Microbial Degradation of Chlorinated Aromatic Compounds

The meta-cleavage pathway
  • Walter Reineke
  • Astrid E. Mars
  • Stefan R. Kaschabek
  • Dick B. Janssen
Part of the Focus on Biotechnology book series (FOBI, volume 3A)


The aerobic microbial degradation of various chloroaromatics usually occurs via chlorocatechols as central intermediates. These are further degraded through the modified ortho-cleavage pathway. Dechlorination takes place during cycloisomerization of chloromuconates and reduction of chloromaleylacetates. In contrast, the degradation of haloaromatics via meta-cleavage was thought to be impossible due to toxicity of cleavage intermediates, whereas the meta route is more effective for methylaromatics. Recently, Pseudomonas putida strain GJ31 was shown to be able to degrade toluene and chlorobenzene simultaneously. Strain GJ31 rapidly degrades chlorobenzene via 3-chlorocatechol using the meta-cleavage pathway without any apparent toxic effects. An unusual chlorocatechol 2,3-dioxygenase oxidizes 3-chlorocatechol. Stoichiometric displacement of chloride then leads to the production of 2-hydroxymuconate, which is a common metabolite of the meta-cleavage pathway. In contrast to other catechol 2,3-dioxgenases, which are subject to inactivation when exposed to 3-chlorocatechol, the chlorocatechol 2,3-dioxygenase is resistant. The gene encoding the chlorocatechol 2,3-dioxygenase (cbzE) of strain GJ31 was cloned and sequenced. CbzE was most similar to catechol 2,3-dioxygenases of the 2.C subfamily of type 1 extradiol dioxygenases. Hybrid enzymes, which were made of CbzE and the 3-methylcatechol 2,3-dioxygenase of strain P. putida UCC2, showed that the resistance of CbzE to suicide inactivation and the substrate specificity were mainly determined by the C-terminal region of the protein. Establishing whether the meta-cleavage pathway of strain GJ31 can function as pathway segment in the construction of novel chloroaromatics-degraders is a future task. Organisms such as P. putida strain GJ31 and its derivatives may be useful for effective treatment of waste streams containing various methyl- and chloroaromatics.


Pseudomonas Putida Environmental Microbiology Chlorine Substituent Hybrid Enzyme Extradiol Dioxygenases 
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  1. Arensdorf J.J and Focht D.D (1994) Formation of chlorocatechol meta cleavage products by a pseudomonad during metabolism of monochlorobiphenyls. Applied and Environmental Microbiology 60:2884–2889.PubMedGoogle Scholar
  2. Arensdorf JJ and Focht DD(1995) A meta cleavage pathway for 4-chlorobenzoate, an intermediate in the metabolism of 4-chlorobiphenyl by Pseudomonas cepacia PI66. Applied and Environmental Microbiology 61:443–447.PubMedGoogle Scholar
  3. Bartels I, Knackmuss H-J and Reineke W (1984) Suicide inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-halocatechols. Applied and Environmental Microbiology 47:500–505.PubMedGoogle Scholar
  4. Chapman P.J (1979) Degradation mechanisms. In: Bourquin AW and Pritchard PH (Eds) Microbial degradation of pollutants in marine environments. EPA-600/9-79-012 (pp. 28–66) Environmental Protection Agency, Gulf Breeze, Fla. USA.Google Scholar
  5. Eltis LD and Bolin JT (1996) Evolutionary relationship among extradiol dioxygenases. Journal of Bacteriology 178:5930–5937.PubMedGoogle Scholar
  6. Heiss G, Stolz A, Kuhm AE, Müller C, Klein J, Altenbuchner J and Knackmuss H-J (1995) Characterization of a 2,3-dihydroxybiphenyl dioxygenase from the naphthalenesulfonate-degrading bacterium strain BN6. Journal of Bacteriology 177:5865–5871.PubMedGoogle Scholar
  7. Higson FK and Focht DD (1992) Utilization of 3-chloro-2-methylbenzoic acid by Pseudomonas cepacia MB2 through the meta fission pathway. Environmental Microbiology 58:2501–2504.Google Scholar
  8. Hirose J, Kimura N, Suyama A, Kobayashi A, Hayashida S and Furukawa K (1994) Functional and structural relationship of various extradiol aromatic ring-cleavage dioxygenases of Pseudomonas origin. FEMS Microbiology Letters 118:273–278.PubMedCrossRefGoogle Scholar
  9. Hofer B, Eltis LD, Dowling DN and Timmis KN (1993) Genetic analysis of a Pseudomonas locus encoding a pathway for biphenyl/polychlorinated biphenyl degradation. Gene 130:47–55.PubMedCrossRefGoogle Scholar
  10. Hollender J, Dott W and Hopp J (1994) Regulation of chloro-and methylphenol degradation in Comamonas testosteroni JH5. Applied and Environmental Microbiology 60:2330–2338.PubMedGoogle Scholar
  11. Hollender J, Hopp J and Dott W (1997) Degradation of 4-chlorophenol via the meta cleavage pathway by Comamonas testosteroni JH5. Applied and Environmental Microbiology 63:4567–4572.PubMedGoogle Scholar
  12. Kaschabek SR, Kasberg T, Müller D, Mars AE, Janssen DB and Reineke W (1998) Degradation of chloroaromatics: Purification and characterization of a novel type of chlorocatechol 2,3-dioxygenase of Pseudomonas putida GJ31. Journal of Bacteriology 180:296–302.PubMedGoogle Scholar
  13. Kaschabek SR and Reineke W (1992) Maleylacetate reductase of Pseudomonas sp. strain B13: dechlorination of chloromaleylacetates, metabolites in the degradation of chloroaromatic compounds. Archives of Microbiology 158:412–417.PubMedCrossRefGoogle Scholar
  14. Kaschabek SR and Reineke W (1995) Maleylacetate reductase of Pseudomonas sp. strain B13: Specificity of substrate conversion and halide elimination. Journal of Bacteriology 177:320–325.PubMedGoogle Scholar
  15. Keil H, Lebens MR and Williams PA (1985) TOL plasmid pWW15 contains two nonhomologous, independently regulated catechol 2,3-dioxygenase genes. Journal of Bacteriology 163:248–255.PubMedGoogle Scholar
  16. Kim K-P, Seo D-I, Lee D-H, Kim Y and Kim C-K (1998) Cloning and expression in E. coli of the genes responsible for degradation of 4-chlorobenzoate and 4-chlorocatechol from Pseudomonas sp. strain S-47. Journal of Microbiology (Korea) 36:99–105.Google Scholar
  17. Kim KP, Seo D-I, Min KH, Ka JO, Park YK and Kim C-K (1997) Characterization of catechol 2,3-dioxygenase produced by 4-chlorobenzoate-degrading Pseudomonas sp. S-47. Journal of Microbiology (Korea) 35:295–299.Google Scholar
  18. Klecka GM and Gibson DT (1981) Inhibition of catechol 2,3-dioxygenase from Pseudomonas putida by 3-chlorocatechol. Applied and Environmental Microbiology 41:1159–1165.PubMedGoogle Scholar
  19. Knackmuss H-J (1981) Degradation of halogenated and sulfonated hydrocarbons. In: Leisinger T, Cook AM, Hütter R and Nüesch J (Eds) Microbial degradation of xenobiotics and recalcitrant compounds, pp. 189–212. Academic Press, London, UK. ai]Kukor JJ and Olsen RH (1996) Catechol 2,3-dioxygenases functional in oxygen-limited (hypoxic) environments. Applied and Environmental Microbiology 62:1728–1740.Google Scholar
  20. Mars AE, Kasberg T, Kaschabek SR, van Agteren MH, Janssen DB and Reineke W (1997) Microbial degradation of chloroaromatics: Use of the meta-cleavage pathway for mineralization of chlorobenzene. Journal of Bacteriology 179:4530–4537.PubMedGoogle Scholar
  21. Mars AE, Kingma J, Kaschabek SR, Reineke W and Janssen DB (1999) Conversion of 3-chlorocatechol by various catechol 2,3-dioxygenases and sequence analysis of the chlorocatechol dioxygenase region of Pseudomonas putida GJ31. Journal of Bacteriology 181:1309–1318.PubMedGoogle Scholar
  22. McClure NC and Venables WA (1986) Adaptation of Pseudomonas putida mt-2 to growth on aromatic amines. Journal of General Microbiology 132:2209–2218.PubMedGoogle Scholar
  23. McCullar MV, Brenner V, Adams RH and Focht DD (1994) Construction of a novel polychlorinated biphenyl-degrading bacterium: utilization of 3,4’-dichlorobiphenyl by Pseudomonas acidovorans M3GY. Applied and Environmental Microbiology 60:3833–3839.PubMedGoogle Scholar
  24. Moon J, Min KR, Kim C-K, Min K-H and Kim Y (1996) Characterization of the gene encoding catechol 2,3-dioxygenase of Alcaligenes sp. KF711: overexpression, enzyme purification, and nucleotide sequencing. Archives of Biochemistry and Biophysics 332:248–254.PubMedCrossRefGoogle Scholar
  25. Müller D, Schlömann M and Reineke W (1996) Maleylacetate reductases in chloroaromatic-degrading bacteria using the modified ortho pathway: Comparison of catalytic properties. Journal of Bacteriology 178:298–300.PubMedGoogle Scholar
  26. Nakai C, Kagamiyama H, Nozaki M, Nakazawa T, Inouye S, Ebina Y and Nakazawa A (1993) Complete nucleotide sequence of the metapyrocatechase gene on the TOL plasmid of Pseudomonas putida mt-2. Journal of Biological Chemistry 258:2923–2928.Google Scholar
  27. Nozaki M, Kotani S, Ono K and Senoh S (1970) Metapyrocatechase. HI. Substrate specificity and mode of ring fission. Biochimica et Biophysica Acta 220:213–223.PubMedCrossRefGoogle Scholar
  28. Oldenhuis R, Kuijk K, Lammers A, Janssen DB and Witholt B (1989) Degradation of chlorinated and nonchlorinated aromatic solvents in soil suspensions by pure bacterial cultures. Applied Microbiology and Biotechnology 30:211–217.CrossRefGoogle Scholar
  29. Potrawfke T, Timmis KN and Wittich RM (1998) Degradation of 1,2,3,4-tetrachlorobenzene by Pseudomonas chlororaphis RW71. Applied and Environmental Microbiology 64:3798–3806.PubMedGoogle Scholar
  30. Rast HG, Engelhardt G and Wallnofer P (1980) 2,3-Cleavage of substituted catechols in Nocardia sp. DSM 43251 (Rhodococcus rubrus). Zentralblatt für Bakteriologie, Mikrobiologie und Hygiene, 1. Abteilung, Original C 1:224–236.Google Scholar
  31. Reineke W, Jeenes DJ, Williams PA and Knackmuss H-J (1982) TOL plasmid pWWO in constructed halobenzoate-degrading Pseudomonas strains: Prevention of meta pathway. Journal of Bacteriology 150:195–201.PubMedGoogle Scholar
  32. Riegert U, Heiss G, Fischer P and Stolz A (1998) Distal cleavage of 3-chlorocatechol by an extradiol dioxygenase to 3-chloro-2-hydroxymuconic semialdehyde. Journal of Bacteriology 180:2849–2853.PubMedGoogle Scholar
  33. Rojo F, Pieper DH, Engesser K-H, Knackmuss H-J and Timmis KN (1987) Assemblage of ortho cleavage route for simultaneous degradation of chloro-and methyl aromatics. Science 238:1395–1398.PubMedCrossRefGoogle Scholar
  34. Sala-Trepat JM and Evans WC (1971) The meta cleavage of catechol by Azotobacter species: 4-oxalocrotonate pathway. European Journal of Biochemistry 20:400–413.PubMedCrossRefGoogle Scholar
  35. Sander P, Wittich R-M, Fortnagel P, Wilkes H and Francke W (1991) Degradation of 1,2,4-trichloro-and 1,2,4,5-tetrachlorobenzene by Pseudomonas strains. Applied and Environmental Microbiology 57:1430–1440.PubMedGoogle Scholar
  36. Schmidt E and Knackmuss H-J (1980) Chemical structure and biodegradability of halogenated aromatic compounds. Conversion of chlorinated muconic acids into maleoylacetic acid. Biochemical Journal 192:339–347.PubMedGoogle Scholar
  37. Seo D-I, Lim JY, Kim YC, Min KH and Kim C-K (1997) Isolation of Pseudomonas sp. S-47 and its degradation of 4-chlorobenzoic acid. Journal of Microbiology (Korea) 35:188–192.Google Scholar
  38. Vollmer MD and Schlömann M (1995) Conversion of 2-chloro-cis,cis-muconate and its metabolites 2-chloroand 5-chloromuconolactone by chloromuconate cycloisomerases of pJP4 and pAC27. Journal of Bacteriology 177:2938–2941.PubMedGoogle Scholar
  39. Vollmer MD, Stadler-Fritzsche K and Schlömann M (1993) Conversion of 2-chloromaleylacetate in Alcaligenes eutrophus JMP134. Archives of Microbiology 159:182–188.PubMedCrossRefGoogle Scholar
  40. Vollmer MD, Fischer P, Knackmuss H-J and Schlömann M (1994) Inability of muconate cycloisomerases to cause dehalogenation during conversion of 2-chloro-cis,cis-muconate. Journal of Bacteriology 176:4366–4375.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2002

Authors and Affiliations

  • Walter Reineke
    • 1
  • Astrid E. Mars
    • 2
  • Stefan R. Kaschabek
    • 1
  • Dick B. Janssen
    • 2
  1. 1.Chemische MikrobiologieBergische Universität — Gesamthochschule WuppertalWuppertalGermany
  2. 2.Department of BiochemistryUniversity of GroningenGroningenThe Netherlands

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