1887

Abstract

is unable to utilize glucose as a carbon source due to the absence of the key glycolytic enzyme 6-phosphofructokinase. The genome sequence of NCTC 11168 indicates that homologues of all the appropriate enzymes for gluconeogenesis from phosphoenolpyruvate (PEP) are present, in addition to the anaplerotic enzymes pyruvate carboxylase (PYC), phosphoenolpyruvate carboxykinase (PCK) and malic enzyme (MEZ). Surprisingly, a pyruvate kinase (PYK) homologue is also present. To ascertain the role of these enzymes, insertion mutants in , , and were generated. However, this could not be acheived for , indicating that PCK is an essential enzyme in . The lack of PEP synthase and pyruvate orthophosphate dikinase activities confirmed a unique role for PCK in PEP synthesis. The mutant was unable to grow in defined medium with pyruvate or lactate as the major carbon source, thus indicating an important role for PYC in anaplerosis. Sequence and biochemical data indicate that the PYC of is a member of the αβ, acetyl-CoA-independent class of PYCs, with a 658 kDa subunit containing the biotin moiety. Whereas growth of the mutant was comparable to that of the wild-type, the mutant displayed a decreased growth rate in complex medium. Nevertheless, the and mutants were able to grow with pyruvate, lactate or malate as carbon sources in defined medium. PYK was present in cell extracts at a much higher specific activity [>800 nmol min (mg protein)] than PYC or PCK [<65 nmol min (mg protein)], was activated by fructose 1,6-bisphosphate and displayed other regulatory properties strongly indicative of a catabolic role. It is concluded that PYK may function in the catabolism of unidentified substrates which are metabolized through PEP. In view of the high of MEZ for malate (∼∼∼9 mM) and the lack of a growth phenotype of the mutant, MEZ seems to have only a minor anaplerotic role in .

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-148-3-685
2002-03-01
2019-08-24
Loading full text...

Full text loading...

/deliver/fulltext/micro/148/3/1480685a.html?itemId=/content/journal/micro/10.1099/00221287-148-3-685&mimeType=html&fmt=ahah

References

  1. Best, E. A. & Knauf, V. C. ( 1993; ). Organization and nucleotide sequences of the genes encoding the biotin carboxyl carrier protein and biotin carboxylase protein of Pseudomonas aeruginosa acetyl coenzyme A carboxylase. J Bacteriol 175, 6881-6889.
    [Google Scholar]
  2. Cohen, N. D., Duc, J. A., Beegen, H. & Utter, M. F. ( 1979; ). Quaternary structure of pyruvate carboxylase from Pseudomonas citronellolis. J Biol Chem 254, 9262-9269.
    [Google Scholar]
  3. Diesterhaft, M. D. & Freese, E. ( 1973; ). Role of pyruvate carboyxlase, phosphoenolpyruvate carboxykinase, and malic enzyme during growth and sporulation of Bacillus subtilis. J Biol Chem 248, 6062-6070.
    [Google Scholar]
  4. Driscoll, B. T. & Finan, T. M. ( 1996; ). NADP+-dependent malic enzyme of Rhizobium meliloti. J Bacteriol 178, 2224-2231.
    [Google Scholar]
  5. Ferrero, R. L., Cussac, V., Courcoux, P. & Labigne-Roussel, A. ( 1992; ). Construction of isogenic urease-negative mutants of Helicobacter pylori by allelic exchange. J Bacteriol 174, 4212-4217.
    [Google Scholar]
  6. Fier, H. A. & Suzuki, I. ( 1969; ). Pyruvate carboxylase of Aspergillus niger: kinetic study of a biotin containing carboxylase. Can J Biochem 47, 697-710.
    [Google Scholar]
  7. Goldie, A. H. & Sanwal, B. D. ( 1980; ). Genetic and physiological characterisation of Escherichia coli mutants deficient in phosphoenolpyruvate carboxykinase activity. J Bacteriol 141, 1115-1121.
    [Google Scholar]
  8. Gornicki, P., Scappino, L. A. & Haselkorn, R. ( 1993; ). Genes for two subunits of acetyl coenzyme A carboxylase of Anabaena sp. strain PCC 7120: biotin carboxylase and biotin carboxyl carrier protein. J Bacteriol 175, 5268-5272.
    [Google Scholar]
  9. Goss, J. A., Cohen, N. D. & Utter, M. F. ( 1981; ). Characterisation of the subunit structure of pyruvate carboxylase from Pseudomonas citronellolis. J Biol Chem 256, 11819-11825.
    [Google Scholar]
  10. Gourdon, P., Baucher, M.-F., Lindley, N. D. & Guyonvarch, A. ( 2000; ). Cloning of the malic enzyme gene from Corynebacterium glutamicum and role of the enzyme in lactate metabolism. Appl Environ Microbiol 66, 2981-2987.[CrossRef]
    [Google Scholar]
  11. Jurica, M. S., Mesecar, A., Heath, P. J., Shi, W., Nowak, T. & Stoddard, B. L. ( 1998; ). The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate. Structure 6, 195-210.[CrossRef]
    [Google Scholar]
  12. Kelly, D. J. ( 2001; ). The physiology and metabolism of Campylobacter jejuni and Helicobacter pylori. J Appl Microbiol 90, 16S-24S.[CrossRef]
    [Google Scholar]
  13. Ketley, J. M. ( 1997; ). Pathogenesis of enteric infection by Campylobacter. Microbiology 143, 5-21.[CrossRef]
    [Google Scholar]
  14. Li, S.-J. & Cronan, J. E.Jr ( 1992; ). The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. J Biol Chem 267, 855-863.
    [Google Scholar]
  15. Liao, C.-L. & Atkinson, D. E. ( 1971; ). Regulation of the phosphoenolpyruvate branchpoint in Azotobacter vinelandii: phosphoenolpyruvate carboxylase. J Bacteriol 106, 31-36.
    [Google Scholar]
  16. Marmur, J. ( 1961; ). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208-218.[CrossRef]
    [Google Scholar]
  17. Mattevi, A., Bolognesi, M. & Valentini, G. ( 1996; ). The allosteric regulation of pyruvate kinase. FEBS Lett 389, 15-19.[CrossRef]
    [Google Scholar]
  18. Modak, H. V. & Kelly, D. J. ( 1995; ). Acetyl-CoA-dependent pyruvate carboxylase from the photosynthetic bacterium Rhodobacter capsulatus: rapid and efficient purification using dye-ligand affinity chromatography. Microbiology 141, 2619-2628.[CrossRef]
    [Google Scholar]
  19. Mukhopadhyay, B. & Purwantini, E. ( 2000; ). Pyruvate carboxylase from Mycobacterium smegmatis: stabilisation, rapid purification, molecular and biochemical characterisation and regulation of the cellular level. Biochim Biophys Acta 1475, 191-206.[CrossRef]
    [Google Scholar]
  20. Mukhopadhyay, B., Stoddard, S. F. & Wolfe, R. S. ( 1998; ). Purification, regulation, and molecular and biochemical characterisation of pyruvate carboxylase from Methanobacterium thermoautotrophicum strain ΔH*. J Biol Chem 273, 5155-5166.[CrossRef]
    [Google Scholar]
  21. O’Brien, R. W., Chuang, D. T., Taylor, B. L. & Utter, M. F. ( 1977; ). Novel enzymic machinery for the metabolism of oxaloacetate, phosphoenolpyruvate, and pyruvate in Pseudomonas citronellolis. J Biol Chem 252, 1257-1263.
    [Google Scholar]
  22. Parkhill, J., Wren, B. W., Mungall, K. & 18 other authors ( 2000; ). The genome sequence of the foodborne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403, 665–668.[CrossRef]
    [Google Scholar]
  23. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  24. Scrutton, M. C. & Taylor, B. L. ( 1974; ). Isolation and characterisation of pyruvate carboxylase from Azotobacter vinelandii OP. Arch Biochem Biophys 164, 641-654.[CrossRef]
    [Google Scholar]
  25. Smibert, R. M. ( 1984; ). Genus Campylobacter Sebald and Véron 1963, 907AL. In Bergey’s Manual of Systematic Bacteriology , pp. 111-117. Edited by N. R. Krieg & J. G. Holt. Baltimore, MD:Williams & Wilkins.
  26. Tauxe, R. V. ( 1992; ). Epidemiology of Campylobacter jejuni infections in the United States and other industrialized nations. In Campylobacter jejuni: Current Status and Future Trends, , pp. 9-19. Edited by I. Nachamkin, M. J. Blaser & L. S. Tompkins. Washington, DC:American Society for Microbiology.
  27. van Vliet, A. H. M., Wooldridge, K. G. & Ketley, J. M. ( 1998; ). Iron-responsive gene regulation in a Campylobacter jejuni fur mutant. J Bacteriol 180, 5291-5298.
    [Google Scholar]
  28. Wang, Y. & Taylor, D. E. ( 1990; ). Chloramphenicol resistance in Campylobacter coli: nucleotide sequence, expression, and cloning vector construction. Gene 94, 23-28.[CrossRef]
    [Google Scholar]
  29. Wynn, J. P. & Ratledge, C. ( 1997; ). Malic enzyme is a major source of NADPH for lipid accumulation by Aspergillus nidulans. Microbiology 143, 253-257.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-148-3-685
Loading
/content/journal/micro/10.1099/00221287-148-3-685
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