2015 ; Vol.25-2: 152~161
|Author||Mingxia Qi, Fei Mei, Hui Wang, Ming Sun, Gejiao Wang, Ziniu Yu, Yeonho Je, Mingshun Li|
|Place of duty||State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Hongshan District, Wuhan 430070, P.R. China|
|Title||Function of Global Regulator CodY in Bacillus thuringiensis BMB171 by Comparative Proteomic Analysis|
J. Microbiol. Biotechnol.2015 ;
|Abstract||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.|
|Key_word||CodY, Bacillus thuringiensis, proteomics, poly-β-hydroxybutyrate, branched chain amino acids, FRR|
Arantes O, Lereclus D. 1991. Construction of cloning vectors for Bacillus thuringiensis. Gene 108: 115-119.
Belitsky BR, Sonenshein AL. 2013. Genome-wide identification of Bacillus subtilis CodY binding sites at single nucleotide resolution. Proc. Natl. Acad. Sci. USA 110: 7026-7031.
Bergara F, Ibarra C, Iwamasa J, Patarroyo JC, Aguilera R, Marquez-Magan LM. 2003. CodY is a nutritional repressor of flagellar gene expression in Bacillus subtilis. J. Bacteriol. 185: 3118-3126.
Blum H, Beier H, Gross H. 1987. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8: 93-99.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
Burbulys D, Trach KA, Hoch JA. 1991. Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay. Cell 6: 545-552.
Château A, van Schaik W, Joseph P, Handke LD, McBride SM, Smeets FM, et al. 2013. Identification of CodY targets in Bacillus anthracis by genome-wide in vitro binding analysis. J. Bacteriol. 195: 1204-1213.
Chen D, Xu D, Li M, He J, Gong Y, Wu D, Sun M, Yu Z. 2012. Proteomic analysis of Bacillus thuringiensis ΔphaC mutant BMB171/PHB-1 reveals that the PHB synthetic pathway warrants normal carbon metabolism. Proteomics 75: 5176-5188.
Chen HJ, Tsai TK, Pan SC, Lin JS, Tseng CL, Shaw GC. 2010. The master transcription factor Spo0A is required for poly (3-hydroxybutyrate) (PHB) accumulation and expression of genes involved in PHB biosynthesis in Bacillus thuringiensis. FEMS Microbiol. Lett. 304: 74-81.
Cheng Q, Campbell EA, Naughton AM, Johnson S, Masure HR. 1997. The com locus controls genetic transformation in Streptococcus pneumonia. Mol. Microbiol. 23: 683-692.
Defeu Soufo HJ, Graumann PL. 2005. Bacillus subtilis actin-like protein MreB influences the positioning of the replication machinery and requires membrane proteins MreC/D and other actin-like proteins for proper localization. BMC Cell Biol. 6: 10.
Den Hengst CD, van Hijum SA, Geurts JM, Nauta A, Kok J, Kuipers OP. 2005. The Lactococcus lactis CodY regulon:identification of a conserved cis-regulatory element. J. Biol. Chem. 280: 34332-34342.
Dennis D, Sein V, Martinez E, Augustine B. 2008. PhaP is involved in the formation of a network on the surface of polyhydroxyalkanoate inclusions in Cupriavidus necator H16. J. Bacteriol. 190: 555-563.
Dineen SS, McBride SM, Sonenshein AL. 2010. Integration of metabolism and virulence by Clostridium difficile CodY. J. Bacteriol. 192: 5350-5362.
Domínguez-Escobar J, Chastanet A, Crevenna AH, Fromion V, Wedlich-Söldner R, Carballido-López R. 2011. Processive movement of MreB-associated cell wall biosynthetic complexes in bacteria. Science 333: 225-228.
Edwards DH, Errington J. 1997. The Bacillus subtilis DivIVA protein targets to the division septum and controls the site specificity of cell division. Mol. Microbiol. 24: 905-915.
Fenner C, Traut RR, Mason DT, Wikman-Coffelt J. 1975. Quantification of Coomassie Blue stained proteins in polyacrylamide gels based on analyses of eluted dye. Anal. Biochem. 6: 595-602.
Gong Y, Li M, Xu D, Wang H, He J, Wu D, et al. 2012. Comparative proteomic analysis revealed metabolic changes and the translational regulation of Cry protein synthesis in Bacillus thuringiensis. Proteomics 75: 1235-1246.
Guo M, Feng H, Zhang J, Wang W, Wang Y, Li Y, et al. 2009. Dissecting transcription regulatory pathways through a new bacterial one-hybrid reporter system. Genome Res. 19:1301-1308.
Handke LD, Shivers RP, Sonenshein AL. 2008. Interaction of Bacillus subtilis CodY with GTP. J. Bacteriol. 190: 798-806.
Hirokawa G, Demeshkina N, Iwakura N, Kaji H, Kaji A. 2006. The ribosome recycling step: consensus or controversy? Trends Biochem. Sci. 31: 143-149.
Hirokawa G, Kiel MC, Muto A, Selmer M, Raj VS, Liljas A, et al. 2002. Post-termination complex disassembly by ribosome recycling factor, a functional tRNA mimic. EMBO J. 21:2272-2281.
Hsueh YH, Somers EB, Wong AC. 2008. Characterization of the cody gene and its influence on biofilm formation in Bacillus cereus. Arch. Microbiol. 189: 557-568.
Jiang M, Grau R, Perego M. 2000. Differential processing of propeptide inhibitors of Rap phosphatases in Bacillus subtilis. J. Bacteriol. 182: 303-310.
Law JH, Slepecky RA. 1961. Assay of poly-beta-hydroxybutyric acid. J. Bacteriol. 82: 33-36.
Levdikov VM, Blagova E, Joseph P, Sonenshein AL, Wilkinson AJ. 2006. The structure of CodY, a GTP and isoleucine-responsive regulator of stationary phase and virulence in gram-positive bacteria. J. Biol. Chem. 281: 1136611373.
Li L, Yu ZN. 1999. The transform and expressed property of Bacillus thuringiensis non-plasmid mutant strain BMB171. Chin. J. Appl. Environ. Microbiol. 5: 395-399.
Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25: 402-408.
Majerczyk CD, Dunman PM, Luong TT, Lee CY, Sadykov MR, Somerville GA, et al. 2010. Direct targets of CodY in Staphylococcus aureus. J. Bacteriol. 192: 2861-2877.
Molle V, Nakaura Y, Shivers RP, Yamaguchi H, Losick R, Fujita Y, Sonenshein AL. 2003. Additional targets of the Bacillus subtilis global regulator CodY identified by chromatin immuno-precipitation and genome-wide transcript analysis. J. Bacteriol. 185: 1911-1922.
Ogura M, Tanaka T. 2009. The Bacillus subtilis late competence operon comE is transcriptionally regulated by yutB and under post-transcription initiation. J. Bacteriol. 191: 949-958.
Park HD, Guinn KM, Harrell MI, Liao R, Voskuil MI, Tompa M, et al. 2003. Rv333c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis. Mol. Microbiol. 48: 833-843.
Pötter M, Madkour MH, Mayer F, Steinbüchel A. 2002. Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16. Microbiology 148: 2413-2426.
Ratcliff WC, Kadam SV, Denison RF. 2008. Poly-3hydroxybutyrate (PHB) supports survival and reproduction in starving rhizobia. FEMS Microbiol. Ecol. 65: 391-399.
Ratnayake-Lecamwasam M, Serror P, Wong KW, Sonenshein AL. 2001. Bacillus subtilis CodY represses early-stationary-phase genes by sensing GTP levels. Genes Dev. 15: 1093-1103.
Serror P, Sonenshein AL. 1996. CodY is required for nutritional repression of Bacillus subtilis genetic competence. J. Bacteriol. 178: 5910-5915.
Shivers RP, Sonenshein AL. 2004. Activation of the Bacillus subtilis global regulator CodY by direct interaction with branched-chain amino acids. Mol. Microbiol. 53: 599-611.
Sonenshein AL. 2005. CodY, a global regulator of stationary phase and virulence in gram-positive bacteria. Curr. Opin. Microbiol. 8: 203-207.
Sonenshein AL. 2007. Control of key metabolic intersections in Bacillus subtilis. Nat. Rev. Microbiol. 5: 917-927.
Sonenshein AL. 2000. Control of sporulation initiation in Bacillus subtilis. Curr. Opin. Microbiol. 3: 561-566.
Thomaides HB, Freeman ME, Karoui M, Errington J. 2001. Division site selection protein DivIVA of Bacillus subtilis has a second distinct function in chromosome segregation during sporulation. Genes Dev. 15: 1662-1673.
Villapakkam AC, Handke LD, Belitsky BR, Levdikov VM, Wilkinson AJ, Sonenshein. AL. 2009. Genetic and biochemical analysis of the interaction of Bacillus subtilis CodY with branched-chain amino acids. J. Bacteriol. 191: 6865-6876.
Ween O, Gaustad P, Havarstein LS. 1999. Identification of DNA binding sites for ComE, a key regulator of natural competence in Streptococcus pneumonia. Mol. Microbiol. 33:817-827.
Wieczorek R, Pries A, Steinbüchel A, Mayer F. 1995. Analysis of a 24-kilodalton protein associated with the polyhydroxyalkanoic acid granules in Alcaligenes eutrophus. J. Bacteriol. 177:2425-2435.
Wu D, He J, Gong Y, Chen D, Zhu X, Qiu N, et al. 2011. Proteomic analysis reveals the strategies of Bacillus thuringiensis YBT-1520 for survival under long-term heat stress. Proteomics 11: 2580-2591.
Yokoyama T, Shaikh TR, Iwakura N, Kaji H, Kaji A, Agrawal RK. 2012. Structural insights into initial and intermediate steps of the ribosome-recycling process. EMBO J. 31: 1836-1846.
Zavialov AV, Hauryliuk VV, Ehrenberg M. 2005. Splitting of the post-termination ribosome into subunits by the concerted action of RRF and EF-G. Mol. Cell 18: 675-686.