BOTR A IS A POSITIVE REGULATOR OF BOTULINUM NEUROTOXIN AND ASSOCIATEDNON-TOXIN PROTEIN GENES IN CLOSTRIDIUM-BOTULINUM-A/

The genes of the botulinum neurotoxin A (BoNT) complex are clustered i n a locus consisting of two divergent polycistronic operons, one conta ining the non-toxic, non-haemagglutinin (NTNH) component and bontA gen es, the other containing the haemagglutinin (HA) component genes. The two operons are separated by a gene (botR/A, previously called orf21) encoding a 21 kDa protein. A recombinant Clostridium botulinum A strai n that overexpresses botR/A was constructed by electroporating strain 62 with the vector pAT19 containing bofR/A under the control of its ow n promoter. The transformed strain produced more BoNT/A and associated non-toxic proteins (ANTPs) and the corresponding mRNAs than the non-t ransformed strain. Partial inhibition of botR/A by antisense mRNA resu lted in lower levels of BoNT/A, NTNH and HA70 and the levels of the co rresponding mRNAs. Gel mobility shift assays and immunoprecipitations showed that BotR/A bound to the DNA promoter region upstream from the two BoNT/A complex operons. These results show that botR/A activated t ranscription of the genes encoding BoNT/A and ANTPs in C. botulinum A by interacting directly with the region promoter, and that the homolog ous genes in C. botulinum B, C and D presumably have the same function .