Physical Characterization of Clostridium Botulinum Neurotoxin Genes

DNA fragments encompassing the neurotoxin genes of Clostridium botulinum types B and E have been cloned and their entire nucleotide sequences determined. Similarly, recombinant clones carrying all but the extreme 51 end of the type F gene have obtained and analyzed by nucleotide sequencing. The current sequence does not encompass the first 20, and terminal 60, codons of the structural gene, and is not fully authenticated with regard to PCR-induced errors. Attempts to clone the type G gene were unsuccessful. A total of 20 different strains of Clostridium sporogenes were examined as potential hosts for expression of toxin gene subfragments. In no case was DNA transfer demonstrated, either by electrotransformation or by conjugative mobilization. Attention was switched to employing Clostridium acetobutylicum NCIB 8052 as the recombinant host and efforts focused on obtaining regulated expression of a previously constructed fac promoter. Some progress towards this aim was made by devising a strategy whereby heterologous DNA may be forced to integrate into the C. acetobutylicum chromosome. Clostridium botulinum Neurotoxin genes Gene cloning Nucleotide sequencing DNA transfer Gene expression RA l gene