Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Gene cloning and in vivo characterization of a dibenzothiophene dioxygenase from Xanthobacter polyaromaticivorans


Xanthobacter polyaromaticivorans sp. nov. 127W is a bacterial strain that is capable of degrading a wide range of cyclic aromatic compounds such as dibenzothiophene, biphenyl, naphthalene, anthracene, and phenanthrene even under extremely low oxygen [dissolved oxygen (DO)≤0.2 ppm] conditions (Hirano et al., Biosci Biotechnol Biochem 68:557–564, 2004). A major protein fraction carrying dibenzothiophene degradation activity was purified. Based on its partial amino acid sequences, dbdCa gene encoding alpha subunit terminal oxygenase (DbdCa) and its flanking region were cloned and sequenced. A phylogenetic analysis based on the amino acid sequence demonstrates that DbdCa is a member of a terminal oxygenase component of group IV ring-hydroxylating dioxygenases for biphenyls and monocyclic aromatic hydrocarbons, rather than group III dioxygenases for polycyclic aromatic hydrocarbons. Gene disruption in dbdCa abolished almost of the degradation activity against biphenyl, dibenzothiophene, and anthracene. The gene disruption also impaired degradation activity of the strain under extremely low oxygen conditions (DO≤0.2 ppm). These results indicate that Dbd from 127W represents a group IV dioxygenase that is functional even under extremely low oxygen conditions.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


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

  2. Calvin NM, Hanawalt PC (1988) High-efficiency transformation of bacterial cells by electroporation. J Bacteriol 170:2796–2801

  3. Crawford RL (1976) Pathways of 4-hydroxybenzoate degradation among species of Bacillus. J Bacteriol 127:204–210

  4. Denome SA, Stanley DC, Olson ES, Young KD (1993) Metabolism of debenzothiophene and naphthalene in Pseudomonas strains: complete DNA sequence of an upper naphthalene catabolic pathway. J Bacteriol 175:6890–6901

  5. Gibson JS, Harwood C (2002) Metabolic diversity in aromatic compound utilization by anaerobic microbes. Annu Rev Microbiol 56:345–369

  6. Habe H, Omori T (2003) Genetics of polycyclic aromatic hydrocarbon metabolism in diverse aerobic bacteria. Biosci Biotechnol Biochem 67:225–243

  7. Haddock JD, Gibson DT (1995) Purification and characterization of the oxygenase component of biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400. J Bacteriol 177:5834–5839

  8. Herrick JB, Stuart-Keil KG, Ghiorse WC, Madsen EL (1997) Natural horizontal transfer of a naphthalene dioxygenase gene between bacteria native to a coal tar-contaminated field site. Appl Environ Microbiol 63:2330–2337

  9. Hirano S, Kitauchi F, Haruki M, Imanaka T, Morikawa M, Kanaya S (2004) Isolation and characterization of Xanthobacter polyaromaticivorans sp. nov. 127W that degrades polycyclic and heterocyclic aromatic compounds under extremely low oxygen conditions. Biosci Biotechnol Biochem 68:557–564

  10. Jiang H, Parales RE, Lynch NA, Gibson DT (1996) Site-directed mutagenesis of conserved amino acids in the alpha subunit of toluene dioxygenase: potential mononuclear non-heme iron coordination sites. J Bacteriol 171:3133–3139

  11. Kanaly RA, Harayama S (2000) Biodegradation of high-molecular mass polycyclic aromatic hydrocarbons by bacteria. J Bacteriol 182:2059–2067

  12. Kauppi B, Lee K, Carredano E, Parales RE, Gibson DT, Eklund H, Ramaswamy S (1998) Structure of an aromatic-ring-hydroxylating dioxygenase-naphthalene 1,2-dioxygenase. Structure 6:571–586

  13. Kawasaki H, Suzuki K (1990) Separation of peptides dissolved in a sodium dodecyl sulfate solution by reversed-phase liquid chromatography: removal of sodium dodecyl sulfate from peptides using an ion-exchange precolumn. Anal Biochem 186:264–268

  14. Kesseler M, Dabbs ER, Averhoff B, Gottschalk G (1996) Studies on the isopropylbenzene 2,3-dioxygenase and the 3-isopropylcatechol 2,3-dioxygenase genes encoded by the linear plasmid of Rhodococcus erythropolis BD2. Microbiology 142:3241–3251

  15. Kimura N, Nishi A, Goto M, Furukawa K (1997) Functional analyses of a variety of chimeric dioxygenases constructed from two biphenyl dioxygenases that are similar structurally but different functionally. J Bacteriol 179:3936–3943

  16. Kitauchi F, Hirano S, Haruki M, Imanaka T, Morikawa M, Kanaya S (2005) A comparative study of dibenzothiophene degrading bacteria under a low oxygen condition. J Environ Biotechnol 4:117–120

  17. Kok M, Oldenhuis R, van der Linden MP, Raatjes P, Kingma J, van Lelyveld PH, Witholt B (1989) The Pseudomonas oleovorans alkane hydroxylase gene. Sequence and expression. J Biol Chem 264:5435–5441

  18. Kukor JJ, Olsen RH (1996) Catechol 2,3-dioxygenases functional in oxygen-limited (hypoxic) environments. Appl Environ Microbiol 62:1728–1740

  19. Maeda K, Nojiri H, Shintani M, Yoshida T, Habe H, Omori T (2003) Complete nucleotide sequence of carbazole/dioxin-degrading plasmid pCAR1 in Pseudomonas resinovorans strain CA10 indicates its mosaicity and the presence of large catabolic transposon Tn4676. J Mol Biol 326:21–33

  20. McKay DB, Prucha M, Reineke W, Timmis KN, Pieper DH (2003) Substrate specificity and expression of three 2,3-dihydroxybiphenyl 1,2-dioxygenases from Rhodococcus globerulus strain P6. J Bacteriol 185:2944–2951

  21. Menzie CA, Potocki BB, Santodonato J (1992) Exposure to carcinogenic PAHs in the environment. Environ Sci Technol 26:1278–1284

  22. Merlin C, Springael D, Mergeay M, Toussaint A (1997) Organisation of the bph gene cluster of transposon Tn4371 encoding enzymes for the degradation of biphenyl and 4-chlorobiphenyl compounds. Mol Gen Genet 253:499–506

  23. Nam JW, Nojiri H, Yoshida T, Habe H, Yamane H, Omori T (2001) New classification system for oxygenase components involved in ring-hydroxylating oxygenations. Biosci Biotechnol Biochem 65:254–263

  24. Page RDM (1996) Tree view: an application to display phylogenetic trees on personal computers. Comp Appl Biosci 12:357–358

  25. Parales RE (2003) The role of active-site residues in naphthalene dioxygenase. J Ind Microbiol Biotechnol 30:271–278

  26. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, pp 1.31–1.122

  27. Tan HM, Tang HY, Joannou CL, Abdel-Wahab NH, Mason JR (1993) The Pseudomonas putida ML2 plasmid-encoded genes for benzene dioxygenase are unusual in codon usage and low in G+C content. Gene 130:33–39

  28. 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

  29. Tovey ER, Baldo BA (1989) Protein binding to nitrocellulose, nylon and PVDF membranes in immunoassays and electroblotting. J Biochem Biophys Methods 19:169–183

  30. Werlen C, Kohler HP, van der Meer JR (1996) The broad substrate chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol dehydrogenase of Pseudomonas sp. strain P51 are linked evolutionarily to the enzymes for benzene and toluene degradation. J Biol Chem 271:4009–4016

  31. Wyndham RC, Cashore AE, Nakatsu CH, Peel MC (1994) Catabolic transposons. Biodegradation 5:323–342

  32. Zhou NY, Al-Dulayymi J, Baird MS, Williams PA (2002) Salicylate 5-hydroxylase from Ralstonia sp. strain U2: a monooxygenase with close relationships to and shared electron transport proteins with naphthalene dioxygenase. J Bacteriol 184:1547–1555

  33. Zielinski M, Backhaus S, Hofer B (2002) The principal determinants for the structure of the substrate-binding pocket are located within central core of a biphenyl dioxygenase alpha subunit. Microbiology 148:2439–2448

Download references


This work was supported by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).

Author information

Correspondence to Masaaki Morikawa.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hirano, S., Haruki, M., Takano, K. et al. Gene cloning and in vivo characterization of a dibenzothiophene dioxygenase from Xanthobacter polyaromaticivorans . Appl Microbiol Biotechnol 69, 672–681 (2006).

Download citation


  • PAHs
  • Anthracene
  • Phenanthrene
  • Dibenzothiophene
  • Degradation Activity