1887

Abstract

The metabolic importance of pyruvate oxidase (PoxB), which converts pyruvate directly to acetate and CO, was assessed using an isogenic set of genetically engineered strains of . In a strain lacking the pyruvate dehydrogenase complex (PDHC), PoxB supported acetate-independent aerobic growth when the gene was expressed constitutively or from the IPTG-inducible promoter. Using aerobic glucose-limited chemostat cultures of PDH-null strains, it was found that steady-states could be maintained at a low dilution rate (005 h) when PoxB is expressed from its natural promoter, but not at higher dilution rates (up to at least 025 h) unless expressed constitutively or from the promoter. The poor complementation of PDH-deficient strains by plasmids was attributed to several factors including the stationary-phase-dependent regulation of the natural promoter and deleterious effects of the multicopy plasmids. As a consequence of replacing the PDH complex by PoxB, the growth rate (μ), growth yield ( ) and the carbon conversion efficiency (flux to biomass) were lowered by 33%, 9–25% and 29–39% (respectively), indicating that more carbon has to be oxidized to CO for energy generation. Extra energy is needed to convert PoxB-derived acetate to acetyl-CoA for further metabolism and enzyme analysis indicated that acetyl-CoA synthetase is induced for this purpose. In similar experiments with a PoxB-null strain it was shown that PoxB normally makes a significant contribution to the aerobic growth efficiency of . In glucose minimal medium, the respective growth rates (μ), growth yields ( ) and carbon conversion efficiencies were 16%, 14% and 24% lower than the parental values, and correspondingly more carbon was fluxed to CO for energy generation. It was concluded that PoxB is used preferentially at low growth rates and that benefits from being able to convert pyruvate to acetyl-CoA by a seemingly wasteful route via acetate.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-147-6-1483
2001-06-01
2020-09-22
Loading full text...

Full text loading...

/deliver/fulltext/micro/147/6/1471483a.html?itemId=/content/journal/micro/10.1099/00221287-147-6-1483&mimeType=html&fmt=ahah

References

  1. Abdel-Hamid A. M.. 1999; Molecular and physiological studies on the role of pyruvate oxidase in E. coli PhD thesis, University of Sheffield;
    [Google Scholar]
  2. Bergmeyer H. U., Bernt E.. 1974; Determination of d-glucose with glucose oxidase and peroxidase. In Methods of Enzymatic Analysis pp1205–1215 Edited by Bergmeyer H. U.. New York & London: Academic Press;
    [Google Scholar]
  3. Bongaerts J., Zoske S., Weidner U., Unden G.. 1995; Transcriptional regulation of the proton translocating NADH dehydrogenase genes ( nuoA-N ) of Escherichia coli by electron acceptors, electron donors and gene regulators. Mol Microbiol16:521–534[CrossRef]
    [Google Scholar]
  4. Brooke A. G., Watling E. M., Attwood M. M., Tempest D. W.. 1989; Environmental control of metabolic fluxes in thermo-tolerant methylotrophic Bacillus strains. Arch Microbiol151:268–273[CrossRef]
    [Google Scholar]
  5. Brown T. D. K., Jones-Mortimer M. C., Kornberg H. L.. 1977; The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli . J Gen Microbiol102:327–336[CrossRef]
    [Google Scholar]
  6. Calhoun M. W., Gennis R. B.. 1993; Demonstration of separate genetic loci encoding distinct membrane-bound respiratory NADH dehydrogenases of Escherichia coli . J Bacteriol175:3013–3019
    [Google Scholar]
  7. Calhoun M. W., Oden K. L., Gennis R. B., Neijssel O. M., Teixeira de Mattos M. J.. 1993; Energetic efficiency of Escherichia coli : effects of mutations in components of the aerobic respiratory chain. J Bacteriol175:3020–3025
    [Google Scholar]
  8. Chang Y.-Y., Cronan J. E. Jr. 1982; Mapping non-selectable genes of Escherichia coli by using transposon Tn 10 : location of a gene affecting pyruvate oxidase. J Bacteriol151:1279–1289
    [Google Scholar]
  9. Chang Y.-Y., Cronan J. E. Jr. 1983; Genetic and biochemical analyses of Escherichia coli strains having a mutation in the structural gene ( poxB ) for pyruvate oxidase. J Bacteriol154:757–762
    [Google Scholar]
  10. Chang Y.-Y., Cronan J. E. Jr. 1986; Molecular cloning, DNA sequencing and enzymatic analysis of two Escherichia coli pyruvate oxidase mutants defective in activation by lipids. J Bacteriol167:312–318
    [Google Scholar]
  11. Chang Y. Y., Cronan J. E. Jr. 1997; Thiol chemistry detects three conformations of the lipid binding region of Escherichia coli pyruvate oxidase. Biochemistry36:11564–11573[CrossRef]
    [Google Scholar]
  12. Chang Y.-Y., Wang A.-Y., Cronan J. E. Jr. 1994; Expression of Escherichia coli pyruvate oxidase (PoxB) depends on the sigma factor encoded by the rpoS ( katF ) gene. Mol Microbiol11:1019–1028[CrossRef]
    [Google Scholar]
  13. Davé E., Guest J. R., Attwood M. M.. 1995; Metabolic engineering in Escherichia coli : lowering the lipoyl domain content of the pyruvate dehydrogenase complex adversely affects growth rate and yield. Microbiology141:1839–1849[CrossRef]
    [Google Scholar]
  14. Fellay R., Frey J., Krisch H.. 1987; Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vivo insertional mutagenesis of Gram-negative bacteria. Gene52:147–154[CrossRef]
    [Google Scholar]
  15. Gennis R. B., Hager L. P.. 1976; Pyruvate oxidase. In The Enzymes and Biological Membranes pp493–504 Edited by Martonosi A.. New York: Plenum;
    [Google Scholar]
  16. Gennis R. B., Stewart V.. others 1996; Respiration. In Escherichia coli and Salmonella : Cellular and Molecular Biology . pp217–261 Edited by Neidhardt F. C.. Washington, DC: American Society for Microbiology;
  17. Grabau C., Cronan J. E. Jr. 1984; Molecular cloning of the gene ( poxB ) encoding the pyruvate oxidase of Escherichia coli , a lipid-activated enzyme. J Bacteriol160:1088–1092
    [Google Scholar]
  18. Grabau C., Cronan J. E. Jr. 1986a; Nucleotide sequence and deduced amino acid sequence of Escherichia coli pyruvate oxidase, a lipid-activated flavoprotein. Nucleic Acids Res14:5449–5460[CrossRef]
    [Google Scholar]
  19. Grabau C., Cronan J. E. Jr. 1986b; In vivo function of Escherichia coli pyruvate oxidase specifically requires a functional lipid binding site. Biochemistry25:3748–3751[CrossRef]
    [Google Scholar]
  20. Grabau C., Chang Y. Y., Cronan J. E. Jr. 1989; Lipid binding by Escherichia coli pyruvate oxidase is disrupted by small alterations of the carboxyl-terminal region. J Biol Chem264:12510–12519
    [Google Scholar]
  21. Green J., Guest J. R.. 1994; Regulation of transcription at the ndh promoter of Escherichia coli by FNR and novel factors. Mol Microbiol12:433–444[CrossRef]
    [Google Scholar]
  22. Guest J. R., Quail M. A., Davé E., Cassey B., Attwood M. M.. 1996; Regulatory and other aspects of pyruvate dehydrogenase complex synthesis in Escherichia coli . In Biochemistry and Physiology of Thiamin Diphosphate Enzymes pp326–333 Edited by Bisswanger H.. Schellenberger A.. Prien: Intemann;
    [Google Scholar]
  23. Hamilton C. M., Aldea M., Washburn B. K., Babitzke P., Kushner S. R.. 1989; New method for generating deletions and gene replacements in Escherichia coli . J Bacteriol171:4617–4622
    [Google Scholar]
  24. Henning U., Herz C.. 1964; Ein Struckturgen-Komplex fur den Pyruvat-Dehydrogenase-Komplex von Escherichia coli K12. Z Vererbungsl95:260–275
    [Google Scholar]
  25. Horiuchi T., Tomizawa J.-I., Novick A.. 1962; Isolation and properties of bacteria capable of high rates of β-galactosidase synthesis. Biochim Biophys Acta55:152–163[CrossRef]
    [Google Scholar]
  26. Kaniga K., Compton M. S., Curtiss R.III., Sundaram P.. 1998; Molecular and functional characterization of Salmonella enterica serovar Typhimurium poxA gene: effect on attenuation of virulence and protection. Infect Immun66:5599–5606
    [Google Scholar]
  27. LeMaster D. M., Cronan J. E. Jr. 1982; Biosynthetic production of 13C-labeled amino acids with site-specific enrichment. J Biol Chem257:1224–1230
    [Google Scholar]
  28. Lennox E. S.. 1955; Transduction of linked genetic characters of the host by bacteriophage P1. Virology1:190–206[CrossRef]
    [Google Scholar]
  29. Lessard I. A. D., Pratt S. D., McCafferty D. G., Bussiere D. E., Hutchins C., Wanner B. L., Katz L., Walsh C. T.. 1998; Homologs of the vancomycin resistance d-Ala-D-Ala dipeptidase VanX in Streptomyces toyocaensis , Escherichia coli and Synechocystis : attributes of catalytic efficiency, stereoselectivity and regulation with implications for function. Chem Biol5:489–504[CrossRef]
    [Google Scholar]
  30. Marsh P.. 1986; ptac-85, an E. coli vector for expression of non-fusion proteins. Nucleic Acids Res14:3603[CrossRef]
    [Google Scholar]
  31. Martinez E., Bartolemé B., De la Cruz F.. 1988; pACYC184-derived cloning vectors containing the multiple cloning site and lacZ α reporter gene of pUC8/9 and pUC18/19 plasmids. Gene68:157–162
    [Google Scholar]
  32. Mather M., Blake R., Koland J., Schrock H., Russell P., O’Brien T., Hager L. P., Gennis R. B.. 1982; Escherichia coli pyruvate oxidase: interaction of a peripheral membrane protein with lipids. Biophys J37:87–88[CrossRef]
    [Google Scholar]
  33. Miller J. H.. 1992; Preparation and use of P1 vir lysates. In A Short Course in Bacterial Genetics: a Laboratory Manual and Handbook for E. coli and Related Bacteria . pp268–272 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  34. Notley L., Ferenci T.. 1996; Induction of RpoS-dependent functions in glucose-limited continuous culture: what level of nutrient limitation induces the stationary phase of Escherichia coli ?. J Bacteriol178:1445–1468
    [Google Scholar]
  35. Oden K. L., De Veaux L. C., Vibat C. R. T., Gennis R. B., Cronan J. E. Jr. 1990; Genomic replacement in Escherichia coli K-12 using covalently closed circular plasmid DNA. Gene96:29–36[CrossRef]
    [Google Scholar]
  36. Recny M. A., Grabau C., Hager L. P., Cronan J. E. Jr. 1985; Characterization of the α-peptide released upon protease activation of pyruvate oxidase. J Biol Chem260:14287–14291
    [Google Scholar]
  37. Rose I. A.. 1962; Acetate kinase. In The Enzymes pp115–118 Edited by Boyer P. D.. Lardy H., Myrbäck K.. New York & London: Academic Press;
    [Google Scholar]
  38. Russell G. C., Guest J. R.. 1990; Overexpression of reconstructed pyruvate dehydrogenase complexes and site-directed mutagenesis of a potential active-site histidine residue. Biochem J269:443–450
    [Google Scholar]
  39. Russell G. C., Machado R. S., Guest J. R.. 1992; Overproduction of the pyruvate dehydrogenase multienzyme complex of Escherichia coli and site-directed substitutions in the E1p and E2p subunits. Biochem J287:611–619
    [Google Scholar]
  40. Russell P., Hager L. P., Gennis R. B.. 1977; Lipid activation and protease activation of pyruvate oxidase. J Biol Chem252:7883–7887
    [Google Scholar]
  41. 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]
  42. Tran Q. H., Bongaerts J., Vlad D., Unden G.. 1997; Requirement for the proton-pumping NADH dehydrogenase I of Escherichia coli in respiration of NADH to fumarate and its bioenergetic implications. Eur J Biochem244:155–160[CrossRef]
    [Google Scholar]
  43. Van Dyk T. K., Smulski D. R., Chang Y.-Y.. 1987; Pleiotropic effects of poxA regulatory mutations of Escherichia coli and Salmonella typhimurium , mutations conferring sulfometuron methyl and α-ketobutyrate hypersensitivity. J Bacteriol169:4540–4546
    [Google Scholar]
  44. Wang A. Y., Chang Y. Y., Cronan J. E. Jr. 1991; Role of the tetrameric structure of Escherichia coli pyruvate oxidase in enzyme activation and lipid binding. J Biol Chem266:10959–10966
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-147-6-1483
Loading
/content/journal/micro/10.1099/00221287-147-6-1483
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