Integration of a recombinant chitinase into Bacillus thuringiensis parasporal insecticidal crystal.
Chitinases have been successfully used in combination with Bacillus thuringiensis delta-endotoxins forming crystals in order to enhance their insecticidal activities. In this context, we opted for promoting the chitinase integration into these crystals. Thus, we engineered, for the first time, a fusion protein (CDF) consisting of the chitinase Chi255 and the carboxy-terminal half of Cry1Ac, both from B. thuringiensis subsp. kurstaki. The constructed transcriptional fusion (chi255Δsp-CTcry1Ac) was cloned into a shuttle vector (Escherichia coli/B. thuringiensis) downstream the sporulation-dependent promoters BtI-BtII and upstream the cry1Ac transcription terminator. The resulting plasmid, named pF, was transferred by electroporation to crystalliferous B. thuringiensis strain BNS3 and acrystalliferous strain BNS3Cry-. The functionality of the chimerical chitinase was demonstrated by an improvement of the relative chitinolytic activity of the recombinant strain BNS3/pF by 2.5 folds. Western blot analyses showed that, despite of the instability of CDF when expressed in the acrystalliferous strain, the C-terminal half of Cry1Ac succeeded to allow the integration of the chimerical chitinase into the crystal of BNS3. The recombinant strain BNS3/pF (LC(50) = 144.06 μg g(-1)) was 1.5 times more active against Ephestia kuehniella larvae than the wild strain (LC(50) = 212.10 μg g(-1)).
Driss
Fatma
F
Laboratory of Biopesticides, Centre of Biotechnology of Sfax, P.O. Box 1177, 3018, Sfax, Tunisia.
Rouis
Souad
S
Azzouz
Hichem
H
Tounsi
Slim
S
Zouari
Nabil
N
Jaoua
Samir
S
D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000472:pathogenicity; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018274:Electroporation; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D007306:Insecticides / Q000378:metabolism; D007814:Larva / Q000382:microbiology; D007915:Lepidoptera / Q000382:microbiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016019:Survival Analysis; D014169:Transformation, Bacterial
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D011993:Recombinant Fusion Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D002686:Chitin; D002688:Chitinases