Heterologous expression of a mutated toxin gene from Bacillus thuringiensis subsp. tenebrionis

Using oligonucleotide probes we have isolated a DNA fragment encoding an insecticidal toxin of the coleopteran specific Bacillus thuringiensis subsp. tenebrionis. The gene was altered by site directed mutagenesis at its 5'-end and adapted for general cloning and expression purposes with a linker including a start codon and new restriction sites. The constructs were inserted into several vector plasmids and expressed in Escherichia coli. Expression E. coli was strongly enhanced by the lac-promoter. A fusion protein with phage MS2-polymerase was produced together with a 67 kDa protein also found for normal expression of the toxin gene. Synthesis of the latter protein indicated a second ribosome binding site at the 5'-terminus of the toxin encoding sequence. Toxin-containing proteins were identified by Western blot analysis. The positive cell extracts from E. coli had insecticidal activity on larvae of the Colorado potato beetle. The cloned gene is not homologous to a gene previously cloned by us whose gene products were also toxic to coleopteran larvae.