title: Molecular genetic manipulation of truncated Cry1C protein synthesis in Bacillus thuringiensis to improve stability and yield.
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 [LC(50)] = 13.2 microg/ml) was lower than that of full-length Cry1C (LC(50) = 1.8 microg/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.
authors.affiliation: Department of Entomology, University of California, Riverside, Riverside, California 92521, USA.
authors.forename: H W
authors.initials: HW
authors.lastname: Park
authors.forename: D K
authors.initials: DK
authors.lastname: Bideshi
authors.forename: B A
authors.initials: BA
authors.lastname: Federici
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics* / Q000502:physiology; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000633:toxicity; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D001681:Biological Assay; D003001:Cloning, Molecular; D017931:DNA Primers; D004731:Endotoxins / Q000096:biosynthesis / Q000235:genetics* / Q000633:toxicity; D004926:Escherichia coli; D006460:Hemolysin Proteins; D007814:Larva; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D018411:Spodoptera; D013171:Spores, Bacterial; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D004731:Endotoxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis