Sequencing the botulinum neurotoxin gene and related genes in Clostridium botulinum type E strains reveals orfx3 and a novel type E neurotoxin subtype

Abstract

Three Clostridium botulinum type E strains were sequenced for the botulinum neurotoxin (BoNT) gene cluster, and 11 type E strains, representing a wide biodiversity, were sequenced for the bont/E gene. The total length of the BoNT/E gene cluster was 12,908 bp, and a novel gene (partial) designated orfx3, together with the complete orfx2 gene, was identified in the three type E strains for the first time. Apart from orfx3, the structure and organization of the neurotoxin gene cluster of the three strains were identical to those of previously published ones. Only minor differences (</=3%) in the nucleotide sequences of the gene cluster components were observed among the three strains and the published BoNT/E-producing clostridia. The orfx3, orfx2, orfx1, and p47 gene sequences of the three type E strains shared homologies of 81%, 67 to 76%, 78 to 79%, and 79 to 85%, respectively, with published sequences for type A1 and A2 C. botulinum. Analysis of bont/E from the 14 type E strains and 19 previously published BoNT/E-producing clostridia revealed six neurotoxin subtypes, with a new distinct subtype consisting of three Finnish isolates alone. The amino acid sequence of the subtype E6 neurotoxin differed 3 to 6% from the other subtypes, suggesting that these subtype E6 neurotoxins may possess specific antigenic or functional properties.

FIG. 1.

Schematic representation of the neurotoxin gene cluster in Clostridium botulinum type E, A1, and A2 strains. The gene orientation is shown by arrows. Only orfx3 in the type E gene cluster is a partial gene. Type A1 and A2 neurotoxin gene clusters are derived from C. botulinum A1 strain NCTC 2916 (GenBank accession numbers AY497357, Y14238, and X52066) and Kyoto-F (accession numbers AY497358, AB004778, X96493, X87974, and X73423).

FIG. 2.

The bont/E genes from 14 Clostridium botulinum type E strains sequenced here and the 19 previously published ones were compared using ClustalW software. Six distinct neurotoxin subtypes were described and designated E1 to E6. C. botulinum strains fell into subtypes E1, E2, E3, and E6, while subtypes E4 and E5 consisted of C. butyricum strains alone. Three Finnish isolates formed a previously unidentified neurotoxin subtype, E6.