Identification and quantification of crystal components of Bacillus thuringiensis strains and their contribution to insecticidal activity

Resum

The main aim of this thesis has been the development of a proteomic method that allows a qualitative and quantitative analysis of the proteins that make up the crystal produced by different wildtype Bt strains. The method uses a liquid chromatography system coupled to a mass spectrum (LC-MS/MS) in combination with a multiple reaction monitoring (MRM). To carry out the analysis, it is necessary to know the genome sequence of the Bt strain to determine the potential insecticidal genes that could be part of the parasporal crystal. The use of bioinformatic tools, allow the selection of proteotypic peptides that specifically detect the presence of each of the proteins in the mixture. These proteotypic peptides, isotopically labelled, allow to determine the relative proportion of each protein within the crystal. The method was validated using two artificial mixtures containing three recombinant proteins (Cry1Aa, Cry2Aa and Cry6Aa), in a known relative proportion. The application of the method allowed the detection of the three proteins independently and the quantification of the relative proportion of each of them with great reliability and precision. Once the method was validated, it was applied to determine the crystal composition of the Bt strains used as active ingredients of four of the best sold commercial products worldwide for the control of different insect orders: DiPel® and XenTari® for Lepidoptera, VectoBac® for Diptera, and Novodor® for Coleoptera. The parasporal crystal of the Bt subsp. kurstaki strain ABTS-351 (DiPel®) was comprised by four proteins, including: Cry1Aa (13-22%), Cry1Ab (16-29%), Cry1Ac (6-12%) and Cry2Aa (40-64%); in the Bt subsp. aizawai strain ABTS-1857 (XenTari®) four proteins were also detected: Cry1Aa (26-33%), Cry1Ab (57-60%), Cry1Ca (7-11%) and Cry1Da (3-4%). The Bt subsp. israelensis strain AM65-52 (VectoBac®) synthesized a crystal formed by Cry4Aa (2-4%), Cry4Ba (10-28%), Cry11Aa (10-27%), Cry60Aa (2-4%), Cry60Ba (5-12%) and Cyt1Aa (38-61%). Finally, the active ingredient of Novodor®, the Bt subsp. tenebrionis strain NB-176, contained Cry3Aa (70-75%), Cry23Aa (14-16%) and Cry37Aa (10-14%). Additionally, the activity of the strain ABTS-1857 was determined for three species of the genus Spodoptera, including: S. exigua, S. littoralis and S. frugiperda. S. exigua was the most susceptible species (LC50 = 7.8 ng/μl), followed by S. littoralis (LC50 = 28 ng/μl). S. frugiperda was the most tolerant insect (LC50 = 120.2 ng/μl). To determine the contribution of each individual protein to the overall toxicity of the strain ABTS-1857 against each of the three insect species, recombinant Bt strains producing Cry1Aa, Cry1Ab, Cry1Ca and Cry1Da proteins, were engineered. The results of the bioassays, using the droplet feeding method, revealed a high toxicity of Cry1Ca for S. exigua and S. littoralis larvae and Cry1Da against S. littoralis and S. frugiperda. Artificial mixtures containing two or three proteins produced mortalities attributed to the amounts of Cry1Ca, in the case of S. exigua, of Cry1Ca and Cry1Da, in the case of S. littoralis, and of Cry1Da in the case of S. frugiperda. The obtained activity values for the artificial mixture containing the four proteins, which reflected the natural composition of the crystal, were consistent with those obtained with the natural crystal of the strain ABTS-1857. Increasing amounts of Cry1Da protein, in combination with decreasing quantities of either Cry1Aa or Cry1Ab proteins, produced an enhanced insecticidal toxicity for S. littoralis and S. frugiperda larvae. These results indicated that the method used for the analysis of the Bt crystals is valid for its use in the characterization and standardization of Bt based commercial products, providing the necessary information to express its insecticidal potency.