We have analysed the genes borne on a 6.0 kb Hind111 fragment cloned from the chromosome of Clostridium botulinum type E strain Mashike. This fragment, cloned within plasmid pU9EMH, contains part of the structural gene for botulinum toxin type E neurotoxin as well as the entire structural gene for a nontoxic component of botulinum type E progenitor neurotoxin gene, ent-120. ent-120 is transcribed in the same direction as the neurotoxin gene and consists of one open reading frame encoding 1162 amino acid residues. Western blotting with anti-nontoxic component sera demonstrates that ent-120 encodes a protein of 120 kDa which forms part of the nontoxic component. ent-120 is homologous to an analogous gene found in botulinum type C strains (69.3% identity at the nucleotide level and 56.1 % at the amino acid level). Two stretches of amino acids at the N-terminus of the ent-120 protein are highly homologous to amino acid sequences within the type E neurotoxin. The stop codon of the ent-120 gene is situated 27 nucleotides upstream from the start codon of the neurotoxin gene.
Chromosomal DNA was extracted from toxigenic Clostridum butyricum strain BL6340 isolated from a case of infant botulism. After digestion by EcoRI, a DNA fragment of about 1 kbp was cloned into Escherichia coli using Agtll, and was subcloned into pUC118. The E coli cells transformed with this cloned fragment produced a 33 kDa protein which reacted with monoclonal antibodies recognizing the light chain (Lc) component of botulinum type E toxin. The nucleotide sequence of the cloned fragment was determined. The sequence was similar to that from botulinum type E toxin gene fragments previously determined by our laboratory (strains Mashike, Otaru and Iwanai). Several highly homologous sequences among the botulinum type A, C, E, butyricum and tetanus toxin genes were found in both translated and untranslated regions. These results suggest that the toxin gene of C. butyricum may have evolved by transfer from C. botuI.um.
Chromosomal DNAs were extracted from Clostridium butyricum strain BL6340 and Clostridium botulinum type E strain Mashike. The 6.0 Kbp fragment coding for the entire light chain (L) component and the N-terminus of heavy chain (H) component of botulinum type E toxin was obtained from each extracted DNAs after digestion with HindIII. The entire nucleotide sequences for the light chain components of these cloned genes were determined, and the derived amino acid sequences were compared to each other, and with those of botulinum type A, Cl, D, and tetanus toxins reported previously. The cleavage site of L and H components of type E toxin was presumed to be Arg-422. In a total of 422 amino acid residues of L component, 17 residues were different between butyricum and type E toxins, and all these differences were found within 200 residues of N-terminus of L component. On the contrary, five regions showing highly homologous sequences were found in L components among these six toxins, and one more region between botulinum type E and tetanus toxins.Clostridium butyricum and Clostridium baratii strains which produce botulinum type E and type F neurotoxins, respectively, were isolated from human infant botulism (7, 10). Type E neurotoxin is produced as a single polypeptide chain, and nicked by protease (trypsin) to separate into two fragments, designated light chain (L; Mr, 50,000) and heavy chain (H; Mr, 100,000), following reduction of a disulfide bond connecting these fragments. The toxin inhibits acetylcholine release from nerve endings, but its detailed mechanisms are still unknown. It is postulated that the heavy chain which consists of the C-terminus of the toxin correlated with the binding of toxin to peripheral synapses, and the light chain which consists of the N-terminus of the toxin associated with the activity to block acetylcholine release.
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