A pair of adjacent genes, cry5Ad and orf2-5Ad, encode the typical N- and C-terminal regions of a Cry5Adelta-endotoxin as two separate proteins in Bacillus thuringiensis strain L366.

CSIRO Livestock Industries, Queensland Bioscience Precinct, St Lucia, Qld, Australia.
FEMS Microbiology Letters (Impact Factor: 2.05). 02/2008; 278(1):115-20. DOI: 10.1111/j.1574-6968.2007.00987.x
Source: PubMed

ABSTRACT A new DNA sequence cry5Ad/orf2-5Ad (GenBank accession number EF219060) was isolated from Bacillus thuringiensis strain L366. This DNA sequence contains two ORFs: cry5Ad (a previously unreported member of the cry5A gene family) and orf2-5Ad. cry5Ad is unique among cry5A genes in that it encodes only the N-terminal region of a typical Cry5Adelta-endotoxin. The cry5Ad sequence includes homology blocks 1-5, which are present in most B. thuringiensisdelta-endotoxins. The usual C-terminal region of a Cry5Adelta-endotoxin (including homology blocks 6-8) is encoded by orf2-5Ad. Both proteins encoded by cry5Ad and orf2-5Ad were found in IPTG-induced Escherichia coli, after a copy of cry5Ad/orf2-5Ad was cloned into the pQE32 expression vector and transformed into pREP4 E. coli cells. Both proteins were also found in parasporal crystal inclusions of B. thuringiensis L366. Sequencing of cDNA derived from transformed E. coli cells showed that the two ORFs are transcribed as a single mRNA. Extracts prepared from the recombinant E. coli expressing Cry5Ad and Orf2-5Ad were not toxic to nematode larvae (Haemonchus contortus), indicating that these two proteins are most likely not responsible for the nematocidal activity seen previously in the B. thuringiensis strain L366.

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    ABSTRACT: Insect cadherin proteins localized in the midgut epithelium were identified as receptors for Bacillus thuringiensis insecticidal crystal proteins (Cry toxins). These cadherins facilitated toxin monomer oligomerization and mediated oligomer binding to secondary receptors. It has been reported that Manduca sexta, Helicoverpa armigera, Anopheles gambiae and Diabrotica virgifera cadherin toxin binding regions function as synergists for Cry1A, Cry4Ba and Cry3A toxicity against target insects. In the present study, the toxin binding region fragment of the H. armigera cadherin (hacad1) gene was cloned and fused with the promoter of the cry3Aa gene. The fusion gene pro3Aa-hacad1 and the cry1Ac gene were inserted into shuttle vector pHT304 and introduced into B. thuringiensis acrystalliferous strain BMB171 for coexpression (resulting in recombinant strain BMB1073). SDS-PAGE and mass spectrum analysis showed that BMB1073 could express HaCad1 and Cry1Ac proteins together. Bioassay results demonstrated that insecticidal activities against H. armigera and Spodoptera exigua could be increased 5.1-fold and 6.5-fold, respectively, by BMB1073 compared with the strain which can only express the Cry1Ac protein. Our discovery showed that coexpression of HaCad1 and Cry1Ac toxin in B. thuringiensis enhanced the insecticidal activity of Cry1Ac toward Lepidoptera insects. This finding also revealed a novel strategy for engineering strains and transgenic plants with higher insecticidal activity.
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