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The genes encoding the toxic determinants of Bacillus sphaericus have been expressed in a nontoxic and a toxic strain of Bacillus thuringiensis subsp. israelensis. In both cases, the B. sphaericus toxin proteins were produced at a high level during sporulation of B. thuringiensis and accumulated as crystalline structures. B. thuringiensis transformants expressing B. sphaericus and B. thuringiensis subsp. israelensis toxins did not show a significant enhancement of toxicity against Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae.

A 135 kDa protein gene and two open reading frames (ORF1 and ORF2) have been cloned from a large plasmid of Bacillus thuringiensis israelensis (Bourgouin et al. 1986). The Escherichia coli recombinant clones containing these genes were highly toxic to larvae of Aedes aegypti, Anopheles stephensi and Culex pipiens. From subcloning experiments it was deduced that the 135 kDa polypeptide alone was responsible for the toxic activity on both A. aegypti and An. stephensi larvae. In contrast, the presence of two polypeptides, the 135 kDa protein and the ORF1 product was required for toxicity to C. pipiens larvae. The minimal toxic fragment of the 135 kDa polypeptide has been delineated. The results indicate that a polypeptide of about 65 kDa, corresponding to an amino-terminal part of the 135 kDa protein is sufficient for toxicity. Sequence comparisons indicate that the ORF1 product may correspond to an N-terminal part of a rearranged 130 kDa protein.

A gene encoding a 125-kilodalton (kDa) mosquitocidal delta-endotoxin was cloned from the 72-MDa resident plasmid of Bacillus thuringiensis subsp. israelensis. This gene is similar in its 3' region to the gene encoding the 135-kDa protein previously cloned (C. Bourgouin, A. Klier, and G. Rapoport, Mol. Gen. Genet. 205:390-397, 1986). Escherichia coli recombinant clones harboring the 125-kDa gene were toxic to larvae of the three mosquito species Aedes aegypti, Anopheles stephensi, and Culex pipiens. In addition, the B. thuringiensis subsp. israelensis DNA fragment carrying the 125-kDa protein gene contains two sets of inverted repeat sequences, identified either by the S1 nuclease method or by electron microscopic observation. The structural organization of inverted repeat sequences and of the 125-kDa gene was analyzed and suggests that this B. thuringiensis subsp. israelensis delta-endotoxin gene is located within a transposable element.

The nucleotide sequence of two repeated sequences (RS) in opposite orientations flanking the 125-kDa toxin gene of Bacillus thuringiensis israelensis (C. Bourgouin et al., J. Bacteriol. 170, 3575-3583, 1988) is reported in this paper. The analysis of these sequences indicates that these two RS display characteristic features of bacterial insertion sequences (IS) and are therefore referred to as IS240. IS240 B is 865 bp long and has two perfect terminal-inverted repeats of 16 bp; IS240 A is 99% identical to IS240 B. A long open reading frame encoding a polypeptide of 235 amino acids spans almost the entire sequence of both IS240 elements. Both the sequence of the inverted repeats and the putative transposases are homologous to IS26 of Proteus vulgaris, IS15-delta of Salmonella panama, IS431 of Staphylococcus aureus, and ISS1 of Streptococcus lactis.

The crystalline parasporal inclusions (crystals) of Bacillus thuringiensis israelensis (Bti), which are specifically toxic to mosquito and black fly larvae, contain three main polypeptides of 28 kDa, 68 kDa and 130 kDa. The genes encoding the 28 kDa protein and the 130 kDa protein have been cloned from a large plasmid of Bti. Escherichia-coli recombinant clones containing the 130 kDa protein gene were highly active against larvae of Aedes aegypti and Culex pipiens, while B. subtilis recombinant cells containing the 28 kDa protein gene were haemolytic for sheep red blood cells. A fragment of the Bti plasmid which is partially homologous to the 130 kDa protein gene was also isolated; it probably corresponds to part of a second type of mosquitocidal toxin gene. Furthermore, restriction enzyme analysis suggested that the 130 kDa protein gene is located on the same Bti EcoRI fragment as another kind of Bti mosquitocidal protein gene cloned by Thorne et al. (1986). Hybridization experiments conducted with the 28 kDa protein gene and the 130 kDa protein gene showed that these two Bti genes are probably present in the plasmid DNA of B. thuringiensis subsp. morrisoni (PG14), which is also highly active against mosquito larvae.