Novel Clostridium botulinum Toxin Gene Arrangement with Subtype A5 and Partial Subtype B3 Botulinum Neurotoxin Genes

Botulinum neurotoxin, the most poisonous substance known and a potential biothreat agent (1), causes human and animal botulism worldwide (4, 9). This toxin is encoded by the bont gene as part of a cluster that includes nontoxic accessory genes (10). Two main gene clusters are known: the hemagglutinin-containing (ha+) cluster in toxin serotypes A1, B, C, D, and G and the orfX cluster in toxin serotypes A, E, and F (7, 10). In addition, some Clostridium botulinum type A strains (e.g., NCTC2916) contain a silent type B gene in their chromosome, designated A(B). The silent B toxin genes described to date contain the full length of the active type B gene cluster and one or more mutations that result in an unexpressed type B neurotoxin (3, 8).

Here we report the first infant botulism case resulting from a rare bont/A5 subtype, which we unexpectedly found to be part of a novel neurotoxin type A/B gene arrangement. Our bont/A5 gene sequence differs by 2 bp from the bont/A5 sequence previously identified in four wound botulism cases (2). The California infant strain, designated IBCA94-0216, was characterized by sequencing the toxin complex genes and their flanking regions (GenBank accession number FJ959094). Sequence analysis confirmed that the bont/A5 is located within a hemagglutinin complex (ha+) that contains the genes ha70, ha17, ha33, botR, ntnh, and bont/A5 (Fig. 1). This type of toxin cluster is commonly associated with strains bont/A1 and bont/B but is not found in strains bont/A2, bont/A3, and bont/A4 (7, 10). The IBCA94-0216 neurotoxin gene cluster has an unexpected 76-bp deletion between the genes ha33 and botR. This is the first example of such a deletion.

FIG. 1.

Arrangement of the IBCA94-0216 botulism neurotoxin gene cluster. The IBCA94-0216 partial bont/B sequence begins at what would be base pair position 1362 in the intact type B neurotoxin gene, and it continues downstream for an additional 2,514 bp to the 3′ end of the neurotoxin gene. The missing 5′ 1,361 bp of the bont/B gene (which would code for the neurotoxin light chain), as well as any second cluster of accessory genes, could not be found by extensive PCR testing. The prime symbol designates a partial gene.

Downstream from bont/A5 are two copies of transposases belonging to the is3 family, a structure identical to the downstream flanking sequence of the type A1 Hall neurotoxin gene cluster (Fig. 1) (10). Interestingly, 2,041 bp downstream from the bont/A5 gene and immediately following the second is3 is a partial bont/B gene that is only 65% of a full-length bont/B (6). The partial bont/B gene lacks the nucleotide sequence coding for the light chain of the toxin and begins at nucleotide 1362 of the 3,876-nucleotide full-length toxin gene. Comparisons of this region (1,362 to 3,876 nucleotides) revealed it is most similar to the rare bont/B3 subtype (5). The entire IBCA94-0216 bont/A/B cluster is flanked by a 5′ flagellin gene and a 3′ transposase, as is found in the silent type B gene cluster of strain NCTC2916 (7).

Nucleic acid pairwise identities (using the Kimura two-parameter method in MEGA4 software) of the toxin cluster genes show that the IBCA94-0216 A5 neurotoxin and ntnh genes are most similar to the type A1 Hall neurotoxin and ntnh genes, respectively, while the hemagglutinin genes are most similar to the hemagglutinin genes of type B strains. This mosaic gene arrangement and the presence of the partial type B neurotoxin gene lead us to speculate that some form of genetic rearrangement occurred between type A and type B neurotoxin gene clusters in the creation of the novel type A/B neurotoxin gene arrangement found in strain IBCA94-0216.