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Applied and Environmental Microbiology, March 2002, p. 1137-1145, Vol. 68, No. 3
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.3.1137-1145.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Novel Bacillus thuringiensis Binary Insecticidal Crystal Proteins Active on Western Corn Rootworm, Diabrotica virgifera virgifera LeConte

R. Tracy Ellis, Brian A. Stockhoff, Lisa Stamp,,{dagger} H. Ernest Schnepf, George E. Schwab, Mark Knuth,,{ddagger} Josh Russell, Guy A. Cardineau, and Kenneth E. Narva*

Dow AgroSciences, San Diego, California 92121

Received 22 August 2001/ Accepted 13 December 2001

A new family of insecticidal crystal proteins was discovered by screening sporulated Bacillus thuringiensis cultures for oral activity against western corn rootworm (WCR) larvae. B. thuringiensis isolates PS80JJ1, PS149B1, and PS167H2 have WCR insecticidal activity attributable to parasporal inclusion bodies containing proteins with molecular masses of ca. 14 and 44 kDa. The genes encoding these polypeptides reside in apparent operons, and the 14-kDa protein open reading frame (ORF) precedes the 44-kDa protein ORF. Mutagenesis of either gene in the apparent operons dramatically reduced insecticidal activity of the corresponding recombinant B. thuringiensis strain. Bioassays performed with separately expressed, biochemically purified 14- and 44-kDa polypeptides also demonstrated that both proteins are required for WCR mortality. Sequence comparisons with other known B. thuringiensis insecticidal proteins failed to reveal homology with previously described Cry, Cyt, or Vip proteins. However, there is evidence that the 44-kDa polypeptide and the 41.9- and 51.4-kDa binary dipteran insecticidal proteins from Bacillus sphaericus are evolutionarily related. The 14- and 44-kDa polypeptides from isolates PS80JJ1, PS149B1, and PS167H2 have been designated Cry34Aa1, Cry34Ab1, and Cry34Ac1, respectively, and the 44-kDa polypeptides from these isolates have been designated Cry35Aa1, Cry35Ab1, and Cry35Ac1, respectively.

* Corresponding author. Mailing address: Dow AgroSciences, 5501 Oberlin Dr., San Diego, CA 92121. Phone: (858) 352-4348. Fax: (858) 352-4466. E-mail: kenarva{at}dowagro.com.

{dagger} Present address: 12756 Via Felino, Del Mar, CA 92014.

{ddagger} Present address: 16744 Valle Verde Road, Poway, CA 92064.

Applied and Environmental Microbiology, March 2002, p. 1137-1145, Vol. 68, No. 3
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.3.1137-1145.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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