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.

  • Letter
  • Published:

Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes

Nature Biotechnology volume 23, pages 88–93 (2005)Cite this article

Abstract

Recent awareness that most microorganisms in the environment are resistant to cultivation has prompted scientists to directly clone useful genes from environmental metagenomes1. Two screening methods are currently available for the metagenome approach, namely, nucleotide sequence–based screening2 and enzyme activity–based screening3. Here we have introduced and optimized a third option for the isolation of novel catabolic operons, that is, substrate-induced gene expression screening (SIGEX). This method is based on the knowledge that catabolic-gene expression is generally induced by relevant substrates and, in many cases, controlled by regulatory elements situated proximate to catabolic genes. For SIGEX to be high throughput, we constructed an operon-trap gfp-expression vector available for shotgun cloning that allows for the selection of positive clones in liquid cultures by fluorescence-activated cell sorting. The utility of SIGEX was demonstrated by the cloning of aromatic hydrocarbon–induced genes from a groundwater metagenome library and subsequent genome-informatics analysis.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Schematic representation of the SIGEX scheme with an example of cloning of a phenol-degradative operon fragment from R. eutropha strain E2.
Figure 2: RFLP types obtained by SIGEX.
Figure 3: Characterization of the Bzo71-8 P450.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Daniel, R. The soil metagenome—a rich resource for the discovery of novel natural products. Curr. Opin. Biotechnol. 15, 199–204 (2004).

    Article  CAS  Google Scholar 

  2. Okuta, A., Ohnishi, K. & Harayama, S. PCR isolation of catechol 2,3-dioxygenase gene fragments from environmental samples and their assembly into functional genes. Gene 212, 221–228 (1998).

    Article  CAS  Google Scholar 

  3. Henne, A., Daniel, R., Schmitz, R.A. & Gottschalk, G. Construction of environmental DNA libraries in Escherichia coli and screening for the presence of genes conferring utilization of 4-hydroxybutyrate. Appl. Environ. Microbiol. 65, 3901–3907 (1999).

    Article  CAS  Google Scholar 

  4. Teramoto, M., Futamata, H., Harayama, S. & Watanabe, K. Characterization of a high-affinity phenol hydroxylase from Comamonas testosteroni R5 by gene cloning, and expression in Pseudomonas aeruginosa PAO1c. Mol. Gen. Genet. 262, 552–558 (1999).

    Article  CAS  Google Scholar 

  5. Debarbouille, M., Martin-Verstraete, I., Klier, A. & Rapoport, G. The transcriptional regulator LevR of Bacillus subtilis has domains homologous to both sigma54- and phosphotransferase system-dependent regulators. Proc. Natl. Acad. Sci. USA 88, 2212–2216.

  6. Rogowsky, P.M. et al. Molecular characterization of the vir regulon of Agrobacterium tumefaciens: complete nucleotide sequence and gene organization of the 2863-kbp regulon cloned as a single unit. Plasmid 23, 85–106 (1990).

    Article  CAS  Google Scholar 

  7. Valdivia, R.H. & Falkow, S. Fluorescence-based isolation of bacterial genes expressed within host cells. Science 277, 2007–2011 (1997).

    Article  CAS  Google Scholar 

  8. Ochman, H., Gerber, A.S. & Hartl, D.L. Genetic applications of an inverse polymerase chain reaction. Genetics 120, 621–623 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Hino, S., Watanabe, K. & Takahashi, N. Phenol hydroxylase cloned from Ralstonia eutropha strain E2 exhibits novel kinetic properties. Microbiology 144, 1765–1772 (1998).

    Article  CAS  Google Scholar 

  10. Sambrook, J. & Russell, D.W. Molecular Cloning: A Laboratory Manual, edn. 3 (Cold Spring Harbor Press, Cold Spring Harbor, New York, 2001).

    Google Scholar 

  11. Sze, C.C., Moore, T. & Shingler, V. Growth phase-dependent transcription of the sigma(54)-dependent Po promoter controlling the Pseudomonas-derived (methyl)phenol dmp operon of pVI150. J. Bacteriol. 178, 3727–3735 (1996).

    Article  CAS  Google Scholar 

  12. Yuste, L., Canosa, I. & Rojo, F. Carbon-source-dependent expression of the PalkB promoter from the Pseudomonas oleovorans alkane degradation pathway. J. Bacteriol. 180, 5218–5226 (1998).

    Article  CAS  Google Scholar 

  13. Watanabe, K. et al. Molecular characterization of bacterial populations in petroleum-contaminated groundwater discharged from underground crude-oil-storage cavities. Appl. Environ. Microbiol. 66, 4803–4809 (2000).

    Article  CAS  Google Scholar 

  14. Marques, S. & Ramos, J.L. Transcriptional control of the Pseudomonas putida TOL plasmid catabolic pathways. Mol. Microbiol. 9, 923–929 (1993).

    Article  CAS  Google Scholar 

  15. Parsek, M.R., Shinabarger, D.L., Rothmel, R.K. & Chakrabarty, A.M. Roles of CatR and cis,cis-muconate in activation of the catBC operon, which is involved in benzoate degradation in Pseudomonas putida . J. Bacteriol. 174, 7798–7806 (1992).

    Article  CAS  Google Scholar 

  16. Seoane, A.S. & Levy, S.B. Characterization of MarR, the repressor of the multiple antibiotic resistance (mar) operon in Eschericia coli . J. Bacteriol. 177, 3414–3419 (1995).

    Article  CAS  Google Scholar 

  17. Cowles, C.E., Nichols, N.N. & Harwood, C.S. BenR, a XylS homolog, regulates three different pathways of aromatic acid degradation in Pseudomonas putida . J. Bacteriol. 182, 6339–6346 (2000).

    Article  CAS  Google Scholar 

  18. Rothmel, R.K. et al. Nucleotide sequencing and characterization of Pseudomonas putida catR: a positive regulator of the catBC operon is a member of the LysR family. J. Bacteriol. 172, 922–931 (1990).

    Article  CAS  Google Scholar 

  19. Blehert, D.S., Fox, B.G. & Chambliss, G.H. Cloning and sequence analysis of two Pseudomonas flavoprotein xenobiotic reductases. J. Bacteriol. 181, 6254–6263 (1999).

    Article  CAS  Google Scholar 

  20. Kumagai, H., Yamada, H., Matsui, H., Ohkishi, H. & Ogata, K. Tyrosine phenol lyase I. Purification, crystallization, and properties. J. Biol. Chem. 245, 1767–1772 (1970).

    CAS  PubMed  Google Scholar 

  21. Poon, W.W. et al. Identification of Escherichia coli ubiB, a gene required for the first monooxygenase step in ubiquinone biosynthesis. J. Bacteriol. 182, 5139–5146 (2000).

    Article  CAS  Google Scholar 

  22. Abe, T. et al. Informatics for unveiling hidden genome signatures. Genome Res. 13, 693–702 (2003).

    Article  CAS  Google Scholar 

  23. Larimer, F.W. et al. Complete genome sequence of the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris . Nat. Biotechnol. 22, 55–61 (2004).

    Article  CAS  Google Scholar 

  24. Peterson, J. et al. Cytochrome P-450terp. Isolation and purification of the protein and cloning and sequencing of its operon. J. Biol. Chem. 267, 14193–14203 (1992).

    CAS  PubMed  Google Scholar 

  25. George, P., Bettlestene, J. & Griffith, J.S. in Haematin Enzymes, vol. 1 (eds. Falk, J.E., Lemberg, R., & Morton, R.K.) 105–141 (Pergamon Press Ltd, Oxford 1961).

    Book  Google Scholar 

  26. Peterson, J.A., Lorence, M.C. & Amarneh, B. Putidaredoxin reductase and putidaredoxin. J. Biol. Chem. 265, 6066–6073 (1990).

    CAS  PubMed  Google Scholar 

  27. Silhavy, T.J., Berman, M.L. & Enquist, L.W. Experiments With Gene Fusions (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1984).

    Google Scholar 

  28. Sakikawa, C., Taguchi, H., Makino, Y. & Yoshida, M. On the maximum size of proteins to stay and fold in the cavity of GroEL underneath GroES. J. Biol. Chem. 274, 21251–21256 (1999).

    Article  CAS  Google Scholar 

  29. Peng, X., Misawa, N. & Harayama, S. Isolation and characterization of thermophilic Bacilli degrading cinnamic, 4-coumaric, and ferulic acids. Appl. Environ. Microbiol. 69, 1417–1427 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Yasuo Igarashi and Hiroshi Ikenaga for helpful advice and continuous encouragement and Fusako Numazaki for technical assistance. This work was supported by the New Energy and Industrial Technology Development Organization (NEDO).

Author information

Authors and Affiliations

  1. Laboratory of Applied Microbiology, Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate, 026-0001, Japan

    Taku Uchiyama & Kazuya Watanabe

  2. Department of Population Genetics, National Institute of Genetics and the Graduate University for Advanced Studies, 1111 Yata, Mishima, 411-8540, Shizuoka, Japan

    Takashi Abe & Toshimichi Ikemura

  3. Xanagen Inc., 2-7-14 Higashinakano, Nakano, Tokyo, 164-0003, Japan

    Takashi Abe

Authors
  1. Taku Uchiyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Takashi Abe
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Toshimichi Ikemura
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kazuya Watanabe
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kazuya Watanabe.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Uchiyama, T., Abe, T., Ikemura, T. et al. Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes. Nat Biotechnol 23, 88–93 (2005). https://doi.org/10.1038/nbt1048

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1048

This article is cited by

Search

Advanced 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