1887

Abstract

Among the few regulatory proteins encoded by is HPr kinase/phosphatase (HPrK/P), the key regulator of carbon metabolism in low-GC Gram-positive bacteria. The corresponding gene, , and the gene encoding the target protein HPr, , were overexpressed. analysis of the purified proteins confirmed ATP-dependent phosphorylation of HPr by HPrK/P. In contrast to HPrK/P of , which is by default a phosphatase and needs high ATP concentrations for kinase activity, the enzyme exhibits kinase activity at very low ATP concentrations and depends on P for phosphatase activity. This inverted control of enzymic activity may result from the adaptation to very different ecological niches. While the standard activities of HPrK/P from and other Gram-positive bacteria differ, they are both modulated by the concentration of ATP, P and glycolytic intermediates. Site-directed mutagenesis of a potential ATP-binding site and of the HPrK/P signature sequence resulted in four different activity classes: (i) inactive proteins, (ii) enzymes with reduced kinase and phosphatase activities, (iii) enzymes that had lost phosphatase, but not kinase activity, and (iv) enzymes that exhibited increased phosphatase activity.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-148-10-3277
2002-10-01
2021-07-30
Loading full text...

Full text loading...

/deliver/fulltext/micro/148/10/1483277a.html?itemId=/content/journal/micro/10.1099/00221287-148-10-3277&mimeType=html&fmt=ahah

References

  1. Balasubramanian S, Schneider T, Gerstein M., Regal L. 2000; Proteomics of Mycoplasma genitalium : identification and characterization of unannotated and atypical proteins in a small model genome. Nucleic Acids Res 28:3075–3082
    [Google Scholar]
  2. Bradford M. M. 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
    [Google Scholar]
  3. Brochu D., Vadeboncoeur C. 1999; The HPr(Ser) kinase of Streptococcus salivarius : purification, properties, and cloning of the hprK gene. J Bacteriol 181:709–717
    [Google Scholar]
  4. Deutscher J, Küster E, Bergstedt U, Charrier V., Hillen W. 1995; Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in Gram-positive bacteria. Mol Microbiol 15:1049–1053
    [Google Scholar]
  5. Dossonnet V, Monedero V, Zagorec M, Galinier A, Perez-Martinez G., Deutscher J. 2000; Phosphorylation of HPr by the bifunctional HPr Kinase/P-ser-HPr phosphatase from Lactobacillus casei controls catabolite repression and inducer exclusion but not inducer expulsion. J Bacteriol 182:2582–2590
    [Google Scholar]
  6. Egan W, Barile M., Rottem S. 1986; 31P-NMR studies of Mycoplasma gallisepticum cells using a continuous perfusion technique. FEBS Lett 204:373–376
    [Google Scholar]
  7. Fieulaine S, Morera S, Poncet S, Monedero V, Gueguen-Chaignon V, Galinier A, Janin J, Deutscher J., Nessler S. 2001; X-ray structure of HPr kinase: a bacterial protein kinase with a P-loop nucleotide-binding domain. EMBO J 20:3917–3927
    [Google Scholar]
  8. Fraser C. M, Gocayne J. D, White O. 26 other authors 1995; The minimal gene complement of Mycoplasma genitalium . Science 270:397–403
    [Google Scholar]
  9. Galinier A, Haiech J, Kilhoffer M.-C, Jaquinod M, Stülke J, Deutscher J., Martin-Verstraete I. 1997; The Bacillus subtilis crh gene encodes a HPr-like protein involved in carbon catabolite repression. Proc Natl Acad Sci USA 94:8439–8444
    [Google Scholar]
  10. Galinier A, Kravanja M, Engelmann R, Hengstenberg W, Kilhoffer M.-C, Deutscher J., Haiech J. 1998; New protein kinase and protein phosphatase families mediate signal transduction in bacterial catabolite repression. Proc Natl Acad Sci USA 95:1823–1828
    [Google Scholar]
  11. Hanson K. G, Steinhauer K, Reizer J, Hillen W., Stülke J. 2002; HPr kinase/phosphatase of Bacillus subtilis : expression of the gene and effects of mutations on enzyme activity, growth and carbon catabolite repression. Microbiology 148:1805–1811
    [Google Scholar]
  12. Himmelreich R, Hilbert H, Plagens H, Pirkl E, Li B.-C., Herrmann R. 1996; Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae . Nucleic Acids Res 24:4420–4449
    [Google Scholar]
  13. Himmelreich R, Plagens H, Hilbert H, Reiner B., Herrmann R. 1997; Comparative analysis of the genomes of the bacteria Mycoplasma pneumoniae and Mycoplasma genitalium . Nucleic Acids Res 25:701–712
    [Google Scholar]
  14. Hoischen C, Dijkstra A, Rottem S, Reizer J., Saier M. H. Jr 1993; Presence of protein constituents of the gram-positive bacterial phosphotransferase regulatory system in Acholeplasma laidlawii . J Bacteriol 175:6599–6604
    [Google Scholar]
  15. Hutchinson C. A.III, Peterson S. C, Gill S. R, Cline R. T, White O, Fraser C. M, Smith H. O., Venter J. C. 1999; Global transposon mutagenesis and a minimal mycoplasma genome. Science 286:2165–2169
    [Google Scholar]
  16. Huynh P. L, Jankovic I, Schnell N. F., Brückner R. 2000; Characterization of an HPr kinase mutant of Staphylococcus xylosus . J Bacteriol 182:1895–1902
    [Google Scholar]
  17. Ikeda T. P, Houtz E., LaPorte D. C. 1992; Isocitrate dehydrogenase kinase/phosphatase: identification of mutations which selectively inhibit phosphatase activity. J Bacteriol 174:1414–1416
    [Google Scholar]
  18. Jault J. M, Fieulaine S, Nessler S, Gonzalo P, Di Pietro A, Deutscher J., Galinier A. 2000; The HPr kinase from Bacillus subtilis is a homo-oligomeric enzyme which exhibits strong positive cooperativity for nucleotide and fructose 1,6-bisphosphate binding. J Biol Chem 275:1773–1780
    [Google Scholar]
  19. Kravanja M, Engelmann R, Dossonnet V, Blüggel M, Meyer H. E, Frank R, Galinier A, Deutscher J, Schnell N., Hengstenberg W. 1999; The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase. Mol Microbiol 31:59–66
    [Google Scholar]
  20. Mason P. W, Carbone D. P, Cushman R. A., Waggoner A. S. 1981; The importance of inorganic phosphate in regulation of energy metabolism of Streptococcus lactis . J Biol Chem 256:1861–1866
    [Google Scholar]
  21. Miles R. J. 1992; Catabolism in mollicutes. J Gen Microbiol 138:1773–1783
    [Google Scholar]
  22. Monedero V, Poncet S, Mijakovic I, Fieulaine S, Dossonnet V, Martin-Verstraete I, Nessler S., Deutscher J. 2001; Mutations lowering the phosphatase activity of HPr kinase/phosphatase switch off carbon metabolism. EMBO J 20:3928–3937
    [Google Scholar]
  23. Razin S, Yogev D., Naot Y. 1998; Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev 62:1094–1156
    [Google Scholar]
  24. Reizer J, Hoischen C, Titgemeyer F, Rivolta C, Rabus R, Stülke J, Karamata D, Saier M. H Jr, Hillen W. 1998; A novel protein kinase that controls carbon catabolite repression in bacteria. Mol Microbiol 27:1157–1169
    [Google Scholar]
  25. Sambrook J, Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  26. Saraste M, Sibbald P. R., Wittinghofer A. 1990; The P-loop – a common motif in ATP- and GTP-binding proteins. Trends Biochem Sci 15:430–434
    [Google Scholar]
  27. Schägger H., von Jagow G. 1987; Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379
    [Google Scholar]
  28. Schirmer F, Ehrt S., Hillen W. 1997; Expression, inducer spectrum, domain structure, and function of MopR, the regulator of phenol degradation in Acinetobacter calcoaceticus NCIB8250. J Bacteriol 179:1329–1336
    [Google Scholar]
  29. Steinhauer K, Allen G. S, Hillen W, Stülke J., Brennan R. G. 2002; Crystallization, preliminary X-ray analysis and biophysical characterization of HPr kinase/phosphatase of Mycoplasma pneumoniae . Acta Crystallogr Sect D 58:515–518
    [Google Scholar]
  30. Stueland C. S, Ikeda T. P., LaPorte D. C. 1989; Mutation of the predicted ATP binding site inactivates both activities of isocitrate dehydrogenase/phosphatase. J Biol Chem 264:13775–13779
    [Google Scholar]
  31. Stülke J., Hillen W. 1999; Carbon catabolite repression in bacteria. Curr Opin Microbiol 2:195–201
    [Google Scholar]
  32. Vertommen D, Bertrand L, Sontag B, Di Pietro A, Louckx M. P, Vidal H, Hue L., Rider M. H. 1996; The ATP-binding site in the 2-kinase domain of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. J Biol Chem 271:17875–17880
    [Google Scholar]
  33. Wagner A, Küster-Schöck E., Hillen W. 2000; Sugar uptake and carbon catabolite repression in Bacillus megaterium strains with inactivated ptsHI . J Mol Microbiol Biotechnol 2:587–592
    [Google Scholar]
  34. Walker J. E, Saraste M, Runswick M. J., Gay N. J. 1982; Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J 1:945–951
    [Google Scholar]
  35. Wasinger V. C, Pollack J. D., Humphery-Smith I. 2000; The proteome of Mycoplasma genitalium : Chaps-soluble component. Eur J Biochem 267:1571–1582
    [Google Scholar]
  36. Weiner J.III, Herrmann R., Browning G. F. 2000; Transcription in Mycoplasma pneumoniae . Nucleic Acids Res 28:4488–4496
    [Google Scholar]
  37. Woese C. R. 1987; Bacterial evolution. Microbiol Rev 51:221–271
    [Google Scholar]
  38. Zhu P.-P, Nosworthy N, Ginsburg A, Miyata M, Seok Y.-J., Peterkofsky A. 1997; Expression, purification, and characterization of Enzyme IIAglc of the phosphoenolpyruvate: sugar phosphotransferase system of Mycoplasma capricolum . Biochemistry 36:6947–6953
    [Google Scholar]
  39. Zhu P.-P, Herzberg O., Peterkofsky A. 1998; Topography of the interaction of HPr(Ser) kinase with HPr. Biochemistry 37:11762–11770
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-148-10-3277
Loading
/content/journal/micro/10.1099/00221287-148-10-3277
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error