Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Paper
  • Published:

New Route to Bacterial Production of Indigo

Abstract

Cells of Escherichia coli K–12 containing a cloned fragment of Pseudomonas putida TOL plasmid pWW0 produce the dye indigo. Analysis of the cloned fragment and Tn1000 transposon insertion mutagenesis has identified the xylA gene as being responsible for this phenotype. The xylA gene specifies xylene oxidase, a relaxed specificity enzyme that hydroxylates or monooxygenates toluene and xylenes and their corresponding alcohols. Indole, which is formed from tryptophan by tryptophanase in E. coli, was shown to be a precursor in the reaction sequence leading to indigo formation. These results suggest a novel route for bacterial production of indigo via hydroxylation of indole.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Gray, P.M.M. 1928. The formation of indigotin from indol by soil bacteria. Proc. Royal Soc. London, Ser. B 102:2263–2279.

    Article  Google Scholar 

  2. Oshima, T., Kawai, S. and Egani, F. Oxidation of indole to indigotin by Pseudomonas indoloxidans . 1965. J. Biol. Chem. 58:259–263.

    CAS  Google Scholar 

  3. Ensley, B.D., Ratzkin, B.J., Osslund, T.D., Simon, M.J., Wackett, L.P. and Gibson, D.T. 1983. Expression of naphtalene oxidation genes in Escherichia coli results in the biosynthesis of indigo. Science 222:167–169.

    Article  CAS  Google Scholar 

  4. Worsey, M.J. and Williams, P.A. 1975. Metabolism of toluene and xylenes by Pseudomonas putida (arvilla) mt–2: evidence for a new function of the TOL plasmid. J. Bacteriol. 124:7–13.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Kunz, D.A. and Chapman, P.J. 1981. Isolation and characterization of spontaneously occuring TOL plasmid mutants of Pseudomonas putida HS1. J. Bacteriol. 146:179–191.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Murray, K., Duggleby, C.J., Sala–Trepat, J.M. and Williams, P.A. 1972. The metabolism of benzoate and methylbenzoates via the meta–cleavage pathway by Pseudomonas arvilla mt–2. Eur. J. Biochem 28:301–310.

    Article  CAS  Google Scholar 

  7. Franklin, F.C.H., Bagdasarian, M., Bagdasarian, M.M. and Timmis, K.N., 1981. Molecular and functional analysis of the TOL plasmid pWW0 from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta cleavage pathway. Proc. Natl. Acad. Sci. USA, 78:7458–7462.

    Article  CAS  Google Scholar 

  8. Inouye, S., Nakazawa, A. and Nakazawa, T. 1981. Molecular cloning of TOL genes xy1B and xy1E in Escherichia coli . J. Bacteriol. 145:1137–1143.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Inouye, S., Ebina, Y., Nakazawa, A. and Nakazawa 1984. Nucleotide sequence surrounding transcription initiation site of xy1ABC operon on TOL plasmid of Pseudomonas putida . Proc. Natl. Acad. Sci. USA 81:1688–1691.

    Article  CAS  Google Scholar 

  10. Worsey, M.J., Franklin, F.C.H. and Williams, P.A. 1978. Regulation of the degradative pathway enzymes coded for by the TOL plasmid (pWW0) from Pseudomonas putida mt–2. J. Bacteriol. 134:757–764 (1978).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Maniatis, T., Fritsch, E.F. and Sambrook, J., Molecular Cloning, A Laboratory Manual. 1982. Cold Spring Harbor, New York.

    Google Scholar 

  12. Meulien, P., Downing, R.G. and Broda, P. 1981. Excision of the 40kb segment of the TOL plasmid pWW0 from Pseudomonas putida mt–2 involves direct repeats. Molec. Gen. Genet. 184:97–101.

    Article  CAS  Google Scholar 

  13. Franklin, F.C.H., Lehrbach, P.R., Lurz, R., Rueckert, B., Bagdasarian, M. and Timmis, K.N. 1983. Localization and functional analysis of transportation mutations in regulatory genes of the TOL catabolic pathway. J. Bacteriol. 154:676–685.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Gasson, M.J. and Willetts, N.S. 1977. Further characterization of the F fertility inhibition systems of “unusual” fin+ plasmids. J. Bacteriol. 131:413–420.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Newton, W.A. and Snell, E.E. 1964. Catalytic properties of tryptophanase, a multifunctional pyridoxal phosphate enzyme. Proc. Natl. Acad. Sci. USA 51:382–389.

    Article  CAS  Google Scholar 

  16. Bilezikim, J.P., Kaempfer, O.R. and Magasanik, B. 1967. Mechanism of tryptophanase induction in Escherichia coli . J. Mol. Biol. 27:495–506.

    Article  Google Scholar 

  17. Yanofsky, C.Y., Kelley, R.L. and Horn, V. 1984. Repression is relieved before attenuation in the trp operon of Escherichia coli as tryptophan starvation becomes increasingly severe. J. Bacteriol. 158:1018–1024.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Harayama, S., Lehrbach, P.R. and Timmis, K.N. 1984. Transposon mutagenesis analysis of meta–cleavage pathway operon genes of the TOL plasmid of Pseudomonas putida mt–2. J. Bacteriol. 160:251–255.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Remaut, E., Stanssens, P. and Fiers, W. 1983. Inducible high level synthesis of mature human fibroblast interferon in Escherichia coli . Nucleic Acids Res. 11:4677–4688.

    Article  CAS  Google Scholar 

  20. Remaut, E., Tsao, H. and Fiers, W. 1983. Improved plasmid vectors with a thermoinducible expression and temperature–regulated runaway replication. Gene 22:103–113.

    Article  CAS  Google Scholar 

  21. Witkop, B. and Patrick, J.B. 1951. Addition reactions and Wagner–Meerwein rearrangement in the indoxyl series. J. Amer. Chem. Soc. 73:713–718.

    Article  CAS  Google Scholar 

  22. May, S.W. and Abbott, B.J. 1973. Enzymic epoxidation. II. Comparison between the epoxidation and hydroxylation reactions catalyzed by the w–hydroxylation system of Pseudomonas oleovorans . J. Biol. Chem. 218:1725–1730.

    Google Scholar 

  23. Reineke, W. and Knackmuss, H.–J. . 1978. Chemical structure and biodegradability of halogenated aromatic compounds. Substituent effect on dehydrogenation of 3,5–cyclohexadiene–1,2–diol–1–carboxylic acid. Biochim. Biophys. Acta 542:412–423.

    Article  CAS  Google Scholar 

  24. Lehrbach, P.R., Zeyer, J., Reineke, W., Knackmuss, H.–J. and Timmis, K.N. 1984. Enzyme recruitment in vitro: use of cloned genes to extend the range of haloaromatics degraded by Pseudomonas sp. strains B13. J. Bacteriol. 158:1025–1032.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Timmis, K.N., Lehrbach, P.R., Harayama, S., Don, R.H., Mermod, N., Bas, S., Leppik, R., Weightman, A.J., Reineke, W. and Knackmuss, H.–J. 1985. Analysis and manipulation of plasmid–encoded pathways for the catabolism of aromatic compounds by soil bacteria, p. 719–739 In: Plasmids in Bacteria. Helinski, D. R., Cohen, S. N., Clewell, D. B, Jackson, D. A. and Hollaender, A. (eds). Plenum, New York.

    Chapter  Google Scholar 

  26. Mermod, N., Lehrbach, P.R., Don, R.H. and Timmis, K.N. 1986. Gene cloning and manipulation in Pseudomonads . In: The Bacteria, Vol. 10. Sokatch, J. R. (ed.). Academic Press, NY.

    Google Scholar 

  27. Iida, A., Harayama, S., Iino, T. and Hazelbauer, G.L. 1984. Molecular cloning and characterization of genes required for ribose transport and utilization in Escherichia coli K–12. J. Bacteriol. 158:674–682.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Harayama, S., Oguchi, T. and Iino, T. 1984. Does Tn10 transpose via the cointegrate molecule? Molec. Gen. Genet. 197:62–66.

    Article  CAS  Google Scholar 

  29. Sancar, A. and Rupp, W.D. 1979. Cloning of uvrA, lexC and ssb genes of Escherichia coli . Biochem. Biophys. Res. Comm. 90:123–129.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mermod, N., Harayama, S. & Timmis, K. New Route to Bacterial Production of Indigo. Nat Biotechnol 4, 321–324 (1986). https://doi.org/10.1038/nbt0486-321

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0486-321

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing