Characterization of Cry34/Cry35 Binary Insecticidal Proteins from Diverse Bacillus thuringiensis Strain Collections

• Published in: Applied and Environmental Microbiology Vol. 71; no. 4; pp. 1765 - 1774
• Main Authors: Schnepf, H. Ernest, Lee, Stacey, Dojillo, JoAnna, Burmeister, Paula, Fencil, Kristin, Morera, Lisa, Nygaard, Linda, Narva, Kenneth E, Wolt, Jeff D
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.04.2005
• Subjects: Amino Acid Sequence; Animals; Bacillus thuringiensis; Bacillus thuringiensis - classification; Bacillus thuringiensis - genetics; bacterial insecticides; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacterial Toxins - chemistry; Bacterial Toxins - genetics; Bacterial Toxins - metabolism; bioassays; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biopesticides; Biotechnology; Cloning, Molecular; Coleoptera - growth & development; cry34 gene; Cry34 protein; cry35 gene; Cry35 protein; crystal proteins; Diabrotica virgifera virgifera; DNA, Bacterial - analysis; Endotoxins - chemistry; Endotoxins - genetics; Endotoxins - metabolism; entomopathogenic bacteria; Fundamental and applied biological sciences. Psychology; genes; geographical variation; Hemolysin Proteins; insecticidal properties; insecticidal proteins; Invertebrate Microbiology; Microbiology; Miscellaneous; Mission oriented research; Molecular Sequence Data; nucleotide sequences; Operon; Pest Control, Biological; Polymerase Chain Reaction - methods; Quantitative genetics; Sequence Analysis, DNA; sequence homology; strains; Characterization; Toxin; Bacillales; Gene; Bacillus thuringiensis; Bacteria; Bacillaceae; Biopesticide; Microbial insecticide; Protein
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.71.4.1765-1774.2005

Bacillus thuringiensis crystal proteins of the Cry34 and Cry35 classes function as binary toxins showing activity on the western corn rootworm, Diabrotica virgifera virgifera LeConte. We surveyed 6,499 B. thuringiensis isolates by hybridization for sequences related to cry35A genes, identifying 78 strains. Proteins of the appropriate molecular mass (ca. 44 kDa) for Cry35 were observed in 42 of the strains. Full-length, or nearly full-length, sequences of 34 cry34 genes and 16 cry35 genes were also obtained from cloning, PCR analysis, and DNA sequencing. These included representatives of all known Cry34A, Cry34B, Cry35A, and Cry35B classes, as well as a novel Cry34A/Cry35A-like pair. Bioassay analysis indicated that cry35-hybridizing strains not producing a ca. 14-kDa protein, indicative of Cry34, were not active on corn rootworms, and that the previously identified Cry34A/Cry35A pairs were more active than the Cry34B/Cry35B pairs. The cry35-hybridizing B. thuringiensis strains were found in locales and materials typical for other B. thuringiensis strains. Comparison of the sequences with the geographic origins of the strains showed that identical, or nearly identical, sequences were found in strains from both Australasia and the Americas. Sequence similarity searches revealed that Cry34 proteins are similar to predicted proteins in Photorhabdus luminescens and Dictyostelium discoidium, and that Cry35Ab1 contains a segment similar to beta-trefoil domains that may be a binding motif. The binary Cry34/Cry35 B. thuringiensis crystal proteins thus appear closely related to each other, are environmentally ubiquitous, and share sequence similarities consistent with activity through membrane disruption in target organisms.