Bacteriology & Antibiotics

Bacillus thuringiensis (Bt) is a Gram positive bacterium characterised by the production, during sporulation, of various toxins having insecticidal activities, among which the delta-endotoxins Cry and the vegetative insecticidal proteins Vip. Hundreds of strains of B. thuringiensis, isolated from Tunisia, Qatar and other countries were studied and their bioinsecticides coding genes cry and vip, were cloned and characterized. Among the Tunisian strains, we evidenced the abundance of the kurstaki subspecies active on Lepidoptera and particularly the lepidopteran olive tree pathogenic insect P. oleae, whereas from the Qatari soil samples, we evidenced the abundance of the spherical crystal producing strains, among which subspecies active on Diptera and disease vetors. The bacterium Bacillus thuringiensis produces, at the vegetative stage of its growth, Vip3A proteins with activity against a broad spectrum of lepidopteran insects. We performed the heterologous expression of corresponding coding genes in B. thuringiensis, P. luminescens and E. coli and evidenced an increase of the synthesis of the insecticidal proteins. B. thuringiensis produces also antifungal chitinases. One chitinase was characterized by both its high chitinolytic and antifungal activities. The cloning and sequencing of the corresponding gene chi255, showed is a new chitinase Chi255, presenting several differences from the published chitinases of B. thuringiensis. Heterologous expression in E. coli was performed by cloning the chi255 ORF downstream a strong promoter in the vector pBAD. Identification, by HPLC analysis, of chitin hydrolysis products issued from the activity exhibited by Chi255, revealed that this enzyme is a chitobiosidase. By heterologous expression we succeeded in integrating the enzyme in the B. thuringiensis crystals.