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Applied and Environmental Microbiology, April 2005, p. 1765-1774, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.1765-1774.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

H. Ernest Schnepf,* Stacey Lee,{dagger} JoAnna Dojillo,{ddagger} Paula Burmeister,§ Kristin Fencil, Lisa Morera, Linda Nygaard, Kenneth E. Narva, and Jeff D. Wolt||

Research and Development Laboratories, Dow AgroSciences, Indianapolis, Indiana

Received 20 August 2004/ Accepted 2 November 2004

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.

* Corresponding author. Present address: 7954 Handel Ct., San Diego, CA 92126. Phone: (858) 271-7867. Fax: (858) 352-4470. E-mail: eschnep1{at}san.rr.com.

{dagger} Present address: The Dow Chemical Company, San Diego, CA 92121.

{ddagger} Present address: Elan Pharmaceuticals, Inc., San Diego, CA 92121.

§ Present address: Archemix Corp., Cambridge, MA 02139.

Present address: Tanabe Research Laboratories USA, Inc., San Diego, CA 92121.

|| Present address: Biosafety Initiative for Genetically Modified Agricultural Products, Iowa State University, Ames, IA 50011.

Applied and Environmental Microbiology, April 2005, p. 1765-1774, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.1765-1774.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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