Characterization of a PyrR-deficient Mutant of Bacillus subtilis by a Proteomic Approach
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The Bacillus subtilis pyrimidine biosynthetic (pyr) operon encodes all of the enzymes for the de novo biosynthesis of Uridine monophosphate (UMP) and additional cistrones encoding a uracil permease and the regulatory protein PyrR. The PyrR is a bifunctional protein with pyr mRNA-binding regulatory funtion and uracil phosphoribosyltransferase activity. To study the global regulation by the pyrR deletion, the proteome comparison between Bacillus subtilis DB104 and Bacillus subtilis DB104 ΔpyrR in the minimal medium without pyrimidines was employed. Proteome analysis of the cytosolic proteins from both strains by 2D-gel electrophoresis showed the variations in levels of protein expression. On the silver stained 2D-gel with an isoelectric point (pI) between 4 and 10, about 1,300 spots were detected and 172 spots showed quantitative variations in which 42 high quantitatively variant proteins were identified. The results showed that production of the pyrimidine biosynthetic enzymes (PyrAA, PyrAB, PyrB, PyrC, PyrD, and PyrF) were significantly increased in B. subtilis DB104 ΔpyrR. Besides, proteins associated carbohydrate metabolism, elongation protein synthesis, metabolism of cofactors and vitamins, motility, tRNA synthetase, catalase, ATP–binding protein, and cell division protein FtsZ were overproduced in the PyrR–deficient mutant. Based on analytic results, the PyrR might be involved a number of other metabolisms or various phenomena in the bacterial cell besides the pyrimidine biosynthesis.
Abstract
The Bacillus subtilis pyrimidine biosynthetic (pyr) operon encodes all of the enzymes for the de novo biosynthesis of Uridine monophosphate (UMP) and additional cistrones encoding a uracil permease and the regulatory protein PyrR. The PyrR is a bifunctional protein with pyr mRNA-binding regulatory funtion and uracil phosphoribosyltransferase activity. To study the global regulation by the pyrR deletion, the proteome comparison between Bacillus subtilis DB104 and Bacillus subtilis DB104 ΔpyrR in the minimal medium without pyrimidines was employed. Proteome analysis of the cytosolic proteins from both strains by 2D-gel electrophoresis showed the variations in levels of protein expression. On the silver stained 2D-gel with an isoelectric point (pI) between 4 and 10, about 1,300 spots were detected and 172 spots showed quantitative variations in which 42 high quantitatively variant proteins were identified. The results showed that production of the pyrimidine biosynthetic enzymes (PyrAA, PyrAB, PyrB, PyrC, PyrD, and PyrF) were significantly increased in B. subtilis DB104 ΔpyrR. Besides, proteins associated carbohydrate metabolism, elongation protein synthesis, metabolism of cofactors and vitamins, motility, tRNA synthetase, catalase, ATP–binding protein, and cell division protein FtsZ were overproduced in the PyrR–deficient mutant. Based on analytic results, the PyrR might be involved a number of other metabolisms or various phenomena in the bacterial cell besides the pyrimidine biosynthesis.
참고문헌 (34)
- Aldritt, S. M., "Pyrimidine salvagein Giardia lamblia", J. Exp. Med, 161, pp.437~445 (1985)
- Bradford, M. M, "A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye-binding", Anal. Biochem, 72, pp.248~254 (1976)
- Bonner, E. R., "Molecular recognition of pyr mRNA by the Bacillussubtilis attenuation regulatory protein PyrR", Nucleic AcidsRes, 29, pp.4851~4865 (2001)
- Chander, P., "Structure ofthe nucleotide complex of PyrR, the pyr Attenuation proteinfrom Bacillus caldolyticus, suggests dual regulation by pyrimidineand purine nucleotides", J. Bacteriol, 187, pp.1773~1782 (2005)
- Fields, C. J, "Regulation of pyr geneexpression in Mycobacterium smegmatis by PyrR-dependenttranslational repression", J. Bacteriol, 189, pp.6236~6245 (2007)
- Ghim, S-Y, "The pyr operon of the themophile Bacilluscaldolyticus encodes both de novo and salvage enzymes forUMP synthesis", 박사, pp.1993
- Ghim, S-Y, "The pyrimidine biosynthesisoperon of the thermophile Bacillus caldolyticus includesgenes for uracil phosphoribosyltransferase and uracil permease", J. Bacteriol, 176, pp.3698~3707 (1994)
- Ghim, S-Y., "Molecularcharacterization of pyrimidine biosysthesis genes from thethermophile Bacillus caldolyticus", Microbiol, 140, pp.479~491 (1994)
- Ghim, S-Y, "The pyrimidine biosynthesisoperon of the thermophile Bacillus caldolyticus includesgenes for uracil phosphoribosyltransferase and uracil permease", J. Bacteriol, 176, pp.3698~3707 (1994)
- Ghim, S-Y, "Characterization of cisactingmutations in the first attenuator region of the Bacillussubtilis pyr operon that are defective in pyrimidine-mediatedregulation of expression", J. Bacteriol, 178, pp.2351~2355 (1996)
- Ghim, S-Y, "Mutations in Bacillussubtilis PyrR, the pyr regulatory protein, with defects in regulationby pyrimidines", FEMS Microbiol. Lett, 137, pp.13~18 (1996)
- Grabner, G.K, "Kinetic studies of theuracil phosphoribosyltransferase reaction catalyzed by theBacillus subtilis pyrimidine attenuation regulatory proteinPyrR", J. Biol. Chem, 278, pp.6921~6927 (2003)
- Justesen, J, "identical to pyrH inSalmonella typhimurium: control of expression of the pyrgenes", J. Bacteriol, 123, pp.851~854 (1975)
- Kawamura, F, "Construction of Bacillussubtilis double mutant deficient in extracellular alkaline andneutral proteases", J. Bacteriol, 160, pp.442~444 (1984)
- Lerner, C. G, "Cloning and structureof the Bacillus subtilis aspartate transcarbamylase gene(pyrB)", J. Biol. Chem, 261, pp.11156~11165 (1986)
- Lerner, C. G., "Structure of the Bacillus subtilis pyrimidine biosynthetic(pyr) gene cluster", J. Bacteriol, 169, pp.2202~2206 (1987)
- Lu, Y, "Evidence that the Bacillussubtilis pyrimidine biosynthetic regulatory protein PyrR actsby binding to pyr mRNA at three sites in vivo", J. Bacteriol, 178, pp.5806~5809 (1996)
- Lu, Y, "Transcriptional attenuation ofthe Bacillus subtilis pyr operon by the PyrR regulatory proteinand uridine nucleotides in vitro", J. Bacteriol, 178, pp.7206~7211 (1996)
- Lu, Y., "Function of theRNA secondary structures in the regulation of the Bacillussubtilis pyr operon expression", Proc. Natl. Acad. Sci, 93, pp.14462~14467 (1996)
- Lu, Y., "Roles of thethree transcriptional attenuators of the Bacillus subtilis pyrimidinebiosynthetic operon in the regulation of its expression", J. Bacteriol, 177, pp.1315~1325 (1995)
- Martinussen, J., "Two genes encoding uracil phosphoribosiltransferaseare present in Bacillus subtilis", J. Bacteriol, 177, pp.271~274 (1995)
- Martinussen, J., "The pyrimidine operon pyrRPB-carA from Lactococcuslactis", J. Bacteriol, 183, pp.2785~2794 (2001)
- Neuhard, J, "Purines and pyrimidines,In: Escherichia coli and Salmonella typimurium : Cellularand Molecular Biology", pp.445~473 (1987)
- Nygaard, P, "Purine and pyrimidine salvage pathways.In: Bacillus subtilis and Other Gram-positive Bacteria:Biochemistry, Physiology, and Molecular Genetics", pp.359~378 (1993)
- Oakley, B. R., "A simplifiedultrasensitive silver stain for detecting proteins inpolyacrylamide gels", Anal. Biochem, 105, pp.361~363 (1980)
- Quinn, C. L, "Functional organization and nucleotide sequence of theBacillus subtilis pyrimidine biosynthetic operon", J. Biol.Chem, 266, pp.9113~ (1991)
- Robert, J. T, "Autogenous transcriptional attenuation ofde novo pyrimidine biosynthesis in Bacillus subtilis", 박사, pp.1996
- Savacool, H. K, "Characterization ofthe interaction of Bacillus subtilis PyrR with pyr mRNA bysite-directed mutagenesis of the protein", J. Bacteriol, 184, pp.2521~2528 (2002)
- Shevchenko, A., "Mass spectrometric sequencing of proteins silver-stainedpolyacrylamide gels", Anal. Chem, 68, pp.850~858 (1996)
- Spizizen, J, "Transformation of biochemically deficientstrains of Bacillus subtilis by deoxyribonucleate", Proc. Natl.Acad. Sci. USA, 44, pp.1072~1078 (1958)
- Switzer, R. L, "A widespread andmechanistically versatile regulator of bacterial pyr genes", Paths to Pyrimidines, 6, pp.45~52 (1998)
- Tomchick, D. R., "Adaptation of an enzyme to regulatory function: structureof Bacillus subtilis PyrR, a bifuntional pyr RNA-bindingattenuation protein and uracil phosphoribosyltransferase", Structure, 6, pp.337~350 (1998)
- Turner, R. J., "Purification and characterization of Bacillus subtilisPyrR, a bifunctional pyr mRNA-binding attenuation protein", uracil phosphoribosyltransferase, 1998, pp.273, 5932~5938
- Turner, R. J., "Regulation ofthe Bacillus subtilis pyrimidine biosynthetic (pyr) genecluster by an autogenous transcriptional attenuation mechanism", J. Bacteriol, 176, pp.3708~3722 (1994)