The combinatory effect of Cyt1Aa flexibility and specificity against dipteran larvae improves the toxicity of Bacillus thuringensis kurstaki toxins.

Cyt1A98 is a novel cytolytic protein, from BUPM98 Bacillus thuringiensis strain, characterized by its synergistic activity with B. thuringiensis kurstaki toxins against lepidopteran larvae. In this study, we evidenced that Cyt1A98 improves the toxicity of B. thuringiensis kurstaki toxins against Aedes aegypti larvae. In fact, the strain BNS3pHTcyt1A98 exhibited a larvicidal activity of about 849-fold of that of BNS3pHTBlue against A. aegypti. The molecular and biochemical characterizations, of cyt1A98 gene and its product, were achieved. Cyt1A98 had an LC50 value of about 126.56 mg l-1 against A. aegypti larvae. Compared to Cyt1Aa of B. thuringiensis israelensis, Cyt1A98 amino acid sequence harbours three substitutions of three conserved amino acids among Cyt1Aa family members (Ser42Pro, Pro82Ala, Met188Thr). The Cyt1A98 protein structural analysis evidenced more flexibility than Cyt1Aa. According to the high fluctuation observed for the residue Pro42, the amino acid at position 42 is implicated in the flexibility property of Cyt1Aa especially for the αC and αD helices, involved in the penetration into the cell membrane. The toxicity improvement could be probably due to the higher flexibility combined with the specific affinity toward dipteran larvae. The Cyt1A/B. thuringiensis kurstaki Cry toxins model provides a potential molecular genetic strategy for an efficient bioinsecticide.

Genome sequence analysis of a novel Bacillus thuringiensis strain BLB406 active against Aedes aegypti larvae, a novel potential bioinsecticide.

BLB406 is a novel isolate of Bacillus thuringiensis with a larvicidal activity against Aedes aegypti larvae. It displays original plasmidic and crystal protein patterns. The present work reported molecular and bioinformatic analyses for the genome sequence of BLB406 using MiSeq Illumina next-generation sequencing technology. The reads were assembled by Velvet tool. Using RAST program and PGAAP, the genome of BLB406 strain was shown to contain 6297 genes corresponding to 5924 protein coding sequences. The BLB406 genome investigation with BtToxin_scanner program shows that this strain has an original and different combination of toxins compared to the published ones: five cry genes (cry11, cry22, cry2, cry60, cry64) and two distinct vegetative insecticidal vip4 genes. This combination provides a potential larvicidal and anti-cancer activities to BLB406. It might be a potential solution to some problems such as the narrow insecticidal spectra and insect resistance. The whole BLB406 genome information provides a valuable background for future in silico analyses as well as biotechnological applications in order to increase the production of commercial bioinsecticide based on BLB406 B. thuringiensis strain.

Evidence of two mechanisms involved in Bacillus thuringiensis israelensis decreased toxicity against mosquito larvae: Genome dynamic and toxins stability.

Biopesticides based on Bacillus thuringiensis israelensis are the most used and most successful around the world. This bacterium is characterized by a dynamic genome able to win or lose genetic materials which leads to a decrease in its effectiveness. The detection of such phenomena is of great importance to monitor the stability of B. thuringiensis strains in industrial production processes of biopesticides. New local B. thuringiensis israelensis isolates were investigated. They present variable levels of delta-endotoxins production and insecticidal activities against Aedes aegypti larvae. Searching on the origin of this variability, molecular and biochemical analyses were performed. The obtained results describe two main reasons of the decrease of B. thuringiensis israelensis insecticidal activity. The first reason was the deletion of cry4Aa and cry10Aa genes from the 128-kb pBtoxis plasmid as evidenced in three strains (BLB124, BLB199 and BLB506) among five. The second was the early degradation of Cry toxins by proteases in larvae midgut mainly due to some amino acids substitutions evidenced in Cry4Ba and Cry11Aa δ-endotoxins detected in BLB356. Before biological treatment based on B. thuringiensis israelensis, the studies of microflore in each ecosystem have a great importance to succeed pest management programs.