Regulation of Botulinum Neurotoxin Expression

Botulinal neurotoxin continues to be a concern in food safety. The molecular biology of botulinal neurotoxin gene expression in Clostridium botuli-num is poorly understood. In this study, the transcriptional and translational kinetics of expression of type A botulinal neurotoxin by C. botulinum type A strains Hall A, 62A, and NCTC 2916 were determined during 96 hours of growth. Strains were grown in TPGY and type A Toxin Production Media (TPM). mRNA transcript levels encoding the proteins of the neurotoxin complex were determined using Northern analyses. Neurotoxin concentrations in culture supernatants and lysed cell pellets were determined using ELISA, Western blots, and mouse bioassay. For all three strains, mRNA transcripts were initially detected in early log phase, reached peak levels in stationary phase, and rapidly decreased when the cells began to lyse. Toxin expression varied dependent on the strain and growth medium. For C. botulinum strain Hall A, 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 of growth. For strain 62A, cell lysis and toxin release occurred to a greater extent in TPM compared to TPGY. For strain NCTC 2916, cell lysis occurred to a greater extent in TPM, but toxin release was greater in TPGY. Toxin production was highest in strain NCTC 2916, followed by Hall A and 62A in TPGY. In contrast, toxin production was highest in strain Hall A, followed by NCTC 2916 and 62A in TPM. Proteolytic activation of toxin occurred to a greater extent in cultures grown in TPM compared to TPGY. Mouse toxicity of culture supernatants was also greater when strains were grown in TPM compared to TPGY. These data together with other findings in our laboratory indicate that toxin regulation in C. botulinum type A is a complex process involving genes affecting positive and negative promoter regulation, cell lysis, proteolytic activation of toxin, and lysis of cells with concomitant release of toxin into the media. Further study should lead to a greater understanding of the ability of C. botulinum to grow and produce neurotoxins in foods.