Thuringiensis Mm2 by about 50 . From these studies, we can conclude that specific mineral nutrients, such as Mg, Cu, and Mn, influence ICP expression and that the effects of these elements vary significantly according to the strains applied and ICP types produced. However, the molecular mechanisms of the effects caused by mineral elements have not been reported yet. Bacillus thuringiensis X022 was isolated in this study and showed strong insecticidal activity against Spodoptera exigua and Helicoverpa armigera. Adding an appropriate amount of Cu2+ to the medium evidently improved the Celecoxib synthesis of 130 kDa ICP. We performed two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC S/MS) to explore the regulatory mechanisms of ICP biosynthesis. This study presents new insights into the effects of metal ions on the metabolic processes and ICP yields of B. thuringiensis and provides new data on the regulation of ICP production.grow very well at either lower or higher than pH 8.0, and therefore doesn’t produce much ICP (Fig. 2a, d). SDS-PAGE showed that the production of 130 kDa crystal proteins was increased by 21 and that the optimal Cu2+ concentration ranges from 10-6 to 10-4 mol/L (Fig. 2c, e). emPAI semi-quantitative analysis of ICP (Additional file 1: Table S3) and qRT-PCR (Fig. 3) showed that Cu2+ increases the expression of Cry1Da and Cry1Ca. Toxicity tests demonstrated that Cu2+ can increases the toxicity of B. thuringiensis X022 to S. exigua and H. armigera (Fig. 4). I en et al. [6] found that Cu greatly stimulates the biosynthesis of 135 and 65 kDa toxin components in B. thuringiensis 81 at concentrations ranging from 10-7 to 10-6 mol/L. However, kan et al. [7] demonstrated that Cu does not favor the biosynthesis of Cry11Aa and Cry4Ba in B. thuringiensis subsp. israelensis HD500. The effect of Cu2+ on ICP biosynthesis may vary according to different strains. The present study showed that appropriate concentrations of Cu2+ have positive effects on the expression of Cry1Da and Cry1Ca in B. thuringiensis X022; this effect, however, is not significant on Cry1Ac. Oves et al. [9] found that B. thuringiensis strain OSM29 shows obvious metal-removing potential; the biosorption capacity of strain OSM29 for Cu is 91.8 at 25 mg/L initial metal ion concentration. Thus, Cu2+ can be absorbed by B. thuringiensis and, after entering the cell, bring about changes in metabolic regulation that can affect bacterial growth and ICP synthesis.Effects of Cu2+ on insecticidal activityResults and discussionCharacterization and identification of the novel strain Bacillus thuringiensis PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/16989806 XBacillus thuringiensis strain X022 in the vegetative phase is rod shaped and around 2.5?.8 m ? 0.5?.7 m in size (Fig. 1a). When it enters the sporulation phase, the strain gradually forms spores and diamond-shaped parasporal crystals (Fig. 1b). The 16S rRNA gene sequence of the strain was uploaded in GenBank of the NCBI database under accession number KJ698649. A phylogenetic tree based on the 16S rRNA gene sequence of the strain was constructed (Fig. 1c). Its 16S rRNA gene is highly homologous to that of B. thuringiensis serovar kurstaki str. HD73 (99.80 identity).Effects of pH and Cu2+ on ICP biosynthesisThe effect of initial pH on ICP biosynthesis was investigated with varying pH ranging from 7.5 to 10.0 (the pH was obtained before sterilization). The relative amount of crystal protein produced was tested by SDS-PAGE. Results showed that 130.