Bacillus thuringiensis subsp. israelensis é uma bactéria gram positiva, amplamente utilizada no controle de insetos Diptera, família Culicidae, tais como Aedes aegypti e Culex quinquefasciatus. O sequenciamento de genomas completos de estirpes de B. thuringiensis tem permitido a identificação e caracterização de proteínas inseticidas, assim como a realização de estudos de genômica comparativa, permitindo evidenciar diferenças funcionais e filogenéticas. Neste contexto, o presente estudo teve como objetivo realizar análises genômicas de quatro isolados de B. thuringiensis subsp. israelensis que apresentam diferentes toxicidades para larvas de A. aegypti e C. quinquefasciatus. O sequenciamento dos genomas dos isolados T0124, T0131, T0137 e T0139 foi realizado a partir da plataforma MiSeq-Ilumina, e a montagem e anotação foram feitas utilizando ferramentas do programa de bioinformática Geneious. As análises resultaram em cromossomos com tamanhos aproximados de 5.414 Kb, conteúdo G+C de 35,2% e 5.358 regiões codificantes. Com relação ao conteúdo extracromossomal, foram montados três plasmídeos menores de 5,4; 6,8 e 7,6 Kb, e três plasmídeos maiores de 127, 235 e 359 Kb para todos os isolados, e estes foram enumerados de 1 a 6. A anotação revelou que apenas os plasmídeos de número 4, que correspondem ao pBtoxis, possuem proteínas inseticidas, sendo elas Cry4Aa, Cry4Ba, Cry11Aa, Cry10Aa, Cyt1Aa, Cyt2Ba e Cyt1Ca. A análise comparativa entre os genomas demonstra que os cromossomos T0124, T0131, T0137 e T0139 apresentam sequências colineares e com 99% de identidade, e seus respectivos plasmídeos 1, 2, 3, 4, 5 e 6 compartilham todas as suas regiões codificantes, incluindo aquelas relacionadas a δ-endotoxinas. A comparação de sequências de enterotoxinas, metaloproteases e fosfolipases demonstra que estes também são conservados entre os genomas. Na análise filogenética, os cromossomos T0124, T0131, T0137 eT0139 agruparam-se juntamente com os genomas dos isolados AM65-52 e HD-789 disponíveis no GenBank, sugerindo uma estreita relação genética entre estirpes de B. thuringiensis subsp. israelensis. Os isolados T0124, T0131, T0137 e T0139 apresentam diferentes concentrações letais para larvas de A. aegypti e C. quinquefasciatus, e a adição de dados proteômicos aos dados genômicos, aqui obtidos, poderão auxiliar na compreensão destas diferenças de toxicidade. Contudo as análises comparativas entre os genomas dos isolados T0124, T0131, T0137 e T0139 mostram alto grau de conservação genética a nível de nucleotídeos de B. thuringiensis subsp. israelensis. / Bacillus thuringiensis subsp. israelensis is a gram-positive bacterium widely used to control insects of the order Diptera, such as Aedes aegypti (Diptera: Culicidae) and Culex quinquefasciatus (Diptera: Culicidae). The sequencing of complete genomes of B. thuringiensis strains has allowed the identification and characterization of insecticidal proteins, as well as the comparative genomic studies, allowing the identification of functional and phylogenetic differences. In this context, the present study aimed to perform genomic analysis of four isolates of B. thuringiensis subsp. israelensis having different toxicities for larvae of A. aegypti and C. quinquefasciatus larvae. Sequencing of genomes of the isolates T0124, T0131, T0137 and T0139 was performed from the MiSeq-Ilumina platform, and assembly and annotation were done using tools from the Geneious bioinformatics program. The analyzes resulted in chromosomes with approximate sizes of 5,414 Kb, G + C content of 35.2% and 5,358 coding regions. Regarding extrachromosomal content, three smaller plasmids of 5.4, 6.8 and 7.6 Kb, and three larger plasmids of 127, 235 and 359 Kb were assembled for all the isolates, and these were enumerated from 1 to 6. The annotation revealed that only the plasmids of number 4, corresponding to pBtoxis, possess insecticidal proteins, being Cry4Aa, Cry4Ba, Cry11Aa, Cry10Aa, Cyt1Aa, Cyt2Ba and Cyt1Ca. Comparative analysis between the genomes demonstrates that chromosomes T0124, T0131, T0137 and T0139 have collinear sequences and 99% identity, and their respective plasmids 1, 2, 3, 4, 5 and 6 share all of their coding regions, including those related to δ-endotoxins. Comparison of enterotoxin, metalloprotease and phospholipase sequences shows that these are conserved among the genomes. In the phylogenetic analysis, chromosomes T0124, T0131, T0137 and T0139 were grouped together with the genomes of the isolates AM65-52 and HD-789 available in GenBank, suggesting a close genetic relation between strains of B. thuringiensis subsp. israelensis. The isolates T0124, T0131, T0137 and T0139 present different lethal concentrations to larvae of A. aegypti and C. quinquefasciatus, and the addition of proteomic data to the genomic data obtained here may help to understand these differences in toxicity. However, the comparative analyzes between the genomes of isolates T0124, T0131, T0137 and T0139 show a high degree of genetic conservation at the nucleotide level of B. thuringiensis subsp. israelensis.

Parmi les méthodes alternatives respectueuses de l'environnement pour la lutte contre les ravageurs et la protection des cultures agricoles, les bioinsecticides à base de Bacillus thuringiensis sont les plus largement utilisés. L’activité insecticide de B.thuringiensis réside dans la production d’inclusions cristallines protéiques appelées δ-endotoxines qui sont formées au cours de la sporulation. En préalable à ces travaux, de nouvelles souches de B.thuringiensis ont été isolées du sol libanais et ont montré un potentiel insecticide élevé, supérieur à des souches classiquement utilisées. Dans le cadre du développement d'une production industrielle, au Liban, de biopesticides à base de ces souches de B.thuringiensis, ce travail a visé à optimiser la production des δ-endotoxines afin d'augmenter les rendements et de réduire les coûts de production. A l'échelle industrielle, le milieu de culture des bactéries représente une part importante du coût de production. Ainsi, l'effet, sur la concentration des δ-endotoxines, de substitution des sources synthétiques coûteuses d'azote, de carbone et de minéraux, par un sous-produit de la minoterie de blé (le son de blé) a été étudié. L’efficacité de ce sous-produit agro-industriel comme source complète de nutriments, disponible localement à faible prix, a été prouvée pour des fermentations submergées de deux souches différentes toxiques contre des diptères ou des lépidoptères. La comparaison du milieu son de blé à d’autres milieux (synthétiques ou non), classiquement utilisés en bibliographie, a montré que ce milieu présente de nombreux avantages en termes de productivité, de rendement relatif des δ-endotoxines et de coût. Parallèlement à ce travail, une méthode simple et rapide de purification des cristaux a été développée de façon à obtenir des cristaux protéiques purs à 99 % nécessaires comme standards pour le dosage des cristaux dans le milieu de fermentation. Un autre objectif du travail a été d’analyser l'effet de l'aération sur la cinétique de croissance et de sporulation d'une nouvelle souche de B.thuringiensis var kurstaki. Les différentes conditions d’aération testées en fermenteurs de 2 L, caractérisées par le coefficient volumétrique de transfert de dioxygène (KLa), ont permis de montrer l’importance de ce paramètre et de l’optimiser pour obtenir la meilleure productivité en biopesticides. Le meilleur rendement en protéines de toxines a été obtenu dans un milieu son de blé 6 % (masse/volume) dans des conditions d’aération correspondant à un KLa de 65,5 h-1 alors que la meilleure productivité des protéines de toxines a été atteinte dans le milieu 9 % pour un KLa de 102 h-1. Sur la base de cette étude, le dimensionnement de la production de B.thuringiensis à l’échelle 1000 L a été réalisé. L’unité de production a été construite et les premiers essais réalisés ont permis de produire de façon satisfaisante des lots de biopesticides. L’ajustement des protocoles industriels reste à faire. / Among the safe and environment friendly methods for the fight against pests and agricultural crops protection, Bacillus thuringiensis based biopesticides are the most widely used. The insecticidal activity of B. thuringiensis resides in the production of protein crystal inclusions called δ-endotoxins, which are formed during sporulation. Preliminary to this work, new B. thuringiensis strains were isolated from Lebanese soil and showed higher insecticidal potential than the conventionally used strains. As part of the development of an industrial production, in Lebanon, of biopesticides based on these strains of B.thuringiensis, this work aimed to optimize the production of δ-endotoxins to increase yields and reduce the cost of mass production. At industrial scale, the bacteria culture medium represents a significant part of the production cost. Thus, the effect on the concentration of the δ-endotoxins, of substituting expensive synthetic sources of nitrogen, carbon, and minerals, with a byproduct of wheat mill (wheat bran) was studied. The effectiveness of this agro-industrial by-product available locally at low prices, as complete source of nutrients, in submerged fermentations of two different strains of B.thuringiensis toxic against Lepidoptera and Diptera, has been proven. The comparison of the wheat bran medium to other mediums (synthetic or not), conventionally used in the bibliography, has shown that this medium this medium has many advantages in terms of productivity, relative yield of δ-endotoxins and cost. In parallel to this work, a simple and quick method of crystal purification was developed to obtain pure protein crystals to 99 % required as standards for the determination of crystals concentration in the fermentation medium. Another objective of the study was to analyze the effect of aeration on growth and sporulation kinetics of a new strain of B.thuringiensis var kurstaki. The different aeration conditions tested in fermenters of 2 L, characterized by the volumetric oxygen transfer coefficient (KLa), permitted to show the importance of this parameter and to optimize it to get the best biopesticides productivity. The best yield of toxin proteins relative to the wheat bran ratio in the culture medium was obtained in the 6 % (weight/volume) medium in aeration conditions corresponding to a KLa of 65.5 h-1, while the highest productivity of toxin proteins has been reached in the 9 % medium for KLa of 102 h-1. On this study basis, the scale up of B. thuringiensis production was performed in a bioreactor of 1000 L. The production unit was built and the first tests carried out have produced satisfactory batches of biopesticides. Industrial protocols are still to be adjusted.