Encapsulation of the Bacillus thuringiensis secretable toxins Vip3Aa and Cry1Ia in Pseudomonas fluorescens

Abstract

Vip3A and Cry1I toxins are secreted during the vegetative growth of Bacillus thuringiensis. Vip3A toxins do not share homology to the crystal (Cry) proteins and are active against a different spectrum of lepidopteran species. Cry1I toxins share similarity with the Cry1 protein group but do not accumulate in the parasporal crystal. Since Vip3A and Cry1I toxins are released from the cell, they are excluded from biological formulates based on spores and crystals of B. thuringiensis. As an approach to obtain novel sprayable insecticides containing Vip3 or Cry1I toxins, Vip3Aa and Cry1Ia proteins were expressed in Pseudomonas fluorescens. This bacterium, non-pathogenic to animals or plants, can be used as a protectant agent in insecticide formulations after being subjected to a killing fixation process that strengthens the P. fluorescens cell wall. Vip3Aa and Cry1Ia expression into P. fluorescens and Escherichia coli was achieved by the cloning of the vip3Aa and the cry1Ia genes into the broad range expression vector pMEKm12. Both proteins remained encapsulated in the bacterial cells as shown by SDS–PAGE and Western Blot. The toxicity of Vip3Aa expressed in P. fluorescens against Spodoptera frugiperda (89ng/cm2 of diet) was similar to that of Vip3Aa microencapsulated in P. fluorescens (104ng/cm2 of diet). In bioassays with Lobesia botrana, the LC50 value of Cry1Ia purified from P. fluorescens (10.7μg/ml) was similar to that of Cry1Ia microencapsulated in P. fluorescens (12.6μg/ml) and that of Cry1Ia purified from E. coli (8.5μg/ml). These results support the suitability of P. fluorescens as a heterologous expression system for the development of novel insecticides based on the secretable Vip3Aa and Cry1Ia toxins from B. thuringiensis.