited number of clostridial strains producing this toxin type have been isolated. As a result, analysis of the diversity of genes encoding BoNT/F has been challenging. In this study, the entire bont/F nucleotide sequences were determined from 33 type F botulinum toxin-producing clostridial strains isolated from environmental sources and botulism outbreak investigations. We examined proteolytic and nonproteolytic Clostridium botulinum type F strains, bivalent strains, including Bf and Af, and Clostridium baratii type F strains. Phylogenetic analysis revealed that the bont/F genes examined formed 7 subtypes (F1 to F7) and that the nucleotide sequence identities of these subtypes differed by up to 25%. The genes from proteolytic (group I) C. botulinum strains formed subtypes F1 through F5, while the genes from nonproteolytic (group II) C. botulinum strains formed subtype F6. Subtype F7 was composed exclusively of bont/F genes from C. baratii strains. The region of the bont/F5 gene encoding the neurotoxin light chain was found to be highly divergent compared to the other subtypes. Although the bont/F5 nucleotide sequences were found to be identical in strains harboring this gene, the gene located directly upstream (ntnh/F) demonstrated sequence variation among representative strains of this subtype. These results demonstrate that extensive nucleotide diversity exists among genes encoding type F neurotoxins from strains with different phylogenetic backgrounds and from various geographical sources.

FIG. 1.

Phylogenetic analysis of partial 16S rRNA gene nucleotide sequences (∼1.3 kb) from various botulinum toxin-producing clostridial strains. The phylogenetic tree was generated using the neighbor-joining method. Bootstrap values and genetic distance (bar) are shown. Clusters corresponding to different phylogenetic groups are labeled according to previous reports (5, 20). The toxin serotype of each strain is shown in parentheses. An asterisk indicates that the sequence of the specified strain was obtained from GenBank (accession numbers are as follows: KyotoF, X73844; NCTC7272, X68185; NCTC10281, X68172; CDC1656, EF051572; 2740, EF030542; 003-9, EF030537; Schantz, EF030538; 202F, EF030541; 17B, EF030536; ATCC 43181, EF030540; Alaska E43, EF030539; ATCC 43756, X68175).

FIG. 2.

Phylogenetic analysis of bont/F nucleotide sequences. The phylogenetic tree was generated using the neighbor-joining method. Bootstrap values and genetic distance (bar) are shown. Clusters designated subtypes F1 to F7 are labeled on the right. The toxin serotypes of bivalent strains are shown in parentheses. An asterisk indicates that the bont/F sequence of the specified strain was obtained from GenBank (accession numbers for the strains are as follows: CDC3281, Y13631; 202F, M92906; Af84, FJ968748; ATCC 43756, X68262).

FIG. 3.

Similarity plot comparing representative bont/F nucleotide sequences to bont/F5 from strain CDC54074. Sequences from representative strains are compared in the plot as follows: F1, Langeland; F2, CDC4013; F3, CDC54086; F4, CDC54078; F6, VPI7943; F7, CDC59837. Nucleotide sequences were aligned using CLUSTALW, and the plot was rendered with Simplot using a 200-bp window and a 20-bp step.

FIG. 4.

Analysis of the locations and nucleotide sequences of the ntnh gene in representative subtype F5 strains. (A) Phylogenetic tree of the ntnh nucleotide sequences found in monovalent subtype F5 strains (CDC54085 and CDC54090) and bivalent (Af) subtype F5 strains (CDC54079 and CDC54096). The ntnh gene associated with bont/F5 is indicated as “ntnh/F,” and the ntnh gene associated with a bont/A gene is indicated as “ntnh/A.” ntnh/A from the subtype A2 strain Kyoto (accession number CP001581) is shown as a reference. The tree was generated using the neighbor-joining method. Bootstrap values and genetic distance (bar) are shown. (B) Schematic representation of the arrangement of partial toxin gene clusters in monovalent (labeled “F”) and bivalent (labeled “Af”) subtype F5 strains. The locations of primers used to amplify the ntnh genes for sequencing are shown with arrows. (C) Alignment of the 3′ ends of the ntnh/A and ntnh/F nucleotide sequences found in representative subtype F5 strains CDC54079 and CDC54085.