DNA hybridization with the insecticidal crystal protein gene cryllA (formerly cryBl) of Bacillus thuringiensis supspecies kurstaki has shown that subspecies kurstaki contains a cryllA-related sequence in addition to the cryllA gene (Donovan et al., 1988a). We have cloned the cryllA-related sequence and have determined that the sequence, which has been designated cryllB, is 89% identical to the cryllA gene. Recombinant B. thuringiensis cells harbouring the cloned cryllB gene produced very little CryllB protein. A high level of production of the CryllB protein was achieved by fusing the regulatory region of the crylllA crystal protein gene to the cryllB gene. The CryllB protein was found to be highly toxic to Lymantria dispar, Heliothis virescens and Trichoplusia ni, and was not toxic to Aedes aegypti.

A novel strain of Bacillus thuringiensis was isolated from soybean grain dust from Kansas and found to be toxic to larvae of Leptinotarsa decemlineata (Colorado potato beetle). The strain (EG2158) synthesized two parasporal crystals: a rhomboid crystal composed of a 73116 dalton protein of approximately 30 kDa. Plasmid transfer and gene cloning experiments demonstrated that the 73 kDa protein was encoded on an 88 MDa plasmid and that the protein was toxic to the larvae of Colorado potato beetle (CPB). The sequence of the 73 kDa protein, as deduced from the sequence of its gene (cryC), was found to have regions of similarity with several B. thuringiensis crystal proteins: the lepidopteran-toxic P1 proteins of var. kurstaki and berliner, the lepidopteran- and dipteran-toxic P2 (or CRYB1) protein of var. kurstaki, and the dipteran-toxic 130 kDa protein of var. israelensis. While B. megaterium cells harboring the cryC gene from EG2158 synthesized significant amounts of the 73 kDa CRYC protein, Escherichia coli cells did not. The cryC-containing B. megaterium cells produced rhomboid crystals that were toxic to CPB larvae.

A gene encoding a 72,357-dalton (Da) crystal protein of Bacillus thuringiensis var. israelensis was isolated from a native 75-MDa plasmid by the use of a gene-specific oligonucleotide probe. Bacillus megaterium cells harboring the cloned gene (cryD) produced significant amounts of the 72-kDa protein (CryD), and the cells were highly toxic to mosquito larvae. In contrast, cryD-containing Escherichia coli cells did not produce detectable levels of the 72-kDa CryD protein. The sequence of the CryD protein, as deduced from the sequence of the cryD gene, was found to contain regions of homology with two previously described B. thuringiensis crystal proteins: a 73-kDa coleopteran-toxic protein and a 66-kDa lepidopteran-and dipteran-toxic protein of B. thuringiensis subsp. kurstaki. A second gene encoding the B. thuringiensis subsp. israelensis 28-kDa crystal protein was located approximately 1.5 kilobases upstream from and in the opposite orientation to the cryD gene.Certain varieties of Bacillus thuringiensis synthesize parasporal crystals composed of proteins that have been shown to be toxic to the larvae of specific insects. B. thluringiensis subsp. kurstaki as well as other varieties produces a bipyramidal crystal composed of one or more related proteins of approximately 130 kilodaltons (kDa) which are toxic to lepidopterans (caterpillars) (for recent reviews, see references 2 and 34) and also a cuboidal crystal composed of a 66-kDa protein that is toxic to both lepidopteran and dipteran (mosquito, black fly) insects (6,37). Other subspecies of B. thuringiensis have been identified which produce rhomboid crystals composed of a 73-kDa protein that is specifically toxic to coleopteran (beetle) larvae (12, 16; W. P. Donovan, J. M. Gonzalez, Jr., M. P. Gilbert, and C. Dankocsik, Mol. Gen. Genet., in press).B. thuringiensis subsp. israelensis synthesizes an irregularly shaped parasporal crystal that is highly toxic to certain dipteran larvae (8). The complex crystal is composed of at least three major proteins of approximately 130 kDa, 70 kDa, and 28 kDa. The genes for the 130-kDa and the 28-kDa crystal proteins have been cloned and their nucleotide sequences have been reported (1,25,30,31). These cloning experiments have indicated that the 130-kDa and the 28-kDa B. thuringiensis subsp. israelensis crystal proteins are mosquito toxic. However, other researchers have reported that the 28-kDa protein has little or no mosquitocidal activity (4,5,11,14,15,28

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