1887

Abstract

The response regulator AgmR was identified to be involved in the regulation of the quinoprotein ethanol oxidation system of ATCC 17933. Interruption of the gene by insertion of a kanamycin-resistance cassette resulted in mutant NG3, unable to grow on ethanol. After complementation with the intact gene, growth on ethanol was restored. Transcriptional fusions were used to identify four operons which are regulated by the AgmR protein: the operon encodes the pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase, the operon encodes a soluble cytochrome and an aldehyde dehydrogenase, the operon carries the PQQ biosynthetic genes, and operon encodes a two-component regulatory system which controls transcription of the operon. Transcription of was restored by transformation of NG3 with a pUCP20T derivative carrying the genes under -promoter control. These data indicate that the AgmR response regulator and the two-component regulatory system are organized in a hierarchical manner. Gene PA1977, which appears to form an operon with the gene, was found to be non-essential for growth on ethanol.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26882-0
2004-06-01
2024-04-27
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/6/mic1501851.html?itemId=/content/journal/micro/10.1099/mic.0.26882-0&mimeType=html&fmt=ahah

References

  1. Altschul S. F., Madden T. L., Schaffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. 1997; Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 [CrossRef]
    [Google Scholar]
  2. Ausubel F. A., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K.editors 2002 Current Protocols in Molecular Biology New York: Wiley;
  3. Boyer H. W., Roulland-Dussoix D. 1969; A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 14:459–472
    [Google Scholar]
  4. Cetin E. T., Töreci K., Ang Ö. 1965; Encapsulated Pseudomonas aeruginosa (Pseudomonas mucosus) strains. J Bacteriol 89:1432–1433
    [Google Scholar]
  5. Diehl A., von Wintzingerode F., Görisch H. 1998; Quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa is a homodimer – sequence of the gene and deduced structural properties of the enzyme. Eur J Biochem 257:409–419 [CrossRef]
    [Google Scholar]
  6. Farinha M. A., Kropinski A. M. 1990; Construction of broad-host-range plasmid vectors for easy visible selection and analysis of promoters. J Bacteriol 172:3496–3499
    [Google Scholar]
  7. Figurski D. H., Helinski D. R. 1979; Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76:1648–1652 [CrossRef]
    [Google Scholar]
  8. Görisch H. 2003; The ethanol oxidation system and its regulation in Pseudomonas aeruginosa. Biochim Biophys Acta 1647:98–102 [CrossRef]
    [Google Scholar]
  9. Hanahan D. 1983; Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580 [CrossRef]
    [Google Scholar]
  10. Henikoff S., Wallace J. C., Brown J. P. 1990; Finding protein similarities with nucleotide sequence databases. Methods Enzymol 183:111–132
    [Google Scholar]
  11. Hoang T. T., Karkhoff-Schweizer R. R., Kutchma A. J., Schweizer H. P. 1998; A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86 [CrossRef]
    [Google Scholar]
  12. Kretzschmar U., Schobert M., Görisch H. 2001; The Pseudomonas aeruginosa acsA gene, encoding an acetyl-CoA synthetase, is essential for growth on ethanol. Microbiology 147:2671–2677
    [Google Scholar]
  13. Kretzschmar U., Jeoung J., Rückert A., Görisch H. 2002; Malate : quinone oxidoreductase is essential for growth on ethanol or acetate in Pseudomonas aeruginosa. Microbiology 148:3839–3847
    [Google Scholar]
  14. Krogh A., Larsson B., von Heijne G., Sonnhammer E. L. 2001; Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580 [CrossRef]
    [Google Scholar]
  15. Miller J. M. 1992 A Short Course in Bacterial Genetics, a Laboratory Manual and Handbook for Escherichia coli and Related Bacteria Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  16. Reichmann P., Görisch H. 1993; Cytochrome c550 from Pseudomonas aeruginosa. Biochem J 289:173–178
    [Google Scholar]
  17. Rozen S., Skaletsky H. J. 1998; Primer3. Code available at http://www-genome.wi.mit.edu/genome_software/other/primer3.html
  18. Rupp M., Görisch H. 1988; Purification, crystallisation and characterization of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa. Biol Chem Hoppe-Seyler 369:431–439 [CrossRef]
    [Google Scholar]
  19. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  20. Schobert M. 1999; Molekulargenetische Untersuchungen zum Ethanol-oxidierenden System in Pseudomonas aeruginosa. PhD thesis Technische Universität; Berlin, Germany:
  21. Schobert M., Görisch H. 1999; Cytochrome c550 is an essential component of the quinoprotein ethanol oxidation system in Pseudomonas aeruginosa: cloning and sequencing of the genes encoding cytochrome c550 and an adjacent acetaldehyde dehydrogenase. Microbiology 145:471–481 [CrossRef]
    [Google Scholar]
  22. Schobert M., Görisch H. 2001; A soluble two-component regulatory system controls expression of quinoprotein ethanol dehydrogenase (QEDH) but not expression of cytochrome c550 of the ethanol-oxidation system in Pseudomonas aeruginosa. Microbiology 147:363–372
    [Google Scholar]
  23. Schwartz E., Gerischer U., Friedrich B. 1998; Transcriptional regulation of Alcaligenes eutrophus hydrogenase genes. J Bacteriol 180:3197–3204
    [Google Scholar]
  24. Schweizer H. P. 1991; The agmR gene, an environmentally responsive gene, complements defective glpR, which encodes the putative activator for glycerol metabolism in Pseudomonas aeruginosa. J Bacteriol 173:6798–6806
    [Google Scholar]
  25. Schweizer H. P. 1992; Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol Microbiol 6:1195–1204 [CrossRef]
    [Google Scholar]
  26. Schweizer H. P., Klassen T. R., Hoang T. 1996; Improved methods for gene analysis in Pseudomonas. In Molecular Biology of Pseudomonads pp. 229–237 Edited by Nakazawa T., Furukawa K., Haas D., Silver S. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  27. Schweizer H. P., Po C. 1996; Regulation of glycerol metabolism in Pseudomonas aeruginosa: characterization of the glpR repressor gene. J Bacteriol 178:5215–5221
    [Google Scholar]
  28. Smith A. W., Iglewski B. H. 1989; Transformation of Pseudomonas aeruginosa by electroporation. Nucleic Acids Res 17:10509 [CrossRef]
    [Google Scholar]
  29. Staskawicz B., Dahlbeck D., Keen N., Napoli C. 1987; Molecular characterization of cloned avirulence genes from race 0 to race 1 of Pseudomonas syringae pv. glycinea. J Bacteriol 169:5789–5794
    [Google Scholar]
  30. Stover C. K., Pham X. Q., Erwin A. L.28 other authors 2000; Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406:959–964 [CrossRef]
    [Google Scholar]
  31. Vrionis H. A., Daugulis A. J., Kropinski A. M. 2002; Identification and characterization of the AgmR regulator of Pseudomonas putida: role in alcohol utilization. Appl Microbiol Biotechnol 58:469–475 [CrossRef]
    [Google Scholar]
  32. Yanisch-Perron C., Vieira J., Messing J. 1985; Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33:103–119 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.26882-0
Loading
/content/journal/micro/10.1099/mic.0.26882-0
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