The structure of the delta-endotoxin from Bacillus thuringiensis subsp. tenebrionis that is specifically toxic to Coleoptera insects (beetle toxin) has been determined at 2.5 A resolution. It comprises three domains which are, from the N- to C-termini, a seven-helix bundle, a three-sheet domain, and a beta sandwich. The core of the molecule encompassing all the domain interfaces is built from conserved sequence segments of the active delta-endotoxins. Therefore the structure represents the general fold of this family of insecticidal proteins. The bundle of long, hydrophobic and amphipathic helices is equipped for pore formation in the insect membrane, and regions of the three-sheet domain are probably responsible for receptor binding.

SUMMARY Bacillus thuringiensis produces crystalline protein inclusions with insecticidal or nematocidal properties. These crystal (Cry) proteins determine a particular strain's toxicity profile. Transgenic crops expressing one or more recombinant Cry toxins have become agriculturally important. Individual Cry toxins are usually toxic to only a few species within an order, and receptors on midgut epithelial cells have been shown to be critical determinants of Cry specificity. The best characterized of these receptors have been identified for lepidopterans, and two major receptor classes have emerged: the aminopeptidase N (APN) receptors and the cadherin-like receptors. Currently, 38 different APNs have been reported for 12 different lepidopterans. Each APN belongs to one of five groups that have unique structural features and Cry-binding properties. While 17 different APNs have been reported to bind to Cry toxins, only 2 have been shown to mediate toxin susceptibly in vivo. In contrast, several cadherin-like proteins bind to Cry toxins and confer toxin susceptibility in vitro, and disruption of the cadherin gene has been associated with toxin resistance. Nonetheless, only a small subset of the lepidopteran-specific Cry toxins has been shown to interact with cadherin-like proteins. This review analyzes the interactions between Cry toxins and their receptors, focusing on the identification and validation of receptors, the molecular basis for receptor recognition, the role of the receptor in resistant insects, and proposed models to explain the sequence of events at the cell surface by which receptor binding leads to cell death.

A 120 kDa glycoprotein in the larval midgut membrane of the lepidopteran Manduca sexta, previously identified as a putative receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin, has been purified by a combination of protoxin affinity chromatography and anion exchange chromatography. In immunoblotting experiments, the purified glycoprotein has the characteristics predicted of the receptor: it binds CrylA(c) toxin in the presence of GlcNAc but not GalNAc; it binds the lectin SBA; but it does not bind CrylB toxin. N-terminal and internal amino acid sequences obtained from the protein show a high degree of similarity with the enzyme aminopeptidase N (EC 3.4.11.2). When assayed for aminopeptidase activity, purified receptor preparations were enriched 5.3-fold compared to M. sexta brush border membrane vesicles. We propose that the receptor for CrylA(c) toxin in the brush border membrane of the lepidopteran M. sexta is the metalloprotease aminopeptidase N.

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The rate of commitment of bacterial spores to germinate after short exposure to L-alanine increases exponentially from the time of addition of L-alanine. This absence of a lag facilitates kinetic analysis and allows the dependence of commitment on temperature and pH to be determined. The pH profile of commitment has been compared with that obtained from measurements of absorbance decreases during germination, and the two profiles exhibit differing pK values. It is suggested that because the decrease in A600 of spore suspensions is a late event in germination, it is an unsuitable parameter for studying germination-triggering reactions. Commitment has been shown to be temperature-dependent, with an optimum at approx. 37 degrees C and an activation energy (mu) of 1.08 X 10(5) J/mol. The data obtained from the present studies have been used to develop a model for the triggering of germination.