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Applied and Environmental Microbiology, October 1998, p. 3932-3938, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Optimization of Cry3A Yields in Bacillus thuringiensis by Use of Sporulation-Dependent Promoters in Combination with the STAB-SD mRNA Sequence

Hyun-Woo Park,1 Baoxue Ge,2 Leah S. Bauer,3 and Brian A. Federici1,2,*

Department of Entomology1 and Interdepartmental Graduate Program in Genetics,2 University of California, Riverside, California 92521, and USDA Forest Service, North Central Forest Experiment Station, Michigan State University, East Lansing, Michigan 488233

Received 2 March 1998/Accepted 22 June 1998

The insecticidal activity of Bacillus thuringiensis strains toxic to coleopterous insects is due to Cry3 proteins assembled into small rectangular crystals. Toxin synthesis in these strains is dependent primarily upon a promoter that is active in the stationary phase and a STAB-SD sequence that stabilizes the cry3 transcript-ribosome complex. Here we show that significantly higher yields of Cry3A can be obtained by using dual sporulation-dependent cyt1Aa promoters to drive the expression of cry3Aa when the STAB-SD sequence is included in the construct. The Cry3A yield per unit of culture medium obtained with this expression system was 12.7-fold greater than that produced by DSM 2803, the wild-type strain of B. thuringiensis from which Cry3Aa was originally described, and 1.4-fold greater than that produced by NB176, a mutant of the same strain containing two or three copies of cry3Aa, which is the active ingredient of the commercial product Novodor, used for control of beetle pests. The toxicities of Cry3A produced with this construct or the wild-type strain were similar when assayed against larvae of the cottonwood leaf beetle, Chrysomela scripta. The volume of Cry3A crystals produced with cyt1Aa promoters and the STAB-SD sequence was 1.3-fold that of typical bipyramidal Cry1 crystals toxic to lepidopterous insects. The dual-promoter/STAB-SD system offers an additional method for potentially improving the efficacy of insecticides based on B. thuringiensis.

* Corresponding author. Mailing address: Department of Entomology, University of California, Riverside, CA 92521. Phone: (909) 787-5006. Fax: (909) 787-3086. E-mail: brian.federici{at}ucr.edu.

Applied and Environmental Microbiology, October 1998, p. 3932-3938, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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