1887

Abstract

A promoter-probe vector (pHX200) was constructed using the broad-host-range cosmid pLA2917 and a promoterless ( gene of as the reporter gene. Insertion of the cloned promoter fragment of the methanol dehydrogenase large subunit gene (ethanol idation) in front of the gene in pHX200V-47 resulted in high-level expression of the gene product catechol 2,3-dioxygenase in XX. The specific activity of the enzyme was four times higher in methanol-grown XX culture than in succinate-grown culture. Interestingly, the insertion of the same fragment in the opposite orientation in front of the gene (pHX200V-74) also led to elevated catechol 2,3-dioxygenase activity. This promoter activity was also methanol regulated. A total of 21 methanol-regulated promoter clones were identified that originate from three gene clusters (groups V, VI and VII) on the XX chromosome involved in methanol oxidation. Vector pHX200 and its derivatives were successfully mobilized into cells of three phylogenetically diverse methylotrophic bacteria: AS1, AM1 and sp. DM4. The reporter gene () was functionally expressed in all three bacteria with the aid of a proper promoter. Transcriptional fusions of methanol-regulated promoters with the gene were mobilized into Mox mutants of XX and AM1 to study the roles of methanol oxidation genes, especially regulatory genes. It appeared that vector pHX200 is an efficient promoter probe with wide host-range and an excellent tool for studies of structure and function of promoters/regulators.

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1993-04-01
2021-07-24
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References

  1. Allen L. N., Hanson R. S. 1985; Construction of broad-host-range cosmid cloning vectors: identification of genes necessary for growth of Methylobacterium organophilum XX on methanol. Journal of Bacteriology 161:955–962
    [Google Scholar]
  2. Anderson D. J., Morris C. J., Nunn D. N., Anthony C., Lidstrom M. E. 1990; Nucleotide sequence of the Methylobacterium extorquens AM1 moxF and moxJ genes involved in methanol oxidation. Gene 90:173–176
    [Google Scholar]
  3. Anthony C. 1982 The Biochemistry of Methylotrophs New York: Academic Press.;
    [Google Scholar]
  4. Anthony C. 1986; Bacterial oxidation of methane and methanol. Advances in Microbial Physiology 27:113–210
    [Google Scholar]
  5. Bastien C., Machlin S., Zhang Y., Donaldson K., Hanson R. S. 1989; Organization of genes required for the oxidation of methanol to formaldehyde in three Type II methylotrophs. Applied and Environmental Microbiology 55:3124–3130
    [Google Scholar]
  6. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
    [Google Scholar]
  7. Cox J. M., Day D. J., Anthony C. 1992; The interaction of methanol dehydrogenase and its electron acceptor, cytochrome cL, in methylotrophic bacteria. Biochimica et Biophysica Acta 1119:97–106
    [Google Scholar]
  8. Figurski D. H., Helinski D. R. 1979; Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proceedings of the National Academy of Sciences of the United States of America 77:7347–7357
    [Google Scholar]
  9. Gicquel-Sanzey B., Cossart P. 1982; Homologies between different procaryotic DNA binding regulatory proteins and between their sites of action. EMBO Journal 1:591–595
    [Google Scholar]
  10. Hahn D. R., Solenberg P. J., Baltz R. H. 1991; Tn5099, a xylE promoter probe transposon for Streptomyces spp. Journal of Bacteriology 173:5573–5577
    [Google Scholar]
  11. Ingram C., Brawner M., Youngman P., Westpheling J. 1989; xylE functions as an efficient reporter gene in Streptomyces spp.: use for the study of galP1, a catabolic-controlled promoter. Journal of Bacteriology 171:6617–6624
    [Google Scholar]
  12. Kataeva I. A., Golovleva L. A. 1990; Catechol 2,3-dioxygenases from Pseudomonas aeruginosa 2x. Methods in Enzymology 188:115–121
    [Google Scholar]
  13. King A., Chater K. F. 1986; The expression of the Escherichia coli lacZ gene in Streptomyces. Journal of General Microbiology 132:1739–1752
    [Google Scholar]
  14. Kingston R. E. 1989; Primer extension. In Current Protocols in Molecular Biology pp. 4.8.1–4.8.3., Edited by. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. New York: John Wiley;
    [Google Scholar]
  15. Lidstrom M. E. 1990; Genetics of carbon metabolism in methylo-trophic bacteria. FEMS Microbiology Reviews 87:431–436
    [Google Scholar]
  16. Machlin S. M., Hanson R. S. 1988; Nucleotide sequence and transcriptional start site of the Methylobacterium organophilum XX methanol dehydrogenase structural gene. Journal of Bacteriology 170:4739–4747
    [Google Scholar]
  17. Machlin S. M., Tam P. E., Bastien C. A., Hanson R. S. 1987; Genetic and physical analysis of Methylobacterium organophilum XX genes encoding methanol oxidation. Journal of Bacteriology 170:141–148
    [Google Scholar]
  18. Machlin S. M., Tam P. E., Allen L. N., Hanson R. S. 1988; Genetic studies of methanol metabolism in the facultative methylotroph, Methylobacterium organophilum XX. In Microbial Metabolism and the Carbon Cycle pp. 381–392, Edited by. Hagedorn S. R., Hanson R. S., Kunz D. A. New York: Harwood Academic Publishers.;
    [Google Scholar]
  19. MacLennan D. G., Onsby J. C., Vasey R. B., Cotton N. T. 1971; The influence of dissolved oxygen on Pseudomonas AM1 grown on methanol in continuous culture. Journal of General Microbiology 69:395–404
    [Google Scholar]
  20. Morris C. J., Lidstrom M. E. 1992; Cloning of a methanol-inducible moxF promoter and its analysis in moxB mutants of Methylobacterium extorquens AMlrif. Journal of Bacteriology 174:4444–4449
    [Google Scholar]
  21. Nunn D. N., Lidstrom M. E. 1986a; Isolation and complementation analysis of 10 methanol oxidation mutant classes and identification of the methanol dehydrogenase structure gene of Methylobacterium sp. strain AM1. Journal of Bacteriology 166:581–590
    [Google Scholar]
  22. Nunn D. N., Lidstrom M. E. 1986b; Phenotypic characterization of 10 methanol oxidation mutant classes in Methylobacterium sp. strain AM1. Journal of Bacteriology 166:591–597
    [Google Scholar]
  23. Nunn D. N., Day D., Anthony C. 1989; The second subunit of methanol dehydrogenase of Methylobacterium extorquens AM1. Biochemical Journal 260:857–862
    [Google Scholar]
  24. O’Connor M. 1981; Regulation and genetics in facultative methylotrophic bacteria. In Microbial growth on C1 Compounds pp. 294–300, Edited by. Dalton H. London: Heyden;
    [Google Scholar]
  25. O’connor M. L., Hanson R. S. 1977; Enzyme regulation in Methylobacterium organophilum. Journal of General Microbiology 98:265–272
    [Google Scholar]
  26. Patt T. E., Cole G. C., Hanson R. S. 1976; Methylobacterium, a new genus of facultative methylotrophic bacteria. International Journal of Systematic Bacteriology 26:226–229
    [Google Scholar]
  27. Ray C., Hay R. E., Carter H. L., Moran C. P. JR 1985; Mutations that affect utilization of a promoter in stationary-phase Bacillus subtilis. Journal of Bacteriology 163:610–614
    [Google Scholar]
  28. Ray C., Igo M., Shafer W., Losick R., Moran C. P. JR 1988; Suppression of ctc promoter mutations in Bacillus subtilis. Journal of Bacteriology 170:900–907
    [Google Scholar]
  29. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, Second edition.. Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory.;
    [Google Scholar]
  30. Tsuji K., Tsien H. C., Hanson R. S., Depalma S. R., Scholtz R., LaRoche S. 1990; 16S ribosomal RNA sequence analysis for determination of phylogenetic relationship among methylotrophs. Journal of General Microbiology 136:1–10
    [Google Scholar]
  31. Windass J. D., Worsey M. J., Pioli E. M., Pioli D., Barth P. T., Atherton K. T., Dart E. C., Byrom D., Powell K., Senior P. J. 1980; Improved conversion of methanol to single-cell protein by Methylophilus methylotrophus. Nature; London: 287396–401
    [Google Scholar]
  32. Winstanley C., Morgan J. A. W., Pickup R. W., Johns J. G., Saunders J. R. 1989; Differential regulation of lambda pL and pR promoters by a cI repressor in a broad-host-range thermoregulated plasmid marker system. Applied and Environmental Microbiology 55:771–777
    [Google Scholar]
  33. Zhu Y. S., Kaplan S. 1985; Effects of light, oxygen, and substrates on steady-state levels of mRNA coding for ribulose-1,5-bisphosphate carboxylase and light-harvesting and reaction center polypeptides in Rhodopseudomonas sphaeroides. Journal of Bacteriology 162:925–932
    [Google Scholar]
  34. Zukowski M. M., Gaffney D. F., Speck D., Kauffmann M., Findeli A., Wisecup A., Lecocq J.-P. 1983; Chromogenic identification of genetic regulatory signals in Bacillus subtilis based on expression of a cloned Pseudomonas gene. Proceedings of the National Academy of Sciences of the United States of America 80:1101–1105
    [Google Scholar]
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