Binding studies were performed with two '251-labeled Bacillus thuringiensis -endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One S-endotoxin, Bt2-protoxin, is a 130-kDa recombinant crystalline protein from B. thuringiensis subsp. berliner. It kills larvae of both insect species. The active Bt2-toxin is a 60-kDa proteolytic fragment of the Bt2-protoxin. It binds saturably and with high affinity to brush border membrane vesicles from the midgut of both species. The other 6-endotoxin, Bt4412-protoxin, is a 136-kDa crystalline protein from B. thuringiensis subsp. thuringiensis, which is highly toxic for P. brassicae, but not for M. sexta larvae. Bt4412-toxin, obtained after proteolytic activation of Bt4412-protoxin, shows high-affinity saturable binding to P. brassicae vesicles but not to M. sexta vesicles. The correlation between toxicity and specific binding is further strengthened by competition studies. Other B. thuringiensis 6-endotoxins active against M. sexta compete for binding of 125I-labeled Bt2-toxin to M. sexta vesicles, whereas toxins active against dipteran or coleopteran larvae do not compete. Bt2-toxin and Bt4412-toxin bind to different sites on P. brassicae vesicles.Bacillus thuringiensis produces crystalline parasporal inclusions containing insecticidal proteins called 8-endotoxins. Most S-endotoxins are protoxins, which are proteolytically activated in the insect midgut to smaller active toxins (1). 8-Endotoxins produced by different B. thuringiensis strains may exhibit different insecticidal spectra. Toxins active toward lepidopteran, dipteran, or coleopteran larvae have been described (2-4). Among &endotoxins specific for Lepidoptera, marked differences exist in the relative levels of toxicity toward different species of this order (5-7).Several factors such as the solubilization and proteolytic activation of the crystals in the insect midgut (5, 8) and the presence of specific cell membrane receptors for different 8-endotoxins (9,10) MATERIALS AND METHODSInsect Toxicity Assays. Insect toxicity assays on M. sexta and P. brassicae have been described (12). M. sexta was reared on an artificial diet (13); P. brassicae was reared on fresh cabbage leaves (Brassica oleracea var. gemnifera D.C.).B. thuringiensis 6-Endotoxins. Cloning of bt2, the gene encoding the 130-kDa 8-endotoxin (Bt2-protoxin) from B. thuringiensis subsp. berliner strain 1715, purification of recombinant Bt2-protoxin from Escherichia coli, and generation of the toxic 60-kDa tryptic fragment (Bt2-toxin) has been described by Hofte et al. (12). For further purification Bt2-toxin was precipitated in (NH4)2SO4 (70%) and was then dissolved in Tris buffer (20 mM Tris HCl/200 mM NaCl, pH 8.65) with 5% (vol/vol) glycerol. Streptomycin sulfate was added to a concentration of 0.2% to remove contaminating nucleic acids. The solution was allowed to stand for 60 min at 4°C and the precipitate was spun down in a Sorvall SS34 rot...

A plasmid-encoded crystal protein gene (bt2) has been cloned from Bacillus thuringiensis berliner 1715. In Escherichia coli, it directs the synthesis of the 130-kDa protein (Bt2) which is toxic to larvae of Pieris brassicae and Manduca sexta. Comparison of the deduced amino acid sequence of this Bt2 protein with the B. thuringiensis kurstaki HD1 Dipel, B. thuringiensis kurstaki HD73 and B. thuringiensis sotto crystal protein sequences suggests that homologous recombination between the different genes has occurred during evolution.Treatment of the Bt2 protein with trypsin or chymotrypsin yields a 60-kDa protease-resistant and fully toxic polypeptide. The minimal portion of the Bt2 protein required for toxicity has been determined by analysing the polypeptides produced by deletion derivatives of the bt2 gene. It coincides with the 60-kDa protease-resistant Bt2 fragment and it starts between amino acids 29 and 35 at the N-terminus and terminates between positions 599 and 607 at the C-terminus.

We have investigated the protein composition and the insecticidal spectrum of crystals of 29 Bacillus thuringiensis strains active against lepidopteran larvae. All crystals contained proteins of 130 to 140 kilodaltons (kDa) which could be grouped into three types by the molecular weight of the protoxin and the trypsin-activated core fragment. Proteins of the three types showed a characteristic insecticidal spectrum when tested against five lepidopteran species. Type A crystal proteins were protoxins of 130 or 133 kDa, which were processed into 60-kDa toxins by trypsin. Several genes encoding crystal proteins of this type have been cloned and sequenced earlier. They are highly conserved in the N-terminal half of the toxic fragment and were previously classified in three subtypes (the 4.5-, 5.3-, and 6.6-kilobase subtypes) based on the restriction map of their genes. The present study shows that different proteins of these three subtypes were equally toxic against Manduca sexta and Pieris brassicae and had no detectable activity against Spodoptera littoralis. However, the 4.5-, 5.3-, and 6.6-kilobase subtypes differed in their toxicity against Heliothis virescens and Mamestra brassicae. Type B crystal proteins consisted of 140-kDa protoxins with a 55-kDa tryptic core fragment. These were only active against one of the five insect species tested (P. brassicae). The protoxin and the trypsin-activated toxin of type C were 135-and 63-kDa proteins, respectively. Proteins of this type were associated with high toxicity against S. littoralis and M. brassicae. A panel of 35 monoclonal antibodies was used to compare the structural characteristics of crystal proteins of the three different types and subtypes. Each type of protein could be associated with a typical epitope structure, indicating an unambiguous correlation between antigenic structure and insect specificity.

No abstract

A crystal protein gene (ht2) has been cioned from plasmid DNA of Bacllus thuringiensis (B.t.) Lerliner 1715 and directs the synthesis of a 130 kd protein (Bt2) in E. ccli which is toxic to iarvae of P(cris brasciccc and Manduca sa.cta. Treatment of the Bt2 prote.t. with trypsin or chymotrypsin yields a 60 kd protease resistont fragment which is fuily toxic tards insect iarvae " in vivo" and nsect celi lines in vitro". The minimai portion of the 3t2 proein required for toxicity has been rnapped by deletion analysis and coincides with the 60 kd protease resistant Bt2.-fraqment. Tobacco piant celis have been transformed with chiineric toxin genes uing a Ti plasmid vector. Transformed plarits express a functionai toxin and exhibit resistance against insect larvas. iNTRODUCTION Bacilius rniencic (3. . ) is a gram positivebacteriun which produces ondogenous crystais upon sporulation. The crys tais are composed of protein and are specificaliy toxic against Recebido em 03/09/87