The Journal of Microbiology

, Volume 49, Issue 6, pp 974–980 | Cite as

Molecular characterization of chloranilic acid degradation in Pseudomonas putida TQ07

  • Luis G. Treviño-QuintanillaEmail author
  • Julio A. Freyre-González
  • Rosa A. Guillén-Garcés
  • Clarita Olvera


Pentachlorophenol is the most toxic and recalcitrant chlorophenol because both aspects are directly proportional to the halogenation degree. Biological and abiotic pentachlorophenol degradation generates p-chloranil, which in neutral to lightly alkaline environmental conditions is hydrolyzed to chloranilic acid that present a violet-reddish coloration in aqueous solution. Several genes of the degradation pathway, cadR-cadCDX, as well as other uncharacterized genes (ORF5 and 6), were isolated from a chloranilic acid degrading bacterium, Pseudomonas putida strain TQ07. The disruption by random mutagenesis of the cadR and cadC genes in TQ07 resulted in a growth deficiency in the presence of chloranilic acid, indicating that these genes are essential for TQ07 growth with chloranilic acid as the sole carbon source. Complementation assays demonstrated that a transposon insertion in mutant CAD82 (cadC) had a polar effect on other genes contained in cosmid pLG3562. These results suggest that at least one of these genes, cadD and cadX, also takes part in chloranilic acid degradation. Based on molecular modeling and function prediction, we strongly suggest that CadC is a pyrone dicarboxylic acid hydrolase and CadD is an aldolase enzyme like dihydrodipicolinate synthase. The results of this study allowed us to propose a novel pathway that offers hypotheses on chloranilic acid degradation (an abiotic by-product of pentachlorophenol) by means of a very clear phenotype that is narrowly related to the capability of Pseudomonas putida strain TQ07 to degrade this benzoquinone.


pentachlorophenol chloranilic acid biodegradation Pseudomonas putida pyrone dicarboxylic acid hydrolase LysR regulator 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ahlborg, U.G., T.M. Thunberg, and H.C. Spencer. 1980. Chlorinated phenols: occurrence, toxicity, metabolism, and environmental impact. Crit. Rev. Toxicol. 7, 1–35.PubMedCrossRefGoogle Scholar
  2. Altschul, S., W. Gish, W. Miller, E. Myers, and D. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410.PubMedGoogle Scholar
  3. Buchanan, C.L., H. Connaris, M.J. Danson, C.D. Reve, and D.W. Hough. 1999. An extremely thermostable aldolase from Sulfolobus solfataricus with specificity for non-phosphorilated substrates. Biochem. J. 343, 563–570.PubMedCrossRefGoogle Scholar
  4. Chen, L. and J. Yang. 2008. Biochemical characterization of the tetrachlobenzoquinone reductase involved in the biodegradation of pentachlorophenol. Int. J. Mol. Sci. 9, 198–212.PubMedCrossRefGoogle Scholar
  5. Czaplicka, M. 2006. Photo-degradation of chlorophenols in the aqueous solution. J. Hazard Mater. 134, 45–59.PubMedCrossRefGoogle Scholar
  6. Dai, J., A.L. Sloat, M.W. Wright, and R.A. Manderville. 2005. Role of phenoxyl radicals in DNA adduction by chlorophenol xenobiotics following peroxidase activation. Chem. Res. Toxicol. 18, 771–779.PubMedCrossRefGoogle Scholar
  7. de Lorenzo, V., M. Herrero, V. Jakubzik, and K.N. Timmis. 1990. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J. Bacteriol. 172, 6568–6572.PubMedGoogle Scholar
  8. Devenish, S.R., F.H. Huisman, E.J. Parker, A.T. Hadfield, and J.A. Gerrard. 2009. Cloning and characterisation of dihydrodipicolinate synthase from the pathogen Neisseria meningitidis. Biochim. Biophys. Acta 1794, 1168–1174.PubMedGoogle Scholar
  9. Figurski, D.H. and D.R. Helinki. 1979. Replication of an origin-containing derivate of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. USA 76, 1648–1652.PubMedCrossRefGoogle Scholar
  10. Garrec, G.M.-L., I. Artaud, and C. Capeillère-Blandin. 2001. Purification and catalytic properties of the chlorophenol 4-monooxygenase from Burkholderia cepacia strain AC1100. Biochim. Biophy. Acta 1547, 288–301.CrossRefGoogle Scholar
  11. Henikoff, S., G.W. Haughn, J.M. Calvo, and J.C. Wallace. 1998. A large family of bacterial activator proteins. Proc. Natl. Acad. Sci. USA 85, 6602–6606.CrossRefGoogle Scholar
  12. Jensen, J. 1996. Chlorophenols in the terrestrial environment. Rev. Environ. Contam. 146, 25–51.CrossRefGoogle Scholar
  13. Kersten, P.J., P.J. Chapman, and S. Dagley. 1985. Enzymatic release of halogens or methanol from some substituted protocatechuic acids. J. Bacteriol. 162, 693–697.PubMedGoogle Scholar
  14. Lee, J.Y. and L. Xun. 1997. Purification and characterization of 2,6-dichloro-p-hydroquinone chlorohydrolase from Flavobacterium sp. strain ATCC 39723. J. Bacteriol. 179, 1521–4.PubMedGoogle Scholar
  15. Longoria, A., R. Tinoco, and R. Vázquez-Duhalt. 2009. Chloroperoxidase-mediated transformation of highly halogenated monoaromatic compounds. Chemosphere 72, 485–490.CrossRefGoogle Scholar
  16. Mars, A.E., J. Kingma, S.R. Kaschabek, W. Reineke, and D.B. Janssen 1999. Conversion of 3-chlorocatechol by various catechol 2,3-dioxygenases and sequence analysis of the chlorocatechol dioxygenase region of Pseudomonas putida GJ31. J. Bacteriol. 181, 1309–1318.PubMedGoogle Scholar
  17. Masai, E.S., S. Shinohara, H. Hara, S. Nishikawa, Y. Katayama, and M. Fukuda. 1999. Genetic and biochemical characterization of a 2-pyrone-4,6-dicarboxylic acid hydrolase involved in the protocatechuate 4,5-clavage pathway of Sphingomonas paucimobilis SYK-6. J. Bacteriol. 181, 55–62.PubMedGoogle Scholar
  18. Mileski, G.J., J.A. Bumpus, M.A. Jurek, and S.D. Aust. 1988. Biodegradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium. Appl. Environ. Microbiol. 54, 2885–2889.PubMedGoogle Scholar
  19. Pisabarro, A., M. Malumbres, L.M. Mateos, J.A. Oguiza, and J.F. Martín. 1993. A cluster of three genes (dapA, orf2, and dapB) of Brevibacterium lactofermentum encodes dihydrodipicolinate synthase, dihydrodipicolinate reductase, and a third polypeptide of unknown function. J. Bacteriol. 175, 2743–2749.PubMedGoogle Scholar
  20. Proudfoot, A.T. 2003. Pentachlorophenol poisoning. Toxicol. Rev. 22, 3–11.PubMedCrossRefGoogle Scholar
  21. Remberger, M., P.A. Hynning, and A.H. Neilson. 1991. 2,5-dichloro-3,6-dihydroxybenzo-1,4-quinone: identification of a new organochlorine compound in kraft bleachery effluents. Environ. Sci. Technol. 25, 1903–1907.CrossRefGoogle Scholar
  22. Romine, M.F., L.C. Stillwell, K.-K. Wong, S.J. Thurston, E.C. Sisk, C. Sensen, T. Gaasterland, J.K. Fredrickson, and J.D. Saffer. 1999. Complete sequence of a 184-kilobase catabolic plasmid from Sphingomonas aromaticivorans F199. J. Bacteriol. 181, 1585–1602.PubMedGoogle Scholar
  23. Roy, A., A. Kucukural, and Y. Zhang. 2010. I-TASSER: a unified platform for automated protein structure and function prediction. Nat. Protoc. 5, 725–738.PubMedCrossRefGoogle Scholar
  24. Ruckdeschel, G. and G. Renner. 1986. Effects of pentachlorophenol and some of its known and possible metabolites on fungi. Appl. Environ. Microbiol. 51, 1370–1372.PubMedGoogle Scholar
  25. Saeki, Y., M. Nozaki, and S. Senoh. 1980. Cleavage of pyrogallol by non-heme iron-containing dioxygenases. J. Biol. Chem. 255, 8465–8471.PubMedGoogle Scholar
  26. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular cloning. A laboratory manual. 2nd ed. Cold Spring Harbor Laboratory Press. New York, USA.Google Scholar
  27. Sarr, D.H., C. Kazunga, M.J. Charles, J.G. Pavlovich, and M.D. Aitken. 1995. Decomposition of Tetrachloro-1,4-benzoquinone (p-chloranil) in Aqueous Solution. Environ. Sci. Technol. 29, 2735–2740.CrossRefGoogle Scholar
  28. Schell, M.A. 1993. Molecular biology of the LysR family transcriptional regulators. Annu. Rev. Microbiol. 47, 597–626.PubMedCrossRefGoogle Scholar
  29. Soares da Costa, T.P., A.C. Muscroft-Taylor, R.C. Dobson, S.R. Devenish, G.B. Jameson, and J.A. Gerrard. 2010. How essential is the ‘essential’ active-site lysine in dihydrodipicolinate synthase? Biochimie. 92, 837–845.PubMedCrossRefGoogle Scholar
  30. Spaink, H.P., R.J.H. Okker, C.A. Wijffelman, E. Pees, and B.J. Lugtenberg. 1987. Promoters in the nodulation region of the Rhizobium leguminosarum sym plasmid pRL1JI. Plant Mol. Biol. 9, 27–39.CrossRefGoogle Scholar
  31. Thorsted, P.B., D.P. Macartney, P. Akhtar, A.S. Haines, N. Ali, P. Davidson, T. Stafford, M.J. Pocklington, W. Pansegrau, B.M. Wilkins, and et al. 1998. Complete sequence of the IncPb plasmid R751: implications for evolution and organization of the IncP backbone. J. Mol. Biol. 282, 969–990.PubMedCrossRefGoogle Scholar
  32. Trevinõ-Quintanilla, L.G., L.J. Galán-Wong, B. Rodríguez-Uribe, and G. Soberón-Chávez. 2002. Cloning and characterization of a FAD-monooxygenase gene (cadA) involved in degradation of chloranilic acid (2,5-dichloro-3,6-dihydroxybenzo-1,4-quinone) in Pseudomonas putida TQ07. Appl. Microbiol. Biotechnol. 59, 545–550.PubMedCrossRefGoogle Scholar
  33. Wattiau, P., L. Bastiaens, R. van Herwijnen, L. Daal, J.R. Parsons, M.-E. Renard, D. Springael, and G.R. Cornelis. 2001. Fluorene degradation by Sphingomonas sp. LB126 proceeds through protocatechuic acid: a genetic analysis. Res. Miocrobiol. 152, 861–872.CrossRefGoogle Scholar
  34. Yang, C.F., C.M. Lee, and C.C. Wang. 2006. Isolation and physiological characterization of the pentachlorophenol degrading bacterium Sphingomonas chlorophenolica. Chemosphere 62, 709–714.PubMedCrossRefGoogle Scholar
  35. Yrjälä, K., L. Paulina, and M. Romantschuk. 1997. Novel organization of chatechol meta-pathway genes in Sphingomonas sp. HV3 pSKY4 plasmid. FEMS Microbiol. Lett. 154, 403–408.PubMedCrossRefGoogle Scholar
  36. Zhao, F., K. Mayura, R.W. Hutchinson, R.P. Lewis, R.C. Burghard, and T.D. Phillips. 1995. Developmental toxicity and structure-activity relationship of chlorophenols using human embryonic palatal mesenchymal cells. Toxicol. Lett. 78, 35–42.PubMedCrossRefGoogle Scholar
  37. Zheng, W., H. Yu, X. Wang, and W. Qu. 2011. Systematic review of pentachlorophenol occurrence in the environment and in humans in China: Not a negligible health risk due to the re-emergence of schistosomiasis. Environ. Int. [Epub ahead of print] PMID: 21601283.Google Scholar

Copyright information

© The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg  2011

Authors and Affiliations

  • Luis G. Treviño-Quintanilla
    • 1
    Email author
  • Julio A. Freyre-González
    • 2
  • Rosa A. Guillén-Garcés
    • 1
  • Clarita Olvera
    • 3
  1. 1.Departamento de Tecnología AmbientalUniversidad Politécnica del Estado de MorelosJiutepec, MorelosMéxico
  2. 2.Programa de Dinámica Genómica, Centro de Ciencias GenómicasUniversidad Nacional Autónoma de MéxicoCuernavaca, MorelosMéxico
  3. 3.Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavaca, MorelosMéxico

Personalised recommendations