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

Abstract

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.

Characterization and selection of Bacillus thuringiensis isolates effective against Sitophilus oryzae

“…The spherical formations, common to the studied isolates, suggest that this crystal morphology could be associated to toxicity of this bacterium to Coleoptera, since they also present a 44 kDa protein profile ( Figure 2) referring to protein Cry35Ba, active against Coleoptera ( Ellis et al, 2002;Schnepf et al, 2005;Storer et al, 2006). These authors have described the binary action of 14 kDa protein Cry34 with Cry35; however this study did not confirm their presence in the proteic and genetic profiles.…”

“…This new mode of action was observed in pathogenicity and virulence bioassays, and also in genetically modified maize (Schnepf et al, 2005;Storer et al, 2006). This study characterized several B. thuringiensis isolates that contain the cry35Ba gene among others and show insecticidal effect against S. oryzae larvae and determined the morphological a total protein profiles of the isolates.…”

Efficacy of Bacillus thuringiensis Cry14 toxin against root knot nematode, Meloidogyne javanica

“…This microorganism is found in many different habitats, particularly soils, due to its spore-forming capacity (Schnepf et al 2005). Widespread screening of Bt strains and cry gene sequencing has led to the identification of more than 700 cry gene sequences (Crickmore et al 2011).…”

Ecological Safety Assessment Of Insecticidal Proteins Introduced Into Biotech Crops

“…Even Cry proteins with novel and recently discovered insecticidal activity (e.g., Cry34Ab1/Cry35Ab1) are commonly distributed in nature, along with more familiar Cry proteins. 41 Naturally occurring Bt δ-endotoxins are in the form of protoxins. Insecticidal activity is conveyed when the ingested protoxin undergoes proteolysis in the insect gut to form toxic polypeptides.…”