High-throughput screening for novel Bacillus thuringiensis insecticidal proteins revealed evidence that the bacterium...

Approximately 3000 Bacillus thuringiensis (Bt) isolates were screened to discover novel three-domain (3D) Cry proteins active against Helicoverpa zea (corn earworm). From 400 active isolates found during the primary screening, Cry1Ac and Cry2A, which are known to be active against H. zea, were removed using multiplex-primer PCR and high-throughput column chromatography. This process reduced the number of active cultures to 48. DNA segments encoding Domain III of these 48 cultures were amplified by PCR and sequenced. Sequencing revealed two novel Cry1B-type Domain IIIs. Further sequencing of the flanking regions of these domains revealed that one was part of Cry1Bj (GenBank: KT952325). However, the other Domain III lacked Domains I and II. Instead, this Domain III was associated with two open reading frames, ORF1 and ORF2. ORF1 was identified as an ATP-binding protein, and ORF2 as an ATPase, suggesting that Bt exchanges Domain III among homologous Cry proteins.