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Two genes encoding insecticidal crystal proteins from Bacillus thuringiensis subsp. kurstaki HD-1 were cloned and sequenced. Both genes, designated cryBi and cryB2, encode polypeptides of 633 amino acids having a molecular mass of ca. 71 kilodaltons (kDa). Despite the fact that these two proteins display 87% identity in amino acid sequence, they exhibit different toxin specificities. The cryBI gene product is toxic to both dipteran (Aedes aegypti) and lepidopteran (Manduca sexta) larvae, whereas the cryB2 gene product is toxic only to the latter. DNA sequence analysis indicates that cryBI is the distal gene of an operon which is comprised of three open reading frames (designated orfi, orJ2, and cryBi). The proteins encoded by cryBi and orJ2 are components of small cuboidal crystals found in several subspecies and strains of B. thuringiensis; it is not known whether the orfl or cryB2 gene products are present in cuboidal crystals. The protein encoded by orJ2has an electrophoretic mobility corresponding to a molecular mass of ca. 50 kDa, although the gene has a coding capacity for a polypeptide of ca. 29 kDa. Examination of the deduced amino acid sequence for this protein reveals an unusual structure which may account for its aberrant electrophoretic mobility: it contains a 15-amino-acid motif repeated 11 times in tandem. Escherichia coli extracts prepared from cells expressing only orfi and orJ2 are not toxic to either test insect.The common soil bacterium Bacillus thuringiensis synthesizes proteinaceous crystalline inclusions that are lethal to a variety of insects. Several subspecies have been identified to date, and their host ranges can vary substantially. By far the greatest number produce large bipyramidal crystals which are toxic only to lepidopteran larvae (reviewed in reference 38). These crystals consist of one or more related protoxin polypeptides having a molecular mass of 130 to 140 kilodaltons (kDa); toxins of ca. 68 kDa are derived from the protoxins by proteolytic processing in the larval gut. Some strains that are lethal to lepidopterans also contain small cuboidal crystals (28,40). Bacillus thuringiensis subsp. kurstaki HD-1 is such a strain: it produces bipyramidal crystals composed of two or three protoxin polypeptides encoded by homologous crystal protein genes (16, 17) and cuboidal crystals containing a ca. 65-kDa polypeptide ("P2 toxin"; 41) which is toxic to both lepidopteran and dipteran larvae.We have cloned and sequenced four genes, designated cryBI, cryB2, orfl, and orf2, from a 225-kilobase (kb) plasmid doublet from B. thuringiensis subsp. klirstaki HD-1.Three of these genes (of1, orJ2, and cryBI) are part of an operon; ciyB2 is located separately. Based on immunoblot analyses and toxicity data, cuboidal crystals contain the cryBi and orJ2 gene products; we do not know whether the orfl and cryB2 gene products are present in cuboidal crystals. Interestingly, although the ciyBl and cryB2 genes are closely related, their gene products exhibit different host range specificities. The cryBl...

A classification for crystal protein genes of Bacillus thuringiensis is presented. Criteria used are the insecticidal spectra and the amino acid sequences of the encoded proteins. Fourteen genes are distinguished, encoding proteins active against either Lepidoptera (cryI), Lepidoptera and Diptera (cryII), Coleoptera (cryIII), or Diptera (cryIV). One gene, cytA, encodes a general cytolytic protein and shows no structural similarities with the other genes. Toxicity studies with single purified proteins demonstrated that every described crystal protein is characterized by a highly specific, and sometimes very restricted, insect host spectrum. Comparison of the deduced amino acid sequences reveals sequence elements which are conserved for Cry proteins. The expression of crystal protein genes is affected by a number of factors. Recently, two distinct sigma subunits regulating transcription during different stages of sporulation have been identified, as well as a protein regulating the expression of a crystal protein at a posttranslational level. Studies on the biochemical mechanisms of toxicity suggest that B. thuringiensis crystal proteins induce the formation of pores in membranes of susceptible cells. In vitro binding studies with radiolabeled toxins demonstrated a strong correlation between the specificity of B. thuringiensis toxins and the interaction with specific binding sites on the insect midgut epithelium. The expression of B. thuringiensis crystal proteins in plant-associated microorganisms and in transgenic plants has been reported. These approaches are potentially powerful strategies for the protection of agriculturally important crops against insect damage.

Sau 3A1 partial digestion fragments from Bacillus thuringiensis var. kurstali HD-1 plasmid DNA were ligated into the BamHI site ofthe cloning vector pBR322 and transformed into Escherichia coli strain HB101. Colonies presumed to contain recombinant plasmids were screened for production of an antigen that would react with antibody made against B. thuringiensis crystals. One strain, ES12, was isolated by using this procedure. ES12 contains a plasmid of Mr 11 x 106 that has DNA sequence homology with pBR322 as well as with Mr 30 X 106 and Mr 47 X 106 plasmids of B. thuringiensis. It makes a protein antigen, detected by antibodies to crystal, which has the same electrophoretic mobility as the B. thuringiensis crystal protein. Protein extracts of ES12 are toxic to larvae of the tobacco hornworm Manduca sexta.