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KMID : 0545120150250020152
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Journal of Microbiology and Biotechnology
2015 Volume.25 No. 2 p.152 ~ p.161
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Function of Global Regulator CodY in Bacillus thuringiensis BMB171 by Comparative Proteomic Analysis
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Mingxia Qi
Fei Mei
Ming Sun
Gejiao Wang
Ziniu Yu
Je Yeon-Ho
Mingshun Li
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Abstract
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CodY is a highly conserved protein in low G+C gram-positive bacteria that regulates genes involved in sporulation and stationary-phase adaptation. Bacillus thuringiensis is a grampositive bacterium that forms spores and parasporal crystals during the stationary phase. To our knowledge, the regulatory mechanism of CodY in B. thuringiensis is unknown. To study the function of CodY protein in B. thuringiensis, BMB171codY- was constructed in a BMB171 strain. A shuttle vector containing the ORF of cry1Ac10 was transformed into BMB171 and BMB171codY-, named BMB171cry1Ac and BMB171codY-cry1Ac, respectively. Some morphological and physiological changes of codY mutant BMB171codY-cry1Ac were observed. A comparative proteomic analysis was conducted for both BMB171codY-cry1Ac and BMB171cry1Ac through two-dimensional gel electrophoresis and MALDI-TOF-MS/MS analysis. The results showed that the proteins regulated by CodY are involved in microbial metabolism, including branched-chain amino acid metabolism, carbohydrate metabolism, fatty acid metabolism, and energy metabolism. Furthermore, we found CodY to be involved in sporulation, biosynthesis of poly-β-hydroxybutyrate, growth, genetic competence, and translation. According to the analysis of differentially expressed proteins, and physiological characterization of the codY mutant, we performed bacterial one-hybrid and electrophoretic mobility shift assay experiments and confirmed the direct regulation of genes by CodY, specifically those involved in metabolism of branched-chain amino acids, ribosomal recycling factor FRR, and the late competence protein ComER. Our data establish the foundation for in-depth study of the regulation of CodY in B. thuringiensis, and also offer a potential biocatalyst for functions of CodY in other bacteria.
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KEYWORD
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CodY, Bacillus thuringiensis, proteomics, poly-β-hydroxybutyrate, branched chain amino acids, FRR
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