Cloning and expression in Escherichia coli

Recombinant plasmids containing the mosquitocidal δ-endotoxin gene were constructed by inserting HindIII fragments of the Bacillus thuringiensis var. israelensis 72-75 Md plasmid in to the Escherichia coli vector pUC12. Two recombinants producing the 26000 Da δ-endotoxin (pIP173 and pIP174) were identified by screening clones in an E. coli in vitro transcription-translation system. Both recombinants were 12.4 kb chimaeric plasmids comprising pUC12 and a common 9.7 kb HindIII fragment of the B. thuringiensis plasmid. The 26000 Da polypeptide synthesis in vivo from pIP174 transformed into E. coli JM101 was lethal to mosquito larvae and cytotoxic to mosquito cells in vitro. The biological authenticity of the cloned product was further confirmed by demonstrating that the cytotoxicity of the polypeptide was neutralised by antiserum to the authentic δ-endotoxin or by preincubation with excess toxin receptor. Transcription of the recombinant δ-endotoxin gene in E. coli appears to utilise a Bacillus promotor sequence(s) rather than the pUC12 β-galactosidase promotor.</p

Assignment of the δ-endotoxin gene of Bacillus thuringiensis

Plasmid analysis of Bacillus thuringiensis var. israelensis revealed the presence of at least 9 plasmids. A high frequency of plasmid loss occurred when this organism was grown at elevated temperature (42°C). Analysis of over 100 isolates cured of one or more plasmids by this method revealed that loss of a 72 MDa plasmid was invariably accompanied by loss of the ability to synthesize the insecticidal δ-endotoxin protein. Deletion of any of the other plasmids had no effect on δ-endotoxin production. These results indicate the presence of a plasmid-coded copy of the structural gene for the insecticidal δ-endotoxin in B. thuringiensis var. israelensis.</p