Applied and Environmental Microbiology (2000) 66, 4449-4455

Hyun-Woo Park, Dennis K. Bideshi and Brian A. Federici (2000)
Molecular genetic manipulation of truncated Cry1C protein synthesis in Bacillus thuringiensis to improve stability and yield
Applied and Environmental Microbiology 66 (10), 4449-4455
Abstract: Cry1 protoxins of Bacillus thuringiensis are insecticidal 135-kDa proteins synthesized and assembled into parasporal crystals during sporulation. After ingestion, these crystals dissolve in the midgut and active toxins with molecular masses of about 65-kDa are released from the N-terminal half of the molecule by midgut proteases. Direct synthesis of the toxin-containing N-terminal half of Cry1 molecules using recombinant DNA techniques results in a low level of unstable truncated proteins that do not crystallize. In the present study, inclusions of truncated Cry1C (Cry1C-t) were obtained by combining genetic elements from other endotoxin genes and operons that enhance Cry protein synthesis and crystallization. Increased levels of Cry1C-t synthesis were achieved by using cyt1A promoters to drive expression of the 5' half of cry1C that included in the construct the 5' cry3A STAB-SD mRNA stabilizing sequence and the 3' stem-loop transcription terminator. RNA dot blot analysis showed that the STAB-SD and 3' transcriptional termination sequences were important for stabilization of truncated cry1C (cry1C-t) mRNA. A low level of cry1C-t mRNA was present when only the cyt1A promoters were used to express cry1C-t, but no accumulation of Cry1C-t was detected in Western blots. The orientation of the transcription terminator was important to enhancing Cry1C-t synthesis. Inclusion of the 20- and 29-kDa helper protein genes in cry1C-t constructs further enhanced synthesis. The Cry1C-t protein was toxic to Spodoptera exigua larvae, though the toxicity (50% lethal concentration [LC₅₀] = 13.2 µg/ml) was lower than that of full-length Cry1C (LC₅₀ = 1.8 µg/ml). However, transformation of the HD1 isolate of B. thuringiensis subsp. kurstaki with the cry1C-t construct enhanced its toxicity to S. exigua as much as fourfold.
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Database assignments for author(s): Hyun-Woo Park

Research topic(s) for pests/diseases/weeds:
biocontrol - natural enemies
Research topic(s) for beneficials or antagonists:
genetical engin./transgenic plants
rearing/culturing/mass production

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Pest and/or beneficial records:

Beneficial	Pest/Disease/Weed	Crop/Product	Country	Quarant.
	Spodoptera exigua
Bacillus thuringiensis var. kurstaki (entomopathogen)	Spodoptera exigua
Bacillus thuringiensis Cry1C-toxin (entomopathogen)	Spodoptera exigua