Neurotoxin gene clusters in Clostridium botulinum type Ab strains

Abstract

There is limited knowledge of the neurotoxin gene diversity among Clostridium botulinum type Ab strains. Only the sequences of the bont/A and bont/B genes in C. botulinum type Ab strain CDC1436 and the sequence of the bont/B gene in C. botulinum type Ab strain CDC588 have been reported. In this study, we sequenced the entire bont/A- and bont/B-associated neurotoxin gene clusters of C. botulinum type Ab strain CDC41370 and the bont/A gene of strain CDC588. In addition, we analyzed the organization of the neurotoxin gene clusters in strains CDC588 and CDC1436. The bont/A nucleotide sequence of strain CDC41370 differed from those of the known bont/A subtypes A1 to A4 by 2 to 7%, and the predicted amino acid sequence differed by 4% to 14%. The bont/B nucleotide sequence in strain CDC41370 showed 99.7% identity to the sequence of subtype B1. The bont/A nucleotide sequence of strain CDC588 was 99.9% identical to that of subtype A1. Although all of the C. botulinum type Ab strains analyzed contained the two sets of neurotoxin clusters, similar to what has been found in other bivalent strains, the intergenic spacing of p21-orfX1 and orfX2-orfX3 varied among these strains. The type Ab strains examined in this study had differences in their toxin gene cluster compositions and bont/A and bont/B nucleotide sequences, suggesting that they may have arisen from separate recombination events.

FIG. 1.

Toxin gene cluster arrangements for BoNT type A-producing strains, including Ab, A(B), and Ba strains.

FIG. 2.

Comparison of bont/A nucleotide sequences. The bont/A genes from the seven Clostridium botulinum strains sequenced in this study and 14 previously reported bont/A sequences were compared. The numbers in the dendrogram represent percent identity values. The four previously described subtypes A1 through A4 are apparent. C. botulinum strain CDC41370 was placed in a separate group, different from the known subtypes.

FIG. 3.

Comparison of bont/B nucleotide sequences. The bont/B genes from the seven Clostridium botulinum strains sequenced in this study and 10 previously reported bont/B sequences were compared. The numbers in the dendrogram represent percent similarity values. The five previously described subtypes B1, B2, B3, nonproteolytic B, and bivalent B are apparent. C. botulinum strain CDC41370 clustered within subtype B1. All other bont/B sequences from bivalent strains clustered together in a separate group.

FIG. 4.

SimPlot comparing the bont/A sequences to the Clostridium botulinum strain CDC41370. The plot shows the similarity of the bont/A sequence of strain CDC41370 to subtypes A1 and A2 and suggests recombination events along the length of this gene.

FIG. 5.

Comparison of bont/A gene clusters. The toxin gene clusters include bont/A, ntnh, p47, p21, orfX1, orfX2, and orfX3. The numbers in the dendrogram represent percent identity values. The nucleotide sequence of the toxin gene cluster of strain CDC41370 was more similar to that of the toxin gene cluster of subtype A2 than to that of the other subtypes.