Cloning and characterization of the upstream region of Clostridium botulinum type B neurotoxin gene.

The upstream region of the gene coding for Clostridium botulinum type B (BoNT/B) neurotoxin was cloned and sequenced. There were two open reading frames, which were identified as a nontoxic-nonhemagglutinin component (ntnh/B) and a 22 kDa adjacent open reading frame (orf22/B). Deduced primary structure of ntnh/B showed that it was composed of 1,197 amino acid residues. Pairwise comparisons of the ntnh/B component with other botulinum toxin types showed high degree of homology to ntnh/A (82% identity). Northern blot analysis revealed that toxin gene could be transcribed alone or co-transcribed with the ntnh gene. The orf22/B gene encoding for 178 amino acids (M.W. 21.6 kDa) was located between the 33 kDa hemagglutinin gene and the ntnh gene. Orf22/B also showed high degree of homology to orf22/A (98.9% identity). These results suggested that the upstream region of the BoNT/B gene (containing the ntnh/B and orf22/B genes) might be evolutionarily closely related to the counterparts of the BoNT/A.