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Volume 144, Issue 7

Phenol hydroxylase cloned from Ralstonia eutropha strain E2 exhibits novel kinetic properties Free

Abstract

Ralstonia eutropha strain E2 (previously Alcaligenes sp.) is a phenol-degrading bacterium expressing phenol-oxygenating activity with a low K (the apparent half-saturation constant in Haldane's equation) and an extremely high K (the apparent inhibition constant). To identify the molecular basis for these novel cellular kinetic properties, a 9.5 kb DNA fragment that allowed Pseudomonas aeruginosa PAO1c (Phl Cat) to grow on phenol as the sole carbon source was cloned from strain E2 into plasmid pRO1614. PAO1c harbouring this plasmid (designated pROE217) transformed phenol to catechol, indicating that this fragment contains gene(s) for phenol hydroxylase. The cloned genes consist of eight complete ORFs, designated poxRABCDEFG. The products are homologous to those of dmpRKLMNOPQ of Pseudomonas sp. CF600, sharing 30--65% identity: this suggests that the phenol hydroxylase is a multicomponent enzyme. The kinetic constants for phenol-oxygenating activity of PA01c(pROE217) were determined, and these were compared with those of strain E2. The kinetic constants of PAO1c derivatives expressing different phenol hydroxylases were also determined. A comparison of these kinetic data suggests that phenol hydroxylase, the first enzyme in the phenol-degradative pathway, determines K and K values for the cellular phenol-oxygenating activity. It is thus suggested that the phenol hydroxylase cloned from strain E2 exhibits the novel kinetic properties that were observed with intact cells of strain E2.

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Keyword(s): kinetics , phenol hydroxylase , phenol-oxygenating activity and Ralstonia eutropha
© Society for General Microbiology 1998
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1998-07-01
2025-04-28
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References

  1. Abril M.A., Buck M., Ramos J.L. 1991; Activation of the Pseudomonas TOL plasmid upper pathway operon: identification of binding sites for the positive regulator XylR and for integration host factor protein.. Biol Chem 266:15832–15838
     [Google Scholar]
  2. Chakrabarty A. M., Mylroie J. R., Friello D. A., Vacca J. G. 1975; Transformation of Pseudomonas putida and Escherichia coli with plasmid-linked drug-resistance factor DNA.. Proc Natl Acad Sci USA 723647–3651
     [Google Scholar]
  3. Dixon R. 1986; The xylABC promoter from Pseudomonas putida TOL plasmid is activated by nitrogen regulatory genes in Escherichia coli.. Mol Gen Genet 203:129–136
     [Google Scholar]
  4. Ehrt S., Schirmer F., Hillen W. 1995; Genetic organization, nucleotide sequence and regulation of expression of genes encoding phenol hydroxylase and catechol 1,2-dioxygenase in Acinetobacter calcoaceticus NCIB8250.. Mol Microbiol 18:13–20
     [Google Scholar]
  5. Folsom B.R., Chapman P.J., Pritchard P.H. 1990; Phenol and trichloroethylene degradation by Pseudomonas cepacia G4: kinetics and interactions between substrates.. Appl Environ Microbiol 56:1279–1285
     [Google Scholar]
  6. Fox B.G., Shanklin J., Somerville C., Münck E. 1993; Stearylacyl carrier protein △9 desaturase from Ricinus communis is a diiron-oxo protein.. Proc Natl Acad Sci USA 902486–2490
     [Google Scholar]
  7. Friedman D.I. 1988; Integration host factor: a protein for all reasons.. Cell 55:545–554
     [Google Scholar]
  8. Fujita M., Ike M., Hioki J., Kataoka K., Takeo M. 1995; Trichloroethylene degradation by genetically engineered bacteria carrying cloned phenol catabolic genes.. J Ferment Bioeng 79:100–106
     [Google Scholar]
  9. Hashimoto S., Iwahori K., Iwagami A. 1992; Phenoldegrading activity of Acinetobacter calcoaceticus AH strain.. Hakkokogaku 70:267–271
     [Google Scholar]
  10. Herrmann H., Müller C., Schmidt I., Mahnke J., Petruschka L., Hahnke K. 1995; Localization and organization of phenol degradation genes of Pseudomonas putida strain H.. Mol Gen Genet 247:240–246
     [Google Scholar]
  11. Holloway B.W., Krishnapillai V., Morgan A.F. 1979; Chromosomal genetics of Pseudomonas.. Microbiol Rev 43:73–102
     [Google Scholar]
  12. Holtel A., Timmis K.N., Ramos J.L. 1992; Upstream binding sequences of the XylR activator protein and integration host factor in the XylS gene promoter region of the Pseudomonas TOL plasmid.. Nucleic Acids Res 20:1755–1762
     [Google Scholar]
  13. Kukor J.J., Olsen R.H. 1990; Molecular cloning, characterization, and regulation of a Pseudomonas pickettii PKOl gene encoding phenol hydroxylase and expression of the gene in Pseudomonas aeruginosa PAO1c.. J Bacteriol 172:4624–4630
     [Google Scholar]
  14. Kustu S., Santero E., Keener J., Popham D., Weiss D. 1989; Expression of σ54 (ntrA)-dependent genes is probably united by a common mechanism.. Microbiol Rev 53:367–376
     [Google Scholar]
  15. Machado R.J., Grady L. Jr 1989; Dual substrate removal by an axenic bacterial culture.. Biotechnol Bioeng 33:327–337
     [Google Scholar]
  16. Marmur J. 1961; A procedure for the isolation of deoxyribonucleic acid from microorganisms.. J Mol Biol 3:208–218
     [Google Scholar]
  17. Ng L.C., Shingler V., Sze C.C., Poh C.L. 1994; Cloning and sequencing of the first eight genes of the chromosomally encoded (methyl) phenol degradation pathway from Pseudomonas putidaP35X.. Gene 151:29–36
     [Google Scholar]
  18. Nordlund I., Powlowski J., Shingler V. 1990; Complete nucleotide sequence and polypeptide analysis of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600.. J Bacteriol 172:6826–6833
     [Google Scholar]
  19. Nurk A., Kasak L., Kivisaar M. 1991; Sequence of the gene (pheA) encoding phenol monooxygenase from Pseudomonas sp. EST1001: expression in Escherichia coli and Pseudomonas putida.. Gene 102:13–18
     [Google Scholar]
  20. Olsen R.H., DeBusscher G., McCombie J.M. 1982; Develop-ment of broad-host-range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa PAO chromosome.. J Bacteriol 150:60–69
     [Google Scholar]
  21. Pearson W.R. 1990; Rapid and sensitive sequence comparison with FASTP and FASTA.. Methods Enzymol 183:63–98
     [Google Scholar]
  22. Pearson W.R., Lipman D.J. 1988; Improved tools for biological sequence analysis.. Proc Natl Acad Sci USA 852444–2448
     [Google Scholar]
  23. Polissi A., Harayama S. 1993; In vivo reactivation of catechol 2,3-dioxygenase mediated by a chloroplast-type ferredoxin: a bacterial strategy to expand the substrate specificity of the aromatic degradative pathway.. EMBO J 12:3339–3347
     [Google Scholar]
  24. Powlowski J., Shingler V. 1990; In vitro analysis of polypeptide requirements of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600.. J Bacteriol 172:6834–6840
     [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. Shine J., Dalgarno L. 1975; Determination of cistron specificity in bacterial ribosomes.. Nature 254:34–38
     [Google Scholar]
  27. Shingler V. 1996; Molecular and regulatory check points in phenol degradation by Pseudomonas sp. CF600.. In Molecular Biology of Pseudomonads pp. 153–164 Nakazawa T., Furukawa K., Haas D., Silveri S. Edited by Washington, DC:: American Society for Microbiology.;
     [Google Scholar]
  28. Shingler V., Bartilson M., Moore T. 1993; Cloning and nucleotide sequencing of the gene encoding the positive regulator (DmpR) of the phenol catabolic pathway encoded by pVI150 and identification of DmpR as a member of the NtrC family of transcriptional activators.. J Bacteriol 175:1596–1604
     [Google Scholar]
  29. Staskawicz B., Dahlbeck D., Keen N., Napoli C. 1987; Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea.. J Bacteriol 169:5789–5794
     [Google Scholar]
  30. Takeo M., Maeda Y., Okada H., Miyama K., Mori K., Ike M., Fujita M. 1995; Molecular cloning and sequencing of phenol hydroxylase gene from Pseudomonas putida BH.. J Ferment Bioeng 79:485–488
     [Google Scholar]
  31. Thompson J.D., Higgins D.G., Gibson T.J. 1994; CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice.. Nucleic Acids Res 22:4673–4680
     [Google Scholar]
  32. Watanabe K., Hino S. 1996; Identification of a functionally important population in phenol-digesting activated sludge with antisera raised against isolated bacterial strains.. Appl Environ Microbiol 62:3901–3904
     [Google Scholar]
  33. Watanabe K., Hino S., Onodera K., Kajie S., Takahashi N. 1996; Diversity in kinetics of bacterial phenol-oxygenating activity.. J Ferment Bioeng 81:562–565
     [Google Scholar]
/content/journal/micro/10.1099/00221287-144-7-1765
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Phenol hydroxylase cloned from Ralstonia eutropha strain E2 exhibits novel kinetic properties
Microbiology 144, 1765 (1998); https://doi.org/10.1099/00221287-144-7-1765
/content/journal/micro/10.1099/00221287-144-7-1765
/content/journal/micro/10.1099/00221287-144-7-1765
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