Biology Bulletin Reviews

, Volume 8, Issue 2, pp 155–167 | Cite as

Bacterial Genes of 2,4-Dichlorophenoxyacetic Acid Degradation Encoding α-Ketoglutarate-Dependent Dioxygenase Activity

  • N. V. Zharikova
  • T. R. Iasakov
  • E. Yu. Zhurenko
  • V. V. Korobov
  • T. V. Markusheva


The tfdA gene encodes α-ketoglutarate-dependent dioxygenase, which catalyzes the first step of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation pathway. The entire range of 2,4-D-degrading bacteria is divided into three groups based on their phylogeny, physiological and biochemical features, and isolation source. Each of these groups has its own version of the tfdA gene. The first group is the most studied and consists of fast-growing copiotrophic β- and γ-Proteobacteria isolated from anthropogenic habitats. The bacteria of this group possess the canonical sequence of this gene. Within this group, tfdA forms at least three classes (I, II, and III) of highly homologous gene families. The tfdA gene of Cupriavidus necator JMP134 was recognized as a Class I type. Class II consists of tfdA sequences belonging only to the genus Burkholderia of β-Proteobacteria, whereas Class III includes tfdA sequences belonging to the genera Delftia, Cupriavidus, Variovorax, Achromobacter, Comamonas, Rhodoferax, Halomonas, and Pseudomonas of β- and γ-Proteobacteria. The similarity of full-length nucleotide sequences between Class I and other classes was about 77–78%, and that between Class II and Class III was 93%. The second and third groups of 2,4-D-degrading bacteria are closely related to the genera Sphingomonas and Bradyrhizobium, which belong to α-Proteobacteria. tfdA-Like genes were identified only in four Sphingomonas spp. This fact and their phylogenetically distinct position make it possible to suggest that these genes evolved independently from each other through vertical gene transfer. The tfdAα gene was identified in the third group of bacteria of the genus Bradyrhizobium, which are both able and unable to degrade 2,4-D. Nevertheless, 2,4-D-degrading bacteria of the genera Bradyrhizobium and Sphingomonas have cad genes, which initiate the first step of the chlorophenoxyacetic acid degradation pathway. Based on the data on tfdAα localization in bacteria isolated from pristine ecosystems, a theory has been proposed that the tfdAα gene is ancestral for tfdA. Horizontal transfer and further adaptation to anthropogenic habitats probably led to the emergence of tfdA. However, tfdA and tfdAα may have diverged from a common ancestor, because they show a high similarity (51–57%) and separate distribution in the β-Proteobacteria, γ-Proteobacteria, and α-Proteobacteria, respectively.


2,4-D degrading bacteria cad tfdA tfdAα tfdA-like gene 


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  1. Amy, P.S., Schulke, J.W., Frazier, L.M., and Seidler, R.J., Characterization of aquatic bacteria and cloning of genes specifying partial degradation of 2,4-dichlorophenoxyacetic acid, Appl. Environ. Microbiol., 1985, vol. 49, pp. 1237–1245.PubMedPubMedCentralGoogle Scholar
  2. Baelum, J., Jacobsen, C.S., and Holben, W.E., Comparison of 16S rRNA gene phylogeny and functional tfdA gene distribution in thirty-one different 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid degraders, Syst. Appl. Microbiol., 2010, vol. 33, pp. 67–70.CrossRefPubMedGoogle Scholar
  3. Boronin, A.M. and Tsoi, T.V., Genetic biodegradation systems: organization and expression regulation, Genetika, 1989, vol. 25, pp. 581–594.PubMedGoogle Scholar
  4. Cavalca, L., Hartmann, A., Rouard, N., and Soulas, G., Diversity of tfdC genes: distribution and polymorphism among 2,4-dichlorophenoxyacetic acid degrading soil bacteria, FEMS Microbiol. Ecol., 1999, vol. 29, pp. 45–58.CrossRefGoogle Scholar
  5. Don, R.H. and Weightman, A.J., Transposon mutagenesis and cloning analysis of the pathways for degradation of 2,4-dichlorophenoxyacetic acid and 3-chlorobenzoate in Alcaligenes eutrophus JMP134 (pJP4), J. Bacteriol., 1985, vol. 161, pp. 85–90.PubMedPubMedCentralGoogle Scholar
  6. Eichhorn, E., van der Ploeg, J.R., Kertesz, M.A., and Leisinger, T., Characterization of a-ketoglutaratedependent taurinedioxygenase from Escherichia coli, J. Biol. Chem., 1997, vol. 272, pp. 23031–23036.CrossRefPubMedGoogle Scholar
  7. Fukumori, F. and Hausinger, R.P., Alcaligenes eutrophus JMP134 “2,4-dichlorophenoxyacetate monooxygenase” is an alpha-ketoglutarate-dependent dioxygenase, J. Bacteriol., 1993a, vol. 175, no. 7, pp. 2083–2086.CrossRefPubMedPubMedCentralGoogle Scholar
  8. Fukumori, F. and Hausinger, R.P., Purification and characterization of 2,4-dichlorophenoxyacetate/alphaketoglutarate dioxygenase, J. Biol. Chem., 1993b, vol. 268, no. 32, pp. 24311–24317.PubMedGoogle Scholar
  9. Fulthorpe, R.R. and Wyndham, R.C., Involvement of a chlorobenzoate catabolic transposon, Tn5271, in community adaptation to chlorobiphenyl, chloroaniline, and 2,4-dichlorophenoxyacetic acid in a freshwater ecosystem, Appl. Environ. Microbiol., 1992, vol. 58, pp. 314–325.PubMedPubMedCentralGoogle Scholar
  10. Fulthorpe, R.R., McGowan, C., Maltseva, O.V., et al., 2,4- Dichlorophenoxyacetic acid-degrading bacteria contain mosaics of catabolic genes, Appl. Environ. Microbiol., 1995, vol. 61, pp. 3274–3281.PubMedPubMedCentralGoogle Scholar
  11. Hayashi, S., Sano, T., Suyama, K., and Itoh, K., 2,4- Dichlorophenoxyacetic acid (2,4-D)- and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-degrading gene cluster in the soybean rootnodulating bacterium Bradyrhizobium elkanii USDA94, Microbiol. Res., 2016, vol. 188, pp. 62–71.CrossRefPubMedGoogle Scholar
  12. Hoffmann, D., Kleinsteuber, S., Muller, R.H., and Babel, W., A transposon encoding the complete 2,4-dichlorophenoxyacetic acid degradation pathway in the alkalitolerant strain Delftia acidovorans P4a, Microbiology, 2003, vol. 149, pp. 2545–2556.CrossRefPubMedGoogle Scholar
  13. Hogan, D.A., Smith, S.R., Saari, E.A., et al., Site-directed mutagenesis of 2,4-dichlorophenoxyacetic acid/alphaketoglutaratedioxygenase. Identification of residues involved in metallocenter formation and substrate binding, J. Biol. Chem., 2000, vol. 275, pp. 12400–12409.CrossRefPubMedGoogle Scholar
  14. Hotopp, J.C.D. and Hausinger, R.P., Alternative substrate of 2,4-dichlorophenoxyacetate/a-ketoglutaratedioxygenase, J. Mol. Catal. B: Enzym., 2001, vol. 15, pp. 155–162.CrossRefGoogle Scholar
  15. Itoh, K., Kanda, R., Momoda, Y., et al., Presence of 2,4- D-catabolizing bacteria in a Japanese arable soil that belong to BANA (BradyrhizobiumAgromonasNitrobacterAfipia) cluster in a-Proteobacteria, Microbes Environ., 2000, vol. 15, pp. 113–117.CrossRefGoogle Scholar
  16. Itoh, K., Kanda, R., Sumita, Y., et al., tfdA-Like genes in 2,4-dichlorophenoxyacetic acid-degrading bacteria belonging to the BradyrhizobiumAgromonasNitrobacterAfipia cluster in a-Proteobacteria, Appl. Environ. Microbiol., 2002, vol. 68, no. 7, pp. 3449–3454.CrossRefPubMedPubMedCentralGoogle Scholar
  17. Itoh, K., Tashiro, Y., Uobe, K., et al., Root nodule Bradyrhizobium spp. harbor tfdAa and cadA, homologous with genes encoding 2,4-dichlorophenoxyacetic acid-degrading proteins, Appl. Environ. Microbiol., 2004, vol. 70, no. 4 pp. 2110–2118.PubMedGoogle Scholar
  18. Ka, J.O., Holben, W.E., and Tiedje, J.M., Genetic and phenotypic diversity of 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacteria isolated from 2,4-Dtreated field soils, Appl. Environ. Microbiol., 1994a, vol. 60, pp. 1106–1115.PubMedPubMedCentralGoogle Scholar
  19. Ka, J.O., Holben, W.E., and Tiedje, J.M., Use of gene probes to aid in recovery and identification of functionally dominant 2,4-dichlorophenoxyacetic acid-degrading populations in soil, Appl. Environ. Microbiol., 1994b, vol. 60, pp. 1116–1120.PubMedPubMedCentralGoogle Scholar
  20. Ka, J.O., Holben, W.E., and Tiedje, J.M., Analysis of competition in soil among 2,4-dichlorophenoxyacetic aciddegrading bacteria, Appl. Environ. Microbiol., 1994c, vol. 60, pp. 1121–1128.PubMedPubMedCentralGoogle Scholar
  21. Kamagata, Y., Fulthorpe, R.R., Tamura, K., et al., Pristine environments harbor a new group of oligotrophic 2,4- dichlorophenoxyacetic acid-degrading bacteria, Appl. Environ. Microbiol., 1997, vol. 63, pp. 2266–2272.PubMedPubMedCentralGoogle Scholar
  22. Karhammer, B. and Olsen, R.N., Cloning and characterization of tfdS, the repressor-activator gene of tfdB, from the 2,4-D catabolic plasmid pJP4, J. Bacteriol., 1990, vol. 172, no. 10, pp. 5856–5862.CrossRefGoogle Scholar
  23. Karhammer, B., Kukor, J.J., and Olsen, R.N., Regulation of tfdCDEF by tfdR of 2,4-dichlorophenoxyacetic acid degradation plasmid pJP4, J. Bacteriol., 1990, vol. 172, pp. 2280–2286.CrossRefGoogle Scholar
  24. Kitagawa, W., Takami, S., Miyauchi, K., et al., Novel 2,4- dichlorophenoxyacetic acid degradation genes from oligotrophic Bradyrhizobium sp. strain HW13 isolated from a pristine environment, J. Bacteriol., 2002, vol. 184, pp. 509–518.CrossRefPubMedPubMedCentralGoogle Scholar
  25. Laemmli, C.M., Leveau, J.H., Zehnder, A.J.B., and van der Meer, J.R., Characterization of a second tfd gene cluster for chlorophenol and chlorocatechol metabolism on plasmid pJP4 in Ralstoniaeutropha JMP134 (pJP4), J. Bacteriol., 2000, vol. 182, pp. 4165–4172.CrossRefPubMedPubMedCentralGoogle Scholar
  26. Maltseva, O., McGowan, C., Fulthorpe, R., and Oriel, P., Degradation of 2,4-dichlorophenoxyacetic acid by haloalkaliphilic bacteria, Microbiology, 1996, vol. 142, pp. 1115–1122.CrossRefPubMedGoogle Scholar
  27. Markusheva, T.V., Zhurenko, E.Yu., Zharikova, N.V., et al., Strains-destructors of chlorophenoxy acids of gamma subclass of proteobacteria, Izv. Samar. Nauch. Tsentra, Ross. Akad. Nauk, 2011, vol. 13, no. 5 (2), pp. 194–195.Google Scholar
  28. Matheson, V.G., Forney, L.J., Suwa, Y., et al., Evidence for acquisition in nature of a chromosomal 2,4-dichlorophenoxyacetic acid/(alpha)-ketoglutarate dioxygenase gene by different Burkholderia spp., Appl. Environ. Microbiol., 1996, vol. 62, no. 7, pp. 2457–2463.PubMedPubMedCentralGoogle Scholar
  29. Matrubutham, U. and Harker, A.R., Analysis of duplicated gene sequences associated with tfdR and tfdS in Alcaligenes eutrophus JMP134, J. Bacteriol., 1994, vol. 176, pp. 2348–2353.CrossRefPubMedPubMedCentralGoogle Scholar
  30. McGowan, C., Fulthorpe, R., Wright, A., and Tiedje, J.M., Evidence for interspecies gene transfer in the evolution of 2,4-dichlorophenoxyacetic acid degraders, Appl. Environ. Microbiol., 1998, vol. 64, no. 10, pp. 4089–4092.PubMedPubMedCentralGoogle Scholar
  31. Mel’nikov, N.N., Pestitsidy. Zhimiya, tekhnologiya i primenenie (Pesticides: Chemistry, Synthesis, and Implementation), Moscow: Khimiya, 1987.Google Scholar
  32. Mel’nikov, N.N. and Belan, S.R., Organic chlorine compounds in the environment, Agrokhimiya, 1998, no. 10, pp. 83–93.Google Scholar
  33. Perkins, E.J., Gordon, M.P., Caceres, O., and Lurquia, P.F., Organization and sequence analysis of the 2,4-dichlorophenol hydroxylase and dichlorocatechol oxidative operons of plasmid pJP4, J. Bacteriol., 1990, vol. 172, pp. 2351–2359.CrossRefPubMedPubMedCentralGoogle Scholar
  34. Sakai, Y., Ogawa, N., Fujii, T., et al., 2,4-Dichrolophenoxyacetic acid-degrading genes from bacteria isolated from soil in Japan: spread of Burkholderia cepacia RASC-type degrading genes harbored on large plasmids, Microbes Environ., 2007, vol. 22, pp. 145–156.CrossRefGoogle Scholar
  35. Sakai, Y., Ogawa, N., Shimomura, Y., and Fujii, T., A 2,4- dichlorophenoxyacetic acid degradation plasmid pM7012 discloses distribution of an unclassified megaplasmid group across bacterial species, Microbiology, 2014, vol. 160, pp. 525–536.CrossRefPubMedGoogle Scholar
  36. Schlomann, M., Evolution of chlorocatechol catabolic pathways, Biodegradation, 1994, vol. 5, pp. 301–321.CrossRefPubMedGoogle Scholar
  37. Shimojo, M., Kawakami, M., and Amada, K., Analysis of genes encoding the 2,4-dichlorophenoxyacetic aciddegrading enzyme from Sphingomonas agrestis 58-1, J. Biosci. Bioeng., 2009, vol. 108, no. 1, pp. 56–59.CrossRefPubMedGoogle Scholar
  38. Streber, W.R., Timmis, K.N., and Zenk, M.H., Analysis, cloning, and high-level expression of 2,4-dichlorophenoxyacetate monooxygenase gene tfdA of Alcaligenes eutrophus JMP134, J. Bacteriol., 1987, vol. 169, no. 7, pp. 2950–2955.CrossRefPubMedPubMedCentralGoogle Scholar
  39. Suwa, Y., Wright, A.D., Fukimori, F., et al., Characterization of a chromosomally encoded 2,4-dichlorophenoxyacetic acid/alpha-ketoglutaratedioxygenase from Burkholderia sp. strain RASC, Appl. Environ. Microbiol., 1996, vol. 62, pp. 2464–2469.PubMedPubMedCentralGoogle Scholar
  40. Tonso, N.L., Matheson, V.G., and Holben, W.E., Polyphasic characterization of a suite of bacterial isolates capable of degrading 2,4-D, Microb. Ecol., 1995, vol. 30, pp. 3–24.CrossRefPubMedGoogle Scholar
  41. Top, E.M., Maltseva, O.V., and Forney, L.J., Capture of a catabolic plasmid that encodes only 2,4-dichlorophe noxyacetic acid: a-ketoglutaric acid dioxygenase (TfdA) by genetic complementation, Appl. Environ. Microbiol., 1996, vol. 62, no. 7, pp. 2470–2476.PubMedPubMedCentralGoogle Scholar
  42. Trefault, N., De la Iglesia, R., Molina, A.M., et al., Genetic organization of the catabolic plasmid pJP4 from Ralstonia eutropha JMP134 (pJP4) reveals mechanisms of adaptation to chloroaromatic pollutants and evolution of specialized chloroaromatic degradation pathways, Environ. Microbiol., 2004, vol. 6, no. 7, pp. 655–668.CrossRefPubMedGoogle Scholar
  43. Vallaeys, T., Courde, L., McGowan, C., et al., Phylogenetic analyses indicate independent recruitment of diverse gene cassettes during assemblage of the 2,4-D catabolic pathway, FEMS Microbiol. Ecol., 1999, vol. 28, pp. 373–382.CrossRefGoogle Scholar
  44. van der Meer, J.R., de Vos, W.M., Harayama, S., and Zehnder, A.J.B., Molecular mechanisms of genetic adaptation to xenobiotic compounds, Microbiol. Rev., 1992, vol. 56, pp. 677–694.PubMedPubMedCentralGoogle Scholar
  45. Vedler, E., Koiv, V., and Heinaru, A., Analysis of the 2,4- dichlorophenoxyacetic acid-degradative plasmid pEST4011 of Achromobacter xylosoxidans subsp. denitrificans strain EST4002, Gene, 2000, vol. 255, pp. 281–288.CrossRefPubMedGoogle Scholar
  46. Xia, X.S., Smith, A.R., and Bruce, I.J., Identification and sequencing of a novel insertion sequence, IS1471 in Burkholderia cepacia strain 2a, FEMS Microbiol. Lett., 1996, vol. 144, nos. 2–3, pp. 203–206.CrossRefPubMedGoogle Scholar
  47. Zharikova, N.V., Zhurenko, E.Yu., Korobov, V.V., et al., Biodiversity of bacteria-destructors of chlorinated phenoxy acids, Vestn. Orenb. Gos. Univ., 2009, no. 6, pp. 121–123.Google Scholar
  48. Zharikova, N.V., Iasakov, T.R., Bumazhkin, B.K., et al., Isolation and sequence analysis of pCS36–4CPA, a small plasmid from Citrobacter sp. 36–4CPA, Saudi J. Biol. Sci., 2016. doi 10.1016/j.sjbs.2016.02.014Google Scholar
  49. Zhurenko, E.Yu., Markusheva, T.V., Galkin, E.G., et al., Gluconobacter oxydans IBRB-2T degrades 2,4,5-thrichlorophenoxyacetic acid, Biotechol. Russ., 2003, no. 6, pp. 75–80.Google Scholar

Copyright information

© Pleiades Publishing, Ltd. 2018

Authors and Affiliations

  • N. V. Zharikova
    • 1
  • T. R. Iasakov
    • 1
  • E. Yu. Zhurenko
    • 1
  • V. V. Korobov
    • 1
  • T. V. Markusheva
    • 1
  1. 1.Ufa Institute of BiologyRussian Academy of SciencesUfaRussia

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