Antonie van Leeuwenhoek

, Volume 78, Issue 3–4, pp 253–261 | Cite as

Glucose kinase of Streptomyces coelicolor A3(2): large-scale purification and biochemical analysis

  • Kerstin Mahr
  • Gilles P. van Wezel
  • Cecilia Svensson
  • Ute Krengel
  • Mervyn J. Bibb
  • Fritz TitgemeyerEmail author


Glucose kinase of Streptomyces coelicolor A3(2) is essential for glucose utilisation and is required for carbon catabolite repression (CCR) exerted through glucose and other carbon sources. The protein belongs to the ROK-family, which comprises bacterial sugar kinases and regulators. To better understand glucose kinase function, we have monitored the cellular activity and demonstrated that the choice of carbon sources did not significantly change the synthesis and activity of the enzyme. The DNA sequence of the Streptomyces lividans glucose kinase gene glkA was determined. The predicted gene product of 317 amino acids was found to be identical to S. coelicolor glucose kinase, suggesting a similar role for this protein in both organisms. A procedure was developed to produce pure histidine-tagged glucose kinase with a yield of approximately 10 mg/l culture. The protein was stable for several weeks and was used to raise polyclonal antibodies. Purified glucose kinase was used to explore protein-protein interaction by surface plasmon resonance. The experiments revealed the existence of a binding activity present in S. coelicolor cell extracts. This indicated that glucose kinase may interact with (an)other factor(s), most likely of protein nature. A possible cross-talk with proteins of the phosphotransferase system, which are involved in carbon catabolite repression in other bacteria, was investigated.

carbon catabolite repression glucose kinase Streptomyces 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Angell S, Schwarz E & Bibb MJ (1992) The glucose kinase gene of Streptomyces coelicolor A3(2): its nucleotide sequence, transcriptional analysis and role in glucose repression. Mol. Microbiol. 6: 2833–2844Google Scholar
  2. Angell S, Lewis CG, Buttner MJ & Bibb MJ (1994) Glucose repression in Streptomyces coelicolor A3(2): a likely regulatory role for glucose kinase. Mol. Gen. Genet. 244: 135–143Google Scholar
  3. Baltz RH (1998) Genetic manipulation of antibiotic-producing Streptomyces. Trends Microbiol. 6: 76–83Google Scholar
  4. Bibb M (1996) 1995 Colworth Prize Lecture. The regulation of antibiotic production in Streptomyces coelicolor A3(2). Microbiology 142: 1335–1344Google Scholar
  5. Butler MJ, Deutscher J, Postma PW, Wilson TJ, Galinier A & Bibb MJ (1999) Analysis of a ptsH homologue from Streptomyces coelicolor A3(2). FEMS Microbiol. Lett. 177: 279–288Google Scholar
  6. Chater KF (1993) Genetics of differentiation in Streptomyces. Annu. Rev. Microbiol. 47: 685–713Google Scholar
  7. Chater KF (1998) Taking a genetic scalpel to the Streptomyces colony. Microbiology 144: 1465–1478Google Scholar
  8. Chater KF & Hopwood DA (1983) Streptomyces. In: Sonenshein AL, Hoch JA & Losick R (Eds) Bacillus subtilis and other Gram-positive bacteria (pp 83–99). American Society for Microbiology, Washington DC.Google Scholar
  9. Geourjon C & Deléage G (1995) SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput. Appl. Biosci. 11: 681–684Google Scholar
  10. Hindle Z & Smith CP (1994) Substrate induction and catabolite repression of the Streptomyces coelicolor glycerol operon are mediated through the GylR protein. Mol.Microbiol. 12: 737–745Google Scholar
  11. Hodgson DA (1982) Glucose repression of carbon source uptake and metabolism in Streptomyces coelicolor A3(2) and its perturbation in mutants resistant to 2–deoxyglucose. J Gen Microbiol 128: 2417–2430Google Scholar
  12. Hopwood DA, Bibb MJ, Chater KF, Kieser T, Bruton CJ, Kieser HM, Lydiate DJ, Smith CP, Ward JM & Schrempf H (1985) Genetic anipulation of Streptomyces. A laboratory Manual. In: John Innes Foundation, Norwich.Google Scholar
  13. Hopwood DA, Chater KF & Bibb MJ (1995) Genetics of antibiotic production in Streptomyces coelicolor A3(2), a model streptomycete. Biotechnology 28: 65–102Google Scholar
  14. Ingram C, Delic I & Westpheling J (1995) ccrA1: a mutation in Streptomyces coelicolor that affects the control of catabolite repression. J. Bacteriol. 177: 3579–3586Google Scholar
  15. Kwakman JH & Postma PW (1994) Glucose kinase has a regulatory role in carbon catabolite repression in Streptomyces coelicolor. J. Bacteriol. 176: 2694–2698Google Scholar
  16. Mahr K, Hillen W & Titgemeyer F (2000) Carbon catabolite repression in Lactobacillus pentosus: analysis of the ccpA region. Appl. Environ. Microbiol. 66: 277–283Google Scholar
  17. Mattern SG, Brawner ME & Westpheling J (1993) Identification of a complex operator for galP1, the glucose-sensitive, galactosedependent promoter of the Streptomyces galactose operon. J. Bacteriol. 175: 1213–1220Google Scholar
  18. Meyer D, Schneider-Fresenius C, Horlacher R, Peist R & Boos W (1997) Molecular characterization of glucokinase from Escherichia coli K-12. J. Bacteriol. 179: 1298–1306Google Scholar
  19. Parche S, Schmid R & Titgemeyer F (1999) The phosphotransferase system (PTS) of Streptomyces coelicolor: identification and biochemical analysis of a histidine phosphocarrier protein HPr encoded by the gene ptsH. Eur. J. Biochem. 265: 308–317Google Scholar
  20. Pope MK, Green BD & Westpheling J (1996) The bld mutants of Streptomyces coelicolor are defective in the regulation of carbon utilization, morphogenesis, and cell-cell signalling. Mol. Microbiol. 19: 747–756Google Scholar
  21. Postma PW, Lengeler JW & Jacobson GR (1993) Phosphoenolpyruvate: carbohydrate phosphotransferase systems of bacteria. Microbiol. Rev. 57: 543–594Google Scholar
  22. Reizer J, Hoischen C, Titgemeyer F, Rivolta C, Rabus R, Stulke J, Karamata D, Saier MH, Jr. & Hillen W (1998) A novel protein kinase that controls carbon catabolite repression in bacteria. Mol. Microbiol. 27: 1157–1169Google Scholar
  23. Roossien FF, Brink J & Robillard GT (1983) A simple procedure for the synthesis of [32P]phosphoenolpyruvate via the pyruvate kinase exchange reaction at equilibrium. Biochim. Biophys. Acta. 760: 185–187Google Scholar
  24. Saier MH, Jr. (1989) Protein phosphorylation and allosteric control of inducer exclusion and catabolite repression by the bacterial phosphoenolpyruvate: sugar phosphotransferase system. Microbiol. Rev. 53: 109–120Google Scholar
  25. Saito A, Fujii T, Yoneyama T & Miyashita K (1998) glkA is involved in glucose repression of chitinase production in Streptomyces lividans. J. Bacteriol. 180: 2911–2914Google Scholar
  26. Sambrook J, Fritsch EF & Maniatis T (1989) Molecular cloning: a laboratory manual. 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  27. Seok YJ, Sondej M, Badawi P, Lewis MS, Briggs MC, Jaffe H & Peterkofsky A (1997) High affinity binding and allosteric regulation of Escherichia coli glycogen phosphorylase by the histidine phosphocarrier protein, HPr. J. Biol. Chem. 272: 26511–26521Google Scholar
  28. Skarlatos P & Dahl MK (1998) The glucose kinase of Bacillus subtilis. J. Bacteriol. 180: 3222–3226Google Scholar
  29. Späth C, Kraus A & Hillen W (1997) Contribution of glucose kinase to glucose repression of xylose utilization in Bacillus megaterium. J. Bacteriol. 179: 7603–7605Google Scholar
  30. Stülke J & Hillen W(1999) Carbon catabolite repression in bacteria. Curr. Opin. Microbiol. 2: 195–201Google Scholar
  31. Titgemeyer F, Reizer J, Reizer A & Saier MH, Jr. (1994a) Evolutionary relationships between sugar kinases and transcriptional repressors in bacteria. Microbiology 140: 2349–2354Google Scholar
  32. Titgemeyer F, Walkenhorst J, Cui X, Reizer J & Saier MH, Jr. (1994b) Proteins of the phosphoenolpyruvate:sugar phosphotransferase system in Streptomyces: possible involvement in the regulation of antibiotic production. Res. Microbiol. 145: 89–92Google Scholar
  33. Titgemeyer F, Walkenhorst J, Reizer J, Stuiver MH, Cui X & Saier MH, Jr. (1995) Identification and characterization of phosphoenolpyruvate: fructose phosphotransferase systems in three Streptomyces species. Microbiology 141: 51–58Google Scholar
  34. van Wezel GP, White J, Young P, Postma PW & Bibb MJ (1997) Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2) is controlled by malR, a member of the lacl-galR family of regulatory genes. Mol. Microbiol. 23: 537–549Google Scholar

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • Kerstin Mahr
    • 1
  • Gilles P. van Wezel
    • 2
  • Cecilia Svensson
    • 3
  • Ute Krengel
    • 3
  • Mervyn J. Bibb
    • 4
  • Fritz Titgemeyer
    • 5
    Email author
  1. 1.Lehrstuhl für MikrobiologieFriedrich-Alexander-Universität Erlangen-NürnbergErlangenGermany
  2. 2.Department of Biochemistry, LICLeiden UniversityRA LeidenThe Netherlands
  3. 3.Center for Structural Biology and Department of Molecular BiotechnologyChalmers University of TechnologyGöteborgSweden
  4. 4.Department of GeneticsJohn Innes CentreNorwichUK
  5. 5.Lehrstuhl für MikrobiologieFriedrich-Alexander-Universität Erlangen-NürnbergErlangenGermany

Personalised recommendations