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The large genome of Bacillus thuringiensis was shortened by provoking plasmid loss, and the effect of such loss on the expression of both chromosomal and plasmid-harboured genes was studied. It was evidenced that the genomic material shortening of B. thuringiensis allows for the improvement of the expression of some of the constructive genes. Indeed, it was shown that curing was an efficient tool to obtain mutant strains with better expression of chromosomal genes. In fact, totally cured strains, HD1Cry-B and BNS3Cry, showed higher chitinolytic and/or proteolytic activities than the corresponding wild and/or partially cured ones. Although total curing is drastic for plasmid-harboured gene expression, the authors obtained partially cured strains, Cur255 and CurHD1, with a doubled level of delta-endotoxin production, and a 1.5-fold improvement of the bacteriocin-specific activity, respectively. In addition, the latter strains showed better relative proteolytic activities. Tunisian Ministere de l’Enseignement Superieur et de la Recherche Scientifique (MESRS)

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)). Tunisian Ministere de l’Enseignement Superieur et de la Recherche Scientifique (MESRS)

Asporogenic and oligosporogenic Bacillus thuringiensis mutants having the ability to overproduce insecticidal crystal protein were generated by using nitrous acid (50 mg/ml), as chemical mutagenic agent. Insecticidal crystal proteins produced by asporogenic mutants remained encapsulated within the cells. Delta-endotoxin production by most of mutants was improved compared to the corresponding wild strains BNS3 and a mutant M26. The overproduction by asporogenic and oligosporogenic mutants was attributed to defect in genes involved in sporulation and to random mutations affecting cell metabolism at different pathways and delta-endotoxin synthesis. Sporeless bioinsecticides could be developed based on stable and environmentally safe Bacillus thuringiensis mutants. Tunisian Ministry of Higher Education, Scientific Research and Technology’’ and the AUF ‘‘Agence Universitaire de la Francophonie’’

The Vegetative insecticidal Vip3A proteins display a wide range of insecticidal spectrum against several agricultural insect pests. The fact that the expression of vip3 genes occurs only during the vegetative growth phase of Bacillus thuringiensis is a limiting factor in term of production level. Therefore, extending the synthesis of the Vip proteins to the sporulation phase is a good alternative to reach high levels of toxin synthesis. In this study, we have demonstrated that the maximal production of the secreted Vip3LB (also called Vip3Aa16) during the growth of the wild-type strain B. thuringiensis BUPM 95 is reached at the end of the vegetative growth phase, and that the protein remains relatively stable in the culture supernatant during the late sporulation stages. The vip3LB gene was cloned and expressed under the control of the sporulation dependant promoters BtI and BtII in B. thuringiensis BUPM 106 (Vip3(-)) and BUPM 95 (Vip3(+)) strains. The examination of the culture supernatants during the sporulation phase evidenced the synthesis of Vip3LB and its toxicity against the second-instars larvae of the Lepidopteron insect Spodoptera littoralis for the recombinant BUPM 106. Moreover, there was an increase of the Vip3LB synthesis level and an enhancement of the oral toxicity for the recombinant BUPM 95 resulting from the expression of the vip3LB gene during both the vegetative and sporulation phases and the relative stability of the Vip3LB protein. Tunisian Ministere de l’Enseignement Superieur et de la Recherche Scientifique (MESRS)

The study of 257 crystal-producing Bacillus thuringiensis isolates from bioinsecticide free soil samples collected from different sites in Tunisia, was performed by PCR amplification, using six primer pairs specific for cry1, cry2, cry3, cry4, and vip3A genes, by the investigation of strain plasmid pattern, crystal morphology and delta-endotoxin content and by the assessment of insecticidal activities against the lepidopteran insect Ephestia kuehniella. Based on plasmid pattern study, 11 representative strains of the different classes were subjected to morphological and molecular analyses. The comparison of the PFGE fingerprints confirmed the heterogeneity of these strains. B. thuringiensis kurstaki strains, harbouring at the same time the genes cry1A, cry2, cry1Ia, and vip3A, were the most abundant (65.4%). 33.34% of the new isolates showed particular delta-endotoxin profiles but no PCR products with the used primer sets. B. thuringiensis israelensis was shown to be also very rare among the Tunisian B. thuringiensis isolates diversity. These findings could have considerable impacts for the set up of new pest control biological agents. Tunisian Ministry of Higher Education, Scientific Research and Technology, the National Priorities Research Program (NPRP) of Qatar National Research Fund number 27-6-7-24, the AUF Agence Universitaire de la Francophonie and the NEPAD New Partnership for Africa Development Scopus