Proteomics Analyses Of Bacillus Thuringiensis Subsp. Chinensis Ct-43

Bacillus thuringiensis is the earliest and the most deeply study of entomopathogenic bacteria. Moreover, it is used as environmentally compatible biopesticide worldwide. One of the most significant features of B. thuringiensis is the formation of parasporal crystals consisting of insecticidal crystal proteins (ICPs) during sporulation, constituting up to20-30%of the cell dry weight. Isobaric tags for relative and absolute quantitation (iTRAQ) technology, a new quantitative proteomics technology, is the latest developed technology in recent years, and is widely used. In this study, we investigated the metabolic regulatory mechanisms associated with the high-level accumulation of ICPs by performing the proteomic analysis of B. thuringiensis strain CT-43, using iTRAQ technique. In8samples (two biological replicates) of7h,9h,13h and22h these four growth phases,1756proteins were identified finally. The results showed that the number of differential proteins reached the peak between7h and22h, followed by9h versus22h,7h versus13h and13h versus22h, while it was the least between7h and9h. Therefore it indicated that the physiological metabolic activities should have great differences between the sporulating cells and nonsporulating cells. And these should be the truly reflection of the regulation of the gene expression and metabolic pathways for the sporulation and ICPs high-level expression in Bt. The total identified proteins of4growth phases and in iTRAQ data were further performed the analysis of COG (cluster of orthologous groups of proteins). At the translational level, using the results of13h as an example, the gene number order of the COG group was R (general function prediction only), E (amino acid transport and metabolism), J (translation, ribosomal structure and biogenesis), C (energy production and conversion), K (transcription), S (function unknown), G (carbohydrate transport and metabolism), M (cell envelope biogenesis, outer membrane). Besides, these results suggested that some physiological metabolic activities including amino acid transport and metabolism, transcriptionand translation, carbohydrate and energy metabolism changed noticeably during sporulation. These changes should be in accordance with the real regularity for metabolic proceeding To reveal the regulation mechanism of the metabolic pathways associated with the high-leve expression of ICP genes, the global quantitative proteomics of strain CT-43were comprehensively analysized by isobaric tags for relative and absolute quantitation (iTRAQ) technique, respectively. Our results indicated that the proteases and the metabolism of some amino acids, particularly, the BCAAs (branched-chain amino acids), became more active during sporulation. The accumulated PHB, acetoin and some low-quality substances would be important carbon and energy sources for sporulation and the formatiom of parasporal crystals. The tricarboxylic acid cycle was also significantly modified during sporulation. There was a significant increase in the levels of the enzymes and cytochromes related with energy production via the electron transport system. Finally, most subunits of the FOF1-ATPase were increased more than1.8-fold during sporulation. Our results, to our knowledge, systematically revealed the metabolic regulatory mechanism of the supply of the amino acids, energy substances and energy for the formation of spores and parasporal crystals at translational levels.