Proteomic analysis of the influence of Cu2+ on the crystal protein production of Bacillus thuringiensis X022

Abstract

BackgroundBacillus thuringiensis X022, a novel strain isolated from soil in China, produces diamond-shaped parasporal crystals. Specific mineral nutrients, such as Mg, Cu, and Mn, influence insecticidal crystal proteins (ICP) expression and the effects of these elements vary significantly. However, the molecular mechanisms of the effects caused by mineral elements have yet to be reported.ResultsThe ICP are mainly composed of Cry1Ca, Cry1Ac, and Cry1Da, which have molecular weights of about 130 kDa. ICP production was most efficient when Cu2+ was added at concentrations ranging from 10−6 to 10−4 mol/L at an initial pH of 8.0. Addition of Cu2+ also evidently increased the toxicity of fermentation broth to Spodoptera exigua and Helicoverpa armigera. After analyzing changes in proteome and fermentation parameters caused by Cu2+ addition, we propose that Cu2+ increases PhaR expression and consequently changes the carbon flow. More carbon sources was used to produce intracellular poly-β-hydroxybutyrate (PHB). Increases in PHB as a storage material bring about increases of ICP production.ConclusionsBacillus thuringiensis X022 mainly expresses Cry1Ca, Cry1Ac, and Cry1Da. Cu2+ increases the expression of Cry1Da, Cry1Ca, and also enhances the toxicity of fermentation broth to S. exigua and H. armigera.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0339-9) contains supplementary material, which is available to authorized users.

Comparative Analysis of Genomics and Proteomics in the New Isolated Bacillus thuringiensis X022 Revealed the Metabolic Regulation Mechanism of Carbon Flux Following Cu2+ Treatment

“…The novel isolated strain Bt X022 (CCTCC No. M2014158) was previously described by Liu et al (2015) . Bt X022 cells was cultured overnight at 30°C with agitation at 200 rpm in liquid LB medium, comprised of polypeptone 10 g; yeast extract 5 g and NaCl 10 g/L (pH 7.6).…”

“…The extraction of total bacterial proteins from X022 cells, trypsin digestion and 2D-LC-MS/MS analysis, database search and functional classification processes were previously described by Liu et al (2015) .…”

“…Following digestion of the residual genomic DNA by DNase I (Thermo Fisher Scientific Co. Ltd., Shanghai, China), 1 μg of total RNA was reverse transcribed to cDNA utilizing RevertAid TM First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Co. Ltd., Shanghai, China) according to the manufacturer’s instruction. The two-step real-time RT-PCR analysis were conducted using Power SYBR ® Green PCR Master Mix (Thermo Fisher Scientific Co. Ltd., Shanghai, China) in a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA), as previously described ( Liu et al, 2015 ). qRT-PCR primers used in this study were summarized in Table 2 .…”

“…kurstaki . A study by Liu et al (2015) found that the ICP production of Bt X022 was efficiently increased when a appropriate concentration of Cu 2+ was added. The mechanisms involved were further explored by performing a comparative proteomic analysis of the total proteins for X022 in spore-release period based on LC-MS/MS approach.…”

“…Secondly, the resulting genomic sequence data were assembled and functional annotated using the Rapid Annotation using Subsystem Technology (RAST) server. Moreover, an in-depth comparative analysis of the genomic sequence data and the proteomic results from Liu et al (2015) for strain X022 was further performed; with the object of mining new insecticidal gene and functional protein resources, and further revealing the complicated regulating mechanisms correlated with increased ICP production and insecticidal toxicity of Bt X022.…”