Cu²⁺ Influencing Crystal Proteins Production And Insecticidal Activity Of A Novel Strain Bacillus Thuringiensis X022

Bacillus thuringiensis X022(Bt X022) is a novel strain isolated from the soil in China and it produces diamond parasporal crystals. Referring to the morphology under microscope and16S rRNA gene sequenced, we preliminarily determined that Bt X022belongs to Bacillus thuringiensis subsp.kurstaki. SDS-PAGE showed that the molecular weight of crystal proteins produced by Bt X022strain is about130kDa. The whole proteins of cells in spore release period revealed by2D-LC-MS/MS demonstrate that Bt X022mainly expresses3kinds of crystal proteins: Cry1Ca, CryaAc and Cry1Da. Based on the medium optimized for Bt4.0718by Liu Fei et al, we improved the initial pH and dissolved oxygen condition and also find that130kDa crystal proteins production was more efficient when an appropriate amount of Cu2+was added. SDS-PAGE and biomedical image analysis software MiBio showed that10"6mol/L Cu2+makes the production of130kDa crystal proteins increased by21%and the favorable concentration of Cu2+ranges from10-6to10-4mol/L. The emPAI semi-quantitative of ICPs and RT-qPCR showed that Cu2+mainly increases the expression of Cry1Da and Cry1Ca. Toxicity test showed that Cu2+made the toxicity of Bt X022fermentation broth to spodoptera exigua and helicoverpa armigera increased greatly.The influences of Cu2+on growth parameters of Bt X022strain was detected, which revealed that Cu2+resulted in a prolongated stationary phase, acceleration of cell lysis in decline phase; rapid pH rebound, lower pH value on the plateau and a prolonged pH plateau phase.To explore the reasons for the crystal proteins production increase caused by Cu2+, we performed two-dimensional-liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) to analyze the whole proteins of cells in spore release period of Bt X022strain which was cultivated in two different mudium (Cu:added10-6mol/L Cu2+; CK:the original medium, as control). Functional classification and comparative analysis of the proteome revealed that Cu2+caused the down-regulation of environmental information-processing proteins and glycan biosynthesis and metabolism proteins; emPAI semiquantitative comparative analysis and RT-qPCR found that Cu2+induced up-regulation of some functional proteins, such as PhaR, BDH, EF-G, KAS II, ALDH and et al.With comprehensive analysis of the changes of proteome and fermentation parameters (prolongated stationary phase, acceleration of cell lysis, rapid pH rebound) caused by adding Cu2+, we proposed that Cu2+increased the expression of PhaR and consequently changed the carbon flow in Bt X022strain. It caused the down-regulation of environmental information-processing proteins and glycan biosynthesis and metabolism proteins and thus reduced the production of extracellular polysaccharide and result in acceleration of cell lysis. More carbon sources can be used to synthetise intracellular PHB. The increase of PHB as storage material brings about prolongated stationary phase, rapid pH rebound and also releasing more energy for protein translation and ultimately raised the production of ICPs.