Characterization and Functional Study of PyrR Orthologues from Genome Sequences of Bacteria

세균 게놈 유래성 PyrR Orthologue의 기능 분석

  • 김사열 (경북대학교 미생물학과) ;
  • 조현수 (경북대학교 미생물학과) ;
  • 설경조 (경북대학교 미생물학과) ;
  • 박승환 (한국생명공학연구원 미생물유전체연구실)
  • Published : 2003.06.01


The regulation of pyrimidine nucleotide synthesis has been proved to be controlled by a regulatory protein PyrR-mediated attenuation in the Gram-positive bacteria. After several bacterial genome sequencing projects, we have discovered the PyrR orthologues in the databases for Haemophilus influenzae and Synechocystis and sp. PCC6803 genome sequences. To investigate whether these PyrR orthologue proteins regulate pyrimidine nucleotide synthesis as well as the cases of Bacillus, the PyrR regions of each strains were amplified by PCR and cloned with pUC19 or T-vector in Escherichia coli and with a shuttle vector pHPS9 for E. coli and B. subtilis. For the regulation test of the PyrR orthologues, the aspartate-transcarbamylase (ATCase) assay was carried out. From the results of the ATCase assay, it was confirmed that Synechocystis sp. PCC6803 could not restore by pyrimidines to a B. subtilis, PyrR but H. influenzae PyrR could. For Purification of PyrR orthologue proteins, PyrR orthologue genes were cloned into the expression vector (pET14b). Over-expressed product of PyrR orthologue genes was purified and analyzed by the SDS-PACE. The purified PyrR orthologue proteins from H. influenzae and Synechocystis sp. PCC6803 turned out to be molecular mass of 18 kDa and 21 kDa, respectively. The result of uracil phosphoribosyl transferase (UPRTase) assay with purified PyrR orthologue proteins showed that H. influenzae PyrR protein only has UPRTase activity. In addition, we could predict several regulatory mechanisms that PyrR orthologue proteins regulate pyrimidine de novo synthesis in bacteria, through phylogenetic analysis for PyrR orthologue protein sequences.


PyrR orthologue;UPRTase;ATCase;expression;phylogenetic tree


  1. Anal. Biochem. v.72 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding Bradford,M.M.
  2. J. Bacteriol. v.183 The pyrimidine operon pyrRPB-carA from Lactococcus lactis Martinussen,J.;J.Schallert;B.Andersen;K.Hammer
  3. Science v.269 Whole-genome random sequencing and assembly of Haemophilus influenzae Rd Fleischmann,R.D.(39 coauthors)
  4. J. Bacteriol. v.178 Transcriptional attenuation of the Bacillus subtilis pyr operon by the PyrR regulatory protein and uridine nucleotides in vitro Lu,Y.;R.L.Switzer
  5. Bacillus subtilis and other Gram-positive bacteria Nygaard,P.;A.L.Sonenshein(ed.);J.A.Hoch(ed.);R.Losick(ed.)
  6. Nucleic Acids Res. v.29 Molecular recognition of pyr mRNA by the Bacillus subtilis attenuation regulatory protein PyrR Bonner,E.R.;J.N.D'Elia;B.K.Billips;R.L.Switzer
  7. Ph. D. thesis, University of Copenhagen The pyr operon of the themophile Bacillus caldolyticus encodes both de novo and salvage enzymes for UMP synthesis Ghim,S.Y.
  8. Gene v.33 Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors Yanisch-Perron,C.;J.Vieira;J.Messing
  9. Eur. J. Appl. Microbiol. Biotechnol. v.6 A rapid gas chromatographic method for the determination of poly-3-hydroxybutyric acid in microbial biomass Braunegg,G.;B.Sonnleitner;R.M.Lafferty
  10. J. Bacteriol v.176 Regulation of the Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster by an autogenous transcriptional attenuation mechanism Turner,R.J.;Y.Lu;R.L.Switzer
  11. Gene v.182 Structure and organisation of the pyrimidine biosynthesis pathway genes in Lactobacillus plantarum: a PCR strategy for sequencing without cloning Elagoz,A.;A.Abdi;J.C.Hubert;B.Kammerer
  12. J. Bacteriol v.176 The pyrimidine biosyn-thesis operon of the Bacillus caldolyticus includes genes for uracil phosphoribosyltransferase and uracil permease Ghim,S.Y.;J.Neuhard
  13. DNA Res v.2 Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. I. Sequence features in the 1Mb region from map positions 64% to 92% of the genome Kaneko,T.;A.Tanaka;S.Sato;H.Kotani;T.Sazuka;N.Miyajima;M.Sugiura;S.Tabata
  14. Mol. Biol. Evol. v.4 The neighbor-joining method: a new method reconstructing phylogenetic trees Saitou,N.;M.Nei
  15. J. Bacteriol. v.160 Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases Kawamura,F.;R.H.Doi
  16. J. Bacteriol v.183 Genome sequence and comparative analysis of the solvent-producting bacterium Clostridium acetobutylicum Nolling,J.G.;Breton,M.V;Omelchenko,K.S.;Makarova,Q.Zeng;R.Gibson;H.M.Lee;J.Dubois;D.Qiu;J.Hitti;Y.I.Wolf;R.L.Tatusov;F.Sabathe;L.Doucette-Stamm;P.Soucaille;M.J.Daly;G.N.Bennett;E.V.Koonin;D.R.Smith
  17. Gene v.86 Development of a -galactosidase -comple-mentation system for molecular cloning in Bacillus subtillis Haima,P.;D.Van Sinderen;H.Schotting;S.Bron;G.Vfnema
  18. Nucleic Acids Res v.25 The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools Thompson,J.D.;T.J.Gibson;F.Plewniak;F.Jeanmougin;D.G.Higgins
  19. Nature v.406 Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen Stover,C.K.;X.Q.Pham;A.L.Erwin;S.D.Mizoguchi;P.Warrener;M.J.Hickey;F.S.Brinkman;W.O.Hufnagle;D.J.Kowalik;M.Lagrou;R.L.Garber;L.Goltry;E.Tolentino;S. Westbrock-Wadman;Y.Yuan;L.L.Brody;S.N.Coulter;K.R.Folger;A.Kas;K.Larbig;R.Lim;K.Smith;D.Spencer;G.K.Wong;Z.Wu;I.T.Paulsen;J.Reizer;M.H.Saie;R.E.Hancock;S.Lory;M. V,Olson
  20. Nature v.393 Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence Cole.S.T.(41 coauthors)
  21. J. Bacteriol v.62 Studies of lysogenesis. I. The mode of phage libration by lysogenic Escherichia coli Bertani,G.
  22. Comput. Appl. Biosci. v.10 MEGA: Molecular evolutionary genetics analysis software for microcomputers Kumar,S.;K.Tamura;M.Nei
  23. J. Bacteriol. v.181 The Enterococcus faecalis pyr operon is regulated by autogenous transcriptional attenuation at a single site in the 5'leader Ghim,S.Y.;C.C.Kim;E.R.Bonner;J.N.D'Elia;G.K.Grabner;R.L.Switzer
  24. Paths to pyrimidines v.6 A widespread and mechanistically versatile regulator of bacterial pyr genes Switzer,R.L.;R.J.Turner
  25. Proc. Natl. Acad. Sci v.82 Catabolite-resistant sporulation (crsA) mutations in the Bacillus subtilis suppressed by mutations in spo0 genes Kawamura,F.;L.F.Wang;R.H.Dol
  26. Methods in Enzymology v.167 DNA transformation Porter,R.D.
  27. Molecular cloning: a laboratory manuarl(3rd ed) Sambrook,J.;D.W.Russell
  28. J. Bacteriol v.110 Regulation of the bacterial cell wall: analysis of a mutent of Bacillus subtilis defective in biosynthesis of teichoic acid Boylan,R.J.;N.H.Mendelson;D.Brooks;F.E.Young
  29. J. Exp. Med. v.161 Pyrimidine salvage in Giardia lamblia Aldritt,S.M.;P.Tien;C.C.Wang
  30. J. Bacteriol v.153 Nutritional regulational of degradation of aspartate transcarbamy-lase and of bulk protein in exponentially growing Bacillus subtilis cells. Bond,R.W.;A.S.Field;R.W.Switzer
  31. J. Bacteriol. v.179 Structure and expression of a pyrimidine gene cluster from the extreme thermophile Thermus strain ZO5 Van de Casteele,M.;P.Chen;M.Roovers;C.Legrain;N.Glansdorff
  32. Nature v.227 Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Laemmli,U.K.
  33. Biochim. Biophys. Acta v.881 Purification and some properties of uracil phosphoribosyltranscation from Escherichia coli K12 Rasmussen,U.B.;B.Mygind;P.Nygaard
  34. J. Bacteriol. v.123 pyrR identical to pyrH in Salmonella typhimurium: control of expression of the pyr genes Justesen,J.;J.Neuhard
  35. Proc. Natl. Acad. SCi. USA. v.93 Function of RNA secondary structures in transcriptional attenuation of the Bacillus subtilis pyr operon Lu,Y.;R.J.Turner;R.L.Switzer
  36. Proc. Natl. Acad. Sci. v.44 Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate Spizizen,J.