Analysis of type A botulinum neurotoxin expression in Clostridium botulinum

The kinetics of botulinum toxin gene expression were investigated in C. botulinum type A strains 62A and Hall A-hyper, and type A(B) strain NCTC 2916 during the growth cycle. The analyses were performed in TPGY medium and type A Toxin Production Medium (TPM). For all three strains, mRNA transcripts for the toxin complex genes were initially detected in early log phase, reached peak levels in early stationary phase, and rapidly decreased in mid-to-late stationary phase. Toxin production was highest in strain Hall A-hyper, followed by NCTC 2916 and 62A. For C. botulinum strain Hall A-hyper, cell lysis and toxin release into the supernatant occurred rapidly for cells grown in TPM, while cells grown in TPGY remained in stationary phase with minimal lysis and toxin release through 96 hours. In contrast, strains 62A and NCTC 2916 lysed more extensively than Hall A-hyper in TPGY. DNA sequence analysis and comparison of the BoNT/A gene clusters of strains 62A and Hall A-hyper revealed only two nucleotide differences between the two strains. This suggests that factors leading to different levels of toxin production by the two strains most likely lie outside of the BoNT/A cluster. Botulinum toxin synthesis and activation appears to be a complex process that is highly regulated by nutritional and environmental conditions.; In additional work, the DNA sequence of the upstream region of the botulinum neurotoxin type A1 (BoNT/A1) cluster of strain NCTC 2916 and the BoNT/A2 cluster of strain Kyoto-F was determined. A novel gene, designated orfx3, was identified following the orfx2 gene in both clusters. The BoNT/A1 and BoNT/A2 clusters share a similar gene arrangement, but exhibit differences in the spacing between certain genes. Sequences with similarity to transposases were identified in these intergenic regions, suggesting that these differences arose from an ancestral insertion event. Transcriptional analysis of the BoNT/A2 cluster revealed that the genes of the cluster are primarily transcribed as three polycistronic transcripts. These findings are essential for future analyses of BoNT/A2 complex composition and BoNT/A2 cluster gene expression.