Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition

• Published in: Applied and Environmental Microbiology Vol. 62; no. 5; pp. 1537 - 1543
• Main Authors: Maagd, R.A. de (Centre for Plant Breeding and Reproduction Research, Wageningen, The Netherlands.), Kwa, M.S.G, Klei, H. van der, Yamamoto, T, Schipper, B, Vlak, J.M, Stiekema, W, Bosch, D
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01-05-1996
• Subjects: ADN RECOMBINADO; ADN RECOMBINE; Amino Acid Sequence; Animals; BACILLUS THURINGIENSIS; Bacillus thuringiensis - metabolism; BACTERIA; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacterial Toxins - genetics; Bacterial Toxins - metabolism; Bacteriology; Base Sequence; Biological and medical sciences; Biological control; Centrum voor Plantenveredelings- en Reproduktieonderzoek; CHIMIORECEPTEUR; Control; ENDOTOXINAS; ENDOTOXINE; Endotoxins - genetics; Endotoxins - metabolism; Fundamental and applied biological sciences. Psychology; GENE; GENES; GENIE BIOCHIMIQUE; Hemolysin Proteins; INGENIERIA BIOQUIMICA; INSECTICIDAS; INSECTICIDE; Insects; INTESTIN; INTESTINOS; Laboratorium voor Virologie; Laboratory of Virology; MEMBRANA MUCOSA; Membrane Proteins - metabolism; Microbiology; Molecular Sequence Data; MUQUEUSE; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; PE&RC; Phytopathology. Animal pests. Plant and forest protection; Protein Binding; PROTEINAS; PROTEINAS AGLUTINANTES; PROTEINE; PROTEINE DE LIAISON; Proteins; Protozoa. Invertebrates; QUIMIORECEPTORES; RECOMBINACION; RECOMBINAISON; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Spodoptera - metabolism; Spodoptera - microbiology; SPODOPTERA EXIGUA; TOXICIDAD; TOXICITE; TOXINAS; TOXINE; Entomopathogen; Membrane protein; Toxicity; Insecta; Biological control; Bacillus thuringiensis; Bacillaceae; Endotoxin; Spodoptera exigua; Bacillales; Bacteria; Recombinant protein; Hybrid gene; Bioassay; Gut; Host agent relation; Gene expression; Toxin; Brush border; Biological fixation; Arthropoda; Lepidoptera; Noctuidae; Parasporal body; Invertebrata; Molecular domain; Microbial insecticide
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.62.5.1537-1543.1996

To test our hypothesis that substitution of domain III of Bacillus thuringiensis delta-endotoxin (Cry) proteins might improve toxicity to pest insects, e.g., Spodoptera exigua, in vivo recombination was used to produce a number of cryIA(b)-cryIC hybrid genes. A rapid screening assay was subsequently exploited to select hybrid genes encoding soluble protoxins. Screening of 120 recombinants yielded two different hybrid genes encoding soluble proteins with domains I and II of CryIA(b) and domain III of CryIC. These proteins differed by only, one amino acid residue. Both hybrid protoxins gave a protease-resistant toxin upon in vitro activation by trypsin. Bioassays showed that one of these CgIA(b)-CryIC hybrid proteins (H04) was highly toxic to S. exigua compared with the parental CryIA(b) protein and significantly more toxic than CryIC. In semiquantitative binding studies with biotin-labelled toxins and intact brush border membrane vesicles of S. exigua, this domain III substitution appeared not to affect binding-site specificity. However, binding to a 200-kDa protein by CryIA(b) in preparations of solubilized and blotted brush border membrane vesicle proteins was completely abolished by the domain III substitution. A reciprocal hybrid containing domains I and II of CryIC and domain III of CryIA(b) did bind to the 200-kDa protein, confirming that domain III of CryIA(b) was essential for this reaction. These results show that domain III of CryIC protein plays an important role in the level of toxicity to S. exigua, that substitution of domain III may be a powerful tool to increase the repertoire of available active toxins for pest insects, and that domain III is involved in binding to gut epithelium membrane proteins of S. exigua