Chimeric vip3Aa16TC Gene Encoding the Toxic Core of the Vegetative Insecticidal Protein Enhanced Bacillus thuringiensis Entomopathogenicity

Vip3 insecticidal protein is produced by Bacillus thuringiensis during the vegetative stage. Its proteolysis by the midgut juice of susceptible larvae formed four major products of approximately 66, 45, 33 and 22 kDa. In this study, we cloned the vip3Aa16TC DNA encoding the “Vip3Aa16 toxic core (TC)” of 33 kDa corresponding to the Vip3Aa16 region from amino acid 200 to 456. The vip3Aa16TC chimeric gene carried by the pHT-vip3Aa16TC plasmid was under the control of the sporulation dependent promoters (BtI-BtII) and the Shine Dalgarno sequence of cry1Ac gene as well as the cry1Ia gene terminator. Western-blot analysis of the culture supernatants of the recombinant B. thuringiensis strain detected Vip3Aa16TC after growing for 14 to 56 h proving that this protein can be produced without the Vip3 amino- and carboxy-terminal regions. Interestingly, the preservation of the Vip3Aa16TC toxicity against the polyphagous lepidopteran Spodoptera littoralis makes it a promising polypeptide for the pest biological control

Heterologous expression of Bacillus thuringiensis vegetative insecticidal protein-encoding gene vip3LB in Photorhabdus temperata strain K122 and oral toxicity against the lepidoptera Ephestia kuehniella and Spodoptera littoralis

Correspondance auteur: S. Jaoua E-mail: [email protected] audiencePhotorhabdus temperata and Bacillus thuringiensis are entomopathogenic bacteria exhibiting toxicities against different insect larvae. Vegetative Insecticidal Protein Vip3LB is a Bacillus thuringiensis insecticidal protein secreted during the vegetative growth stage exhibiting lepidopteran specificity. In this study, we focused for the first time on the heterologous expression of vip3LB gene in Photorhabdus temperata strain K122. Firstly, Western blot analyses of whole cultures of recombinant Photorhabdus temperata showed that Vip3LB was produced and appeared lightly proteolysed. Cellular fractionation and proteinase K proteolysis showed that in vitro-cultured recombinant Photorhabdus temperata K122 accumulated Vip3LB in the cell and appeared not to secrete this protein. Oral toxicity of whole cultures of recombinant Photorhabdus temperata K122 strains was assayed on second-instar larvae of Ephestia kuehniella, a laboratory model insect, and the cutworm Spodoptera littoralis, one of the major pests of many important crop plants. Unlike the wild strain K122, which has no effect on the larval growth, the recombinant bacteria expressing vip3LB gene reduced or stopped the larval growth. These results demonstrate that the heterologous expression of Bacillus thuringiensis vegetative insecticidal protein-encoding gene vip3LB in Photorhabdus temperata could be considered as an excellent tool for improving Photorhabdus insecticidal activitie

Screening and identification of a Bacillus thuringiensis strain S1/4 with large and efficient insecticidal activities.

The bacterium Bacillus thuringiensis was recognized for its entomopathogenic activities related to Cry and Cyt proteins forming the δ-endotoxins and some extracellular activities like the vegetative insecticidal proteins (VIP) and Cry1I. These activities may act specifically against diverse organisms and some of them typically characterize each strain. Here, we screened a set of 212 B. thuringiensis strains to search the higher insecticidal activities. These strains had bipyramidal and cubic crystal morphologies and 30% of them showed PCR amplification of vip3 internal region, from which five isolates (S1/4, S17, S122, S123, and S144) showed plasmid profile variability. These five strains contained the cry1I, cry1Aa and/or cry1Ac, cry1Ab and cry2 genes, and S1/4 harbored in addition the cry1C, vip1, and vip2 genes. They produced from 25 to 46 µg δ-endotoxin per 10(7) spores. Their δ-endotoxins displayed distinct lethal concentrations 50% against either Spodoptera littoralis or Ephestia kuehniella larvae with the lowest one for S1/4, which was also active against Tuta absoluta. Fortunately, the analysis of the culture supernatants revealed that S1/4 had the higher toxicity towards these lepidopteron but it did not show any toxicity against the Tribolium castaneum coleopteran larvae; additionally, S1/4 displayed an antibacterial activity. S1/4 is a good candidate for agricultural pest control, as it is more efficient than the reference strain HD1