Unraveling the Composition of Insecticidal Crystal Proteins in Bacillus thuringiensis: a Proteomics Approach

ABSTRACT Bacillus thuringiensis (Bt) is the most widely used active ingredient for biological insecticides. The composition of δ-endotoxins (Cry and Cyt proteins) in the parasporal crystal determines the toxicity profile of each Bt strain. However, a reliable method for their identification and quantification has not been available, due to the high sequence identity of the genes that encode the δ-endotoxins and the toxins themselves. Here, we have developed an accurate and reproducible mass spectrometry-based method (liquid chromatography-tandem mass spectrometry-multiple reaction monitoring [LC-MS/MS-MRM]) using isotopically labeled proteotypic peptides for each protein in a particular mixture to determine the relative proportion of each δ-endotoxin within the crystal. To validate the method, artificial mixtures containing Cry1Aa, Cry2Aa, and Cry6Aa were analyzed. Determination of the relative abundance of proteins (in molarity) with our method was in good agreement with the expected values. This method was then applied to the most common commercial Bt-based products, DiPel DF, XenTari GD, VectoBac 12S, and Novodor, in which between three and six δ-endotoxins were identified and quantified in each product. This novel approach is of great value for the characterization of Bt-based products, not only providing information on host range, but also for monitoring industrial crystal production and quality control and product registration for Bt-based insecticides. IMPORTANCE Bacillus thuringiensis (Bt)-based biological insecticides are used extensively to control insect pests and vectors of human diseases. Bt-based products provide greater specificity and biosafety than broad-spectrum synthetic insecticides. The biological activity of this bacterium resides in spores and crystals comprising complex mixtures of toxic proteins. We developed and validated a fast, accurate, and reproducible method for quantitative determination of the crystal components of Bt-based products. This method will find clear applications in the improvement of various aspects of the industrial production process of Bt. An important aspect of the production of Bt-based insecticides is its quality control. By specifically quantifying the relative proportion of each of the toxins that make up the crystal, our method represents the most consistent and repeatable evaluation procedure in the quality control of different batches produced in successive fermentations. This method can also contribute to the design of specific culture media and fermentation conditions that optimize Bt crystal composition across a range of Bt strains that target different pestiferous insects. Quantitative information on crystal composition should also prove valuable to phytosanitary product registration authorities that oversee the safety and efficacy of crop protection products.

Growth and Thermal Properties of Mg-Doped Lithium Isotope Niobate (Mg:7LiNbO3) Crystal

An Mg-doped isotope lithium niobate (Mg:7LiNbO3) crystal was successfully grown from 7LiOH, Nb2O5, and MgO using the Crozchralski method. The weight of the as-grown crystal with good quality was about 40 g. The crystal structure was determined as an R3c space group using the X-ray powder diffraction (XRPD) method, and the crystal composition (Li%) determined using the Raman mode linewidth method was 49.29%. The average transmittance of the crystal in the range of 500–2500 nm was approximately 72%. Various thermal properties, including the specific heat (Cp), the thermal expansion coefficient (α), the thermal diffusion coefficient (λ), and the thermal conductivity (κ), were carefully determined and calculated, and the value divergences among Mg:7LiNbO3, the undoped isotope lithium niobate (7LiNbO3), and natural lithium niobate (LiNbO3) crystals were mainly related to the differences in microstructure caused by the crystal composition.

Broad Band Nonlinear Crystals of Multicomponent Chalcogenides for the Mid-Ir Applications

The main trends in change of functional characteristics are considered as crystal composition is varied.