title: Carboxy-terminal extension effects on crystal formation and insecticidal properties of Colorado potato beetle-active Bacillus thuringiensis delta-endotoxins.
abstract: Many Bacillus thuringiensis crystal proteins, particularly those active against lepidopteran insects, have carboxy-terminal extensions that mediate bipyramidal crystal formation. These crystals are only soluble at high (>10.0) pH in reducing conditions such as generally found in the lepidopteran midgut. Most of the Colorado potato beetle (CPB)-active toxins lack such an extension, yet some toxins with a carboxy-terminal extension have cryptic activity against this insect, revealed only after in vitro solubilization. Crystal formation, morphology, protein content, and activity against CPB were compared for two sets of proteins, the Cry1-hybrid SN19 and Cry3Aa, both with and without a carboxy-terminal extension. Cry3Aa, with or without extension, formed flat square or rectangular crystals. SN19 (with extension) and its derivative without extension formed irregular inclusion bodies. All Cry3Aa and SN19 crystals and inclusion bodies were almost equally active before and after in vitro presolubilization and could be solubilized in diluted CPB midgut extract. In contrast, bipyramidal crystals of Cry1Ba were insoluble under these conditions. Our results suggest that bipyramidal crystal formation typical for proteins with a carboxy-terminal extension may preclude activity against CPB, but that interfering with this crystal formation can increase the activity.
authors.affiliation: Department of Plant Physiology and Molecular Biology, University of Plovdiv, Plovdiv, Bulgaria.
authors.forename: Samir
authors.initials: S
authors.lastname: Naimov
authors.forename: Elena
authors.initials: E
authors.lastname: Martens-Uzunova
authors.forename: Mieke
authors.initials: M
authors.lastname: Weemen-Hendriks
authors.forename: Stefan
authors.initials: S
authors.lastname: Dukiandjiev
authors.forename: Ivan
authors.initials: I
authors.lastname: Minkov
authors.forename: Ruud A
authors.initials: RA
authors.lastname: de Maagd
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry* / Q000235:genetics; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity* / Q000648:ultrastructure; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D001517:Coleoptera / Q000187:drug effects* / Q000254:growth & development; D003460:Crystallization; D004731:Endotoxins / Q000235:genetics / Q000633:toxicity*; D004926:Escherichia coli / Q000235:genetics; D006460:Hemolysin Proteins; D002479:Inclusion Bodies / Q000378:metabolism / Q000648:ultrastructure; D007306:Insecticides / Q000737:chemistry / Q000633:toxicity*; D008855:Microscopy, Electron, Scanning; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D011993:Recombinant Fusion Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis