Comparative Proteomic Analysis Revealed Metabolic Changes And The Translational Regulation Of Cry Protein Synthesis In Bacillus Thuringiensis YBT-1520

Bacillus thuringiensis is a Gram-positive endospore forming bacterium that produces insecticidal crystal proteins (ICPs) which are principal bio-pesticides used in agricultural and sanitary pest controls. ICPs could accumulate and account for up to20to30%of the dry weight of sporulated cells. Although the transcription studies of ICPs’ genes were performed a lot, very little is known about the translational regulation of ICPs’ synthesis. The comparative proteomic analysis of B. thuringiensis strain YBT-1520that grew in a minimal medium was performed to understand the translational regulation of ICPs synthesis. The different pH gradient of2-DE gels included pH4-7, pH5-8, pH3-10, and pH7-11were tested before the formal experiment. The pH4-7gradient was observed well separation of whole cell protein in YBT-1520. Cell samples for protein and for total RNA isolation were collected at the time points of17h,20h and26h with corresponding OD600at0.284,0.522and0.724respectively. The three replication pH4-7gradient2-DE gels for differrent growth phases of YBT-1520were constructed. Eight to nine hundreds protein spots could be separated in pH4-7gradient2-DE gels which were stained by coomassie brilliant blue G-250. Two hundreds and four proteins changed at least two folds in spot volumes on2-DE gels between mid-log phase and stationary phase. Among them,101proteins were chosen for MALDI-TOF-MS analysis, and72of which were further identified by the MASCOT Peptide Mass Fingerprinting (PMF) search. The transcription levels of carbohydrate metabolism related genes were also studied by real-time RT-PCR. Significant changes in carbohydrate, fatty acid, acetyl-CoA, and amino acid metabolisms in the different phases were revealed in this study. Tricarboxylic acid cycle (TCA cycle), the part of glycolysis and the synthesis of most amino acids were depressed from log to stationary phases. Acetyl-CoA mainly entered into TCA cycle, PHB, fatty acid, and acetate synthesis in log phase but only converted to fatty acid in stationary phase. Metabolic changes indicated that the amino acid precursors of ICPs were mainly oBtained from the protein degradation in vivo. The energy produced from fatty acid oxidation and glycolysis in stationary phase could meet the need of ICPs synthesis. The expression changes of EF-Tu, YfiA, GroEL and GatB suggested the direct translational regulation of ICPs’synthesis in B. thuringiensis. This work was the first comparative proteomic study of B. thuringiensis grew in defined medium at different growth phases. The research contributes to better understand the relationship between metabolic changes and ICPs synthesis in B. thuringiensis. The analysis of metabolic changes provides a new sight in the translational regulation of ICPs’synthesis.