Skip to main content
SpringerLink
Log in

Cloning and characterization of a gene cluster involved in tetrahydrofuran degradation in Pseudonocardia sp. strain K1

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

A gene cluster involved in the utilization of tetrahydrofuran by Pseudonocardia sp. strain K1 was cloned and sequenced. Analysis of a 9.2-kb DNA fragment revealed eight ORFs. The genes designated as thmADBC encode the components of a putative monooxygenase exhibiting a high similarity to different binuclear-iron-containing multicomponent monooxygenases. thmA encodes the derived 545-amino-acid oxygenase α-subunit, thmD the 360-amino-acid reductase component, thmB the 346-amino-acid oxygenase β-subunit, and thmC the 117-amino-acid coupling protein. Upstream of the thm genes, an additional ORF (sad) was identified coding for a protein with high similarity to various aldehyde dehydrogenases. A succinate semialdehyde dehydrogenase activity was specifically expressed in tetrahydrofuran-grown cells. N-terminal sequence analysis of the purified protein revealed that it is encoded by sad. Northern blot analysis indicated that transcription of the thm genes and sad was specifically induced during growth on tetrahydrofuran. Mono-, di- and polycistronic transcripts of these genes were detected. Primer-extension analysis identified transcriptional start sites 37, 61, and 41 bp upstream of the translation start of sad, thmA, and thmB, respectively. Additional ORFs were identified upstream (orfY) and downstream (orfZ and aldH) of the thm genes. Furthermore, the data indicated that the analyzed gene cluster was present as a single copy and located on a plasmid.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1.
Fig. 2a, b.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.
Fig. 7a–c.

Similar content being viewed by others

References

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  PubMed  Google Scholar 

  • Becker D, Schräder T, Andreesen JR (1997) Two-component, flavin-dependent pyrrole-2-carboxylate monooxygenase from Rhodococcus sp. Eur J Biochem 249:739–747

    CAS  PubMed  Google Scholar 

  • Beltrametti F, Marcono AM, Bestetti G, Colombo C, Galli E, Ruzzi M, Zennaro E (1997) Sequencing and functional analysis of styrene catabolism genes from Pseudomonas fluorescens ST. Appl Environ Microbiol 63:2232–2239

    CAS  PubMed  Google Scholar 

  • Bernhardt B, Diekmann H (1991) Degradation of dioxane, tetrahydrofuran and other cyclic ethers by an environmental Rhodococcus strain. Appl Microbiol Biotechnol 36:120–123

    CAS  PubMed  Google Scholar 

  • Bernhardt FH, Bill E, Trautwein AX, Twilfer H (1988) 4-Methoxybenzoate monooxygenase from Pseudomonas putida: isolation, biochemical properties, substrate specificity, and reaction mechanism of the enzyme components. Methods Enzymol 161:281–294

    CAS  PubMed  Google Scholar 

  • Bertoni G, Martino M, Galli E, Barbieri P (1998) Analysis of the gene cluster encoding toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1. Appl Environ Microbiol 64:3626–3632

    CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Busch U, Preac-Mursic V, Wilske B (1996) Strain typing of Borrelia burgdorferi sensu lato by linear plasmid profile analysis. QIAGEN News 5:18–19

    Google Scholar 

  • Cardy DLN, Laidler V, Salmond GPC, Murrell JC (1991) Molecular analysis of the methane monooxygenase (MMO) gene cluster of Methylosinus trichosporium OB3b. Mol Microbiol 5:335–342

    CAS  PubMed  Google Scholar 

  • Chambliss KL, Caudle DL, Hinson DD, Moomaw CR, Slaughter CA, Jacobs C, Gibson KM (1995) Molecular cloning of the mature NAD+-dependent succinic semialdehyde dehydrogenase from rat and human. J Biol Chem 270:461–467

    Article  PubMed  Google Scholar 

  • Draper AJ, Manda A, Parkinson A (1997) Inhibition of coumarin 7-hydroxylase activity in human liver microsomes. Arch Biochem Biophys 341:47–61

    Article  CAS  PubMed  Google Scholar 

  • Eaton RW (1997) p-Cymene catabolic pathway in Pseudomonas putida F1: Cloning and characterization of DNA encoding conversion of p-cumene to p-cumate. J Bacteriol 179:3171–3180

    CAS  PubMed  Google Scholar 

  • 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

    CAS  PubMed  Google Scholar 

  • Eltis LD, Karlson U, Timmis KN (1993) Purification and characterization of cytochrome P-450 from Rhodococcus rhodochrous. Eur J Biochem 213:211–216

    CAS  PubMed  Google Scholar 

  • Fox BG, Froland WA, Dege JE, Lipscomb JD (1989) Methane monooxygenase from Methylosinus trichsporium OB3b. Purification and properties of a three-component system with high specific activity from a type II methanotroph. J Biol Chem 264:10023–10033

    CAS  PubMed  Google Scholar 

  • Horinouchi M, Kasuga K, Noriji H, Yamane H, Omori T (1997) Cloning and characterization of genes encoding an enzyme which oxidizes dimethyl sulfide in Acinetobacter sp. strain 20B. FEMS Microbiol Lett 155:99–105

    Article  CAS  PubMed  Google Scholar 

  • Johnson GR, Olsen RH (1995) Nucleotide sequence analysis of genes encoding a toluene/benzene-2-monooxygenase from Pseudomonas sp. strain JS150. Appl Environ Microbiol 61:3336–3346

    CAS  PubMed  Google Scholar 

  • Karlson U, Dwyer DF, Hooper SW, Moore ERB, Timmis KN, Eltis LD (1993) Two independently regulated cytochrome P-450 in a Rhodococcus rhodochrous strain that degrades 2-ethoxyphenol and 4-methoxybenzate. J Bacteriol 175:1467–1474

    CAS  PubMed  Google Scholar 

  • Kohlweyer U, Thiemer B, Schräder T, Andreesen JR (2000) Tetrahydrofuran degradation by a newly isolated culture of Pseudonocardia sp. strain K1. FEMS Microbiol Lett 186:301–306

    Article  CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    PubMed  Google Scholar 

  • Larkin MJ, De Mot R, Kulakov LA, Nagy I (1998) Applied aspects of Rhodococcus genetics. Antonie van Leeuwenhoek 74:133–153

    CAS  PubMed  Google Scholar 

  • Lipscomb JD (1994) Biochemistry of the soluble methane monooxygenase. Annu Rev Microbiol 48:371–399

    Article  CAS  PubMed  Google Scholar 

  • McFall E, Newmann EB (1996) Amino acids as carbon sources. In: Neidhardt FC (ed) Escherichia coli and Salmonella. ASM Press, Washington DC, pp358–379

  • Miura A, Dalton H (1995) Purification and characterization of the alkene monooxygenase from Nocardia corallina B-276. Biosci Biotech Biochem 59:853–859

    CAS  Google Scholar 

  • Moody DE (1991) The effect of tetrahydrofuran on biological systems: does a hepatotoxic potential exist? Drug Chem Toxicol 14:319–342

    CAS  PubMed  Google Scholar 

  • Nagy I, Schoofs G, Compernolle F, Proost P, Vanderleyden J, De Mot R (1995). Degradation of the thiocarbamate herbicide EPTC (S-ethyl dipropylcarbamothioate) and biosafening by Rhodococcus sp. strain NI86/21 involves an inducible cytochrome P-450 system and aldehyde dehydrogenase. J Bacteriol 177:676–687

    CAS  PubMed  Google Scholar 

  • Newman LM, Wackett LP (1995). Purification and characterization of toluene 2-monooxygenase from Burkholderia cepacia G4. Biochemistry 34:14066–14076

    CAS  PubMed  Google Scholar 

  • Nielsen AK, Gerdes K, Degn H, Murrell JC (1996) Regulation of bacterial methane oxidation: transcription of soluble methane monooxygenase operon of Methylococcus capsulatus (Bath) is repressed by copper ions. Microbiology 142:1289–1296

    CAS  Google Scholar 

  • Nielsen AK, Gerdes K, Murrell JC (1997) Copper-dependent reciprocal transcriptional regulation of methane monooxygenase genes in Methylococcus capsulatus and Methylosinus trichosporium. Mol Microbiol 25:399–409

    CAS  PubMed  Google Scholar 

  • 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

    CAS  PubMed  Google Scholar 

  • Normark S, Bergstrom S, Edlund T, Grundstrom T, Jaurin B, Lindberg FP, Olsson O (1983) Overlapping genes. Annu Rev Genet 17:499–525

    CAS  PubMed  Google Scholar 

  • Painter HA, King EF (1985) Ring test program 1983–84. Assessment of biodegradability of chemicals in water by manometric respirometry. Comm. Eur. Communities (Rep.), EUR 9962 (CA 103:183290)

    Google Scholar 

  • Parales RE, Adamus JE, White N, May HD (1994) Degradation of 1,4-dioxane by an actinomycete in pure culture. Appl Environ Microbiol 60:4527–4530

    CAS  PubMed  Google Scholar 

  • Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444–2448

    CAS  PubMed  Google Scholar 

  • Platt T (1986) Transcription termination an the regulation of gene expression. Annu Rev Biochem 55:339–372

    Article  CAS  PubMed  Google Scholar 

  • Rosenzweig AC, Frederick CA, Lippard SJ, Nordlund P (1993) Crystal structure of a bacterial non-haem iron hydroxylase that catalyses the biological oxidation of methane. Nature 366:537–543

    Google Scholar 

  • Rosenzweig AC, Brandstetter H, Whittington DA, Nordlund P, Lippard SJ, Frederick CA (1997) Crystal structures of the methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): Implication for substrate gating and component interactions. Proteins 29:141–152

    Article  CAS  PubMed  Google Scholar 

  • Saeki H, Furuhashi K (1994) Cloning and characterization of a Nocardia corallina B-276 gene cluster encoding alkene monooxygenase. J Ferment Bioeng 78:399–406

    CAS  Google Scholar 

  • Saito H, Miura KI (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72:619–629

    CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR. (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    PubMed  Google Scholar 

  • Schneider BL, Ruback S, Kiupakis AK, Kasbarian H, Pybus C, Reitzer L (2002) The Escherichia coli gab DTPC operon: specific γ-aminobutyrate catabolism and nonspecific induction. J Bacteriol 184:6976–6986

    Article  CAS  PubMed  Google Scholar 

  • Schräder T, Zarnt G, Andreesen JR (2001) NAD(P)–dependent aldehyde dehydrogenases induced during growth of Ralstonia eutropha strain Bo on tetrahydrofurfuryl alcohol. J Bacteriol 183:7408–7411

    Article  PubMed  Google Scholar 

  • Shingler V, Franklin FCH, Tsuda M, Holroyd D, Bagdasaria M (1989) Molecular analysis of a plasmid-encoded phenol hydroxylase from Pseudomonas CF600. J Gen Microbiol 135:1083–1092

    CAS  PubMed  Google Scholar 

  • Small FJ, Ensign SA (1997) Alkene monooxygenase from Xanthobacter strain Py2. J Biol Chem 272:24913–24920

    Article  CAS  PubMed  Google Scholar 

  • Stainthorpe AC, Lees V, Salmond GPC, Dalton H, Murrell JC (1990) The methane monooxygenase gene cluster of Methycoccus capsulatus (Bath). Gene 91:27–34

    Article  CAS  PubMed  Google Scholar 

  • Stirling DI, Dalton H (1980) Oxidation of dimethyl ether, methyl formate and bromomethane by Methylococcus capsulatus (Bath). J Gen Mircobiol 116:277–283

    CAS  Google Scholar 

  • Strohl WR (1992) Compilation and analysis of DNA sequences associated with apparent streptomycete promoters. Nucleic Acids Res 20:961–974

    CAS  PubMed  Google Scholar 

  • Thiemer B, Andreesen JR, Schräder T (2001) The NADH-dependent reductase of multicomponent tetrahydrofuran monooxygenase contains a covalently bound FAD. Eur J Biochem 268:3774–3782

    Article  CAS  PubMed  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) Clustal-W-improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    PubMed  Google Scholar 

  • Trower MK, Sariaslani FS, O'Keefe DPO (1989) Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus. J Bacteriol 171:1781–1787

    CAS  PubMed  Google Scholar 

  • Van Hylckama Vlieg JET, Leemhuis H, Lutje Spelberg JH, Janssen DB (2000) Characterization of the gene cluster involved in isoprene metabolism in Rhodococcus sp. strain AD45 J. Bacteriol 182:1956–1963

    Article  Google Scholar 

  • Westerheide L, Pascaly M, Krebs B (2000) Methane monooxygenase and its related biomimetic models. Curr Opin Chem Biol 4:235–241

    Article  CAS  PubMed  Google Scholar 

  • White GF, Russell NJ, Tidswell EC (1996) Bacterial scission of ether bonds. Microbiol Rev 60:216–232

    CAS  PubMed  Google Scholar 

  • Wösten MMSM (1998) Eubacterial sigma-factors. FEMS Microbiol Rev 22:127–150

    PubMed  Google Scholar 

  • Yen KW, Karl MR, Blatt LM, Simon MJ, Winter RB, Fausset PR, Lu HS, Harcourt AA, Chen KK (1991) Cloning and characterization of a Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase. J Bacteriol 173:5315–5327

    CAS  PubMed  Google Scholar 

  • Zarnt G, Schräder T, Andreesen JR (2001) Catalytic and molecular properties of the quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase from Ralstonia eutropha strain Bo. J Bacteriol 183:1954–1960

    Article  CAS  PubMed  Google Scholar 

  • Zhou NY, Jenkins A, Chan Kwo Chion CKN, Leak DJ (1999) The alkene monooxygenase from Xanthobacter strain Py2 is closely related to aromatic monooxygenases and catalyzes aromatic monohydroxylation of benzene, toluene, and phenol. Appl Environ Microbiol 65:1589–1595

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr. Peter Rücknagel (Max-Plank-Gesellschaft, Forschungsstelle Enzymologie der Proteinfaltung, Halle) for N-terminal sequence analysis. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Graduiertenkolleg: Adaptive physiologisch-biochemische Reaktionen auf ökologisch relevante Wirkstoffe) and the Fonds der Chemischen Industrie.

Author information

Authors and Affiliations

  1. Institut für Mikrobiologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 , Halle, Germany

    Barbara Thiemer & Jan R. Andreesen

  2. GL-Biotech GmbH, Fahrenheitstrasse 1, 28359 , Bremen, Germany

    Thomas Schräder

Authors
  1. Barbara Thiemer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jan R. Andreesen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Thomas Schräder
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Thomas Schräder.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Thiemer, B., Andreesen, J.R. & Schräder, T. Cloning and characterization of a gene cluster involved in tetrahydrofuran degradation in Pseudonocardia sp. strain K1. Arch Microbiol 179, 266–277 (2003). https://doi.org/10.1007/s00203-003-0526-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00203-003-0526-7

Keywords

Search

Navigation