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A 26-mer oligonucleotide probe was synthesized (based on the determined amino acid sequence of the Nterminus of the Closlridium botulinum type A neurotoxin, BoNT/A) and used in Southern blot analysis to construct a restriction map of the region of the clostridial genome encompassing BoNT/A. The detailed information obtained enabled the cloning of the structural gene as three distinct fragments, none of which were capable of directing the expression of a toxic molecule. The central portion was cloned as a 2-kb PvuII -TuqI fragment and the remaining regions of the light chain and heavy chain as a 2.4-kb Sea1 -TuqI fragment and a 3.4-kb HpaI-f'~vuII fragment, respectively. The nucleotide sequence of all three fragments was determined and an open reading frame identified, composed of 1296 codons corresponding to a polypeptide of 149502 Da. The deduced amino acid sequence exhibited 33% similarity to tetanus toxin, with the most highly conserved regions occurring between the N-termini of the respective heavy chains. Conservation of Cys residues flanking the position at which the toxins are cleaved to yield the heavy chain and light chain allowed the tentative identification of those residues which probably form the disulphide bridges linking the two toxin subfragments.Toxigenic strains of Clostridium botulinum, an anaerobic spore-forming bacteria, produce one or more of seven immunologically distinct neurotoxins. They act primarily at the neuromuscular junction, causing paralysis by inhibiting the release of the neurotransmitter acetylcholine [I]. The botulinum neurotoxins are synthesised as a single polypeptide chain but in their most active form comprise of two asymmetric polypeptide units; a light chain (50-59 kDa) and a heavy chain (85 -105 kDa), linked by at least one disulphide bridge [2].Although botulinum neurotoxins exhibit high degrees of similarity (when compared to each other and related tetanus and diphtheria toxins) in their molecular masses and modes of action, they still remain poorly characterised at the molecular level. Proposals for the mechanism of toxic action implicates three separate functional domains. The light chain has the pharmacological activity, while the N-terminal and C-terminal of the heavy chain mediate channel formation and toxin binding, respectively. These heavy-chain, toxin-binding functional domains have been demonstrated with diphtheria toxin [3 -51 and with tetanus toxin [6 -81. Channel forming activities have been demonstrated for the N-terminus of the heavy chain with C. botulinum type A [9, 101 and there is circumstantial Correspondence to N.

A method is presented for the introduction of plasmids into Clostridium acetobutylicum ATCC 8052 by electroporation. A plasmid shuttle vector, pMTL500E, which contains the erythromycin resistance gene and replication machinery of plasmid pAMβ1, was constructed and introduced into C. acetobutylicum by electroporation. The vector was then used to introduce a 2.2 kb ClaI/SphI chromosomal fragment from C. pasteurianum into a leucine requiring mutant of C. acetobutylicum, SBA9, where complementation of auxotrophy was observed. Plasmid DNA indistinguishable from that introduced, on the basis of agarose gel electrophoresis, was observed in transformants containing either plasmid.

A method is presented for the introduction of plasmids into Clostridium acetobutylicum ATCC 8052 by electroporation. A plasmid shuttle vector, pMTL500E, which contains the erythromycin resistance gene and replication machinery of plasmid pAMβ1, was constructed and introduced into C. acetobutylicum by electroporation. The vector was then used to introduce a 2.2 kb ClaI/SphI chromosomal fragment from C. pasteurianum into a leucine requiring mutant of C. acetobutylicum, SBA9, where complementation of auxotrophy was observed. Plasmid DNA indistinguishable from that introduced, on the basis of agarose gel electrophoresis, was observed in transformants containing either plasmid.

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