Induction of ortho- and meta-cleavage pathways in Pseudomonas in biodegradation of high benzoate concentration: MS identification of catabolic enzymes

Genomics and Proteomics

Abstract

The degradation pathways of benzoate at high concentration in Pseudomonas putida P8 were directly elucidated through mass spectrometric identification of some key catabolic enzymes. Proteins from P. putida P8 grown on benzoate or succinate were separated using two-dimensional gel electrophoresis. For cells grown on benzoate, eight distinct proteins, which were absent in the reference gel patterns from succinate-grown cells, were found. All the eight proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as catabolic enzymes involved in benzoate degradation. Among them, CatB (EC5.5.1.1), PcaI (EC2.8.3.6), and PcaF (EC2.3.1.174) were the enzymes involved in the ortho-cleavage pathway; DmpC (EC1.2.1.32), DmpD (EC3.1.1.-), DmpE (EC4.2.1.80), DmpF (EC1.2.1.10), and DmpG (EC4.1.3.-) were the meta-cleavage pathway enzymes. In addition, enzyme activity assays showed that the activities of both catechol 1,2-dioxygenase (C12D; EC1.13.11.1) and catechol 2,3-dioxygenase (C23D; EC1.13.11.2) were detected in benzoate-grown P. putida cells, undoubtedly suggesting the simultaneous expression of both the ortho- and the meta-cleavage pathways in P. putida P8 during the biodegradation of benzoate at high concentration.

Keywords

Benzoate biodegradation Ortho-cleavage pathway Meta-cleavage pathway Pseudomonas putida 2-DE MALDI-TOF MS 

Notes

Acknowledgments

This research work was supported by a research grant from the Singapore Ministry of Education Academic Research Fund (R-279-000-181-112). The authors gratefully acknowledge the National University of Singapore for providing the research scholarship to Bin Cao.

References

  1. Aldrich TL, Frantz B, Gill JF, Kilbane JJ, Chakrabarty AM (1987) Cloning and complete nucleotide-sequence determination of the catB gene encoding cis,cis-muconate lactonizing enzyme. Gene 52:185–195CrossRefGoogle Scholar
  2. Alexander M (1981) Biodegradation of chemicals of environmental concern. Science 211:132–138CrossRefGoogle Scholar
  3. Ampe F, Lindley ND (1996) Flux limitations in the ortho pathway of benzoate degradation of Alcaligenes eutrophus: metabolite overflow and induction of the meta pathway at high substrate concentrations. Microbiology 142:1807–1817CrossRefGoogle Scholar
  4. Bhushan B, Halasz A, Hawari M (2005) Biotransformation of CL-20 by a dehydrogenase enzyme from Clostridium sp. EDB2. Appl Microbiol Biotechnol 69:448–455CrossRefGoogle Scholar
  5. Bouwer EJ, Zehnder AJB (1993) Bioremediation of organic compounds—putting microbial metabolism to work. Trends Biotechnol 11:360–367CrossRefGoogle Scholar
  6. Caldwell ME, Suflita JM (2000) Detection of phenol and benzoate as intermediates of anaerobic benzene biodegradation under different terminal electron-accepting conditions. Environ Sci Technol 34:1216–1220CrossRefGoogle Scholar
  7. Cao B, Loh KC (2008) Catabolic pathways and cellular responses of Pseudomonas putida P8 during growth on benzoate with proteomics approach. Biotechnol Bioeng. doi:10.1002/bit.21991
  8. Denef VJ, Park J, Tsoi TV, Rouillard JM, Zhang H, Wibbenmeyer JA, Verstraete W, Gulari E, Hashsham SA, Tiedje JM (2004) Biphenyl and benzoate metabolism in a genomic context: outlining genome-wide metabolic networks in Burkholderia xenovorans LB400. Appl Environ Microbiol 70:4961–4970CrossRefGoogle Scholar
  9. Denef VJ, Patrauchan MA, Florizone C, Park J, Tsoi TV, Verstraete W, Tiedje JM, Eltis LD (2005) Growth substrate- and phase-specific expression of biphenyl, benzoate, and C-1 metabolic pathways in Burkholderia xenovorans LB400. J Bacteriol 187:7996–8005CrossRefGoogle Scholar
  10. Feist CF, Hegeman GD (1969) Phenol and benzoate metabolism by Pseudomonas putida: regulation of tangential pathways. J Bacteriol 100:1121–1123Google Scholar
  11. Gescher J, Zaar A, Mohamed M, Schagger H, Fuchs G (2002) Genes coding for a new pathway of aerobic benzoate metabolism in Azoarcus evansii. J Bacteriol 184:6301–6315CrossRefGoogle Scholar
  12. Hamzah RY, Albaharna BS (1994) Catechol ring-cleavage in Pseudomonas cepacia—the simultaneous induction of ortho and meta pathways. Appl Microbiol Biotechnol 41:250–256CrossRefGoogle Scholar
  13. Harwood CS, Parales RE (1996) The beta-ketoadipate pathway and the biology of self-identity. Annu Rev Microbiol 50:553–590CrossRefGoogle Scholar
  14. Iwaki H, Hasegawa Y (2007) Degradation of 2-nitrobenzoate by Burkholderia terrae strain KU-15. Biosci Biotechnol Biochem 71:145–151CrossRefGoogle Scholar
  15. Jimenez JI, Minambres B, Garcia JL, Diaz E (2002) Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440. Environ Microbiol 4:824–841CrossRefGoogle Scholar
  16. Kim SI, Song SY, Kim KW, Ho EM, Oh KH (2003) Proteomic analysis of the benzoate degradation pathway in Acinetobacter sp. KS-1. Res Microbiol 154:697–703CrossRefGoogle Scholar
  17. Kim SI, Kim JY, Yun SH, Kim JH, Leem SH, Lee C (2004) Proteome analysis of Pseudomonas sp. K82 biodegradation pathways. Proteomics 4:3610–3621CrossRefGoogle Scholar
  18. Kim YH, Cho K, Yun S-H, Kim JY, Kwon K-H, Yoo JS, Kim SI (2006) Analysis of aromatic catabolic pathways in Pseudomonas putida KT 2440 using a combined proteomic approach: 2-DE/MS and cleavable isotope-coded affinity tag analysis. Proteomics 6:1301–1318CrossRefGoogle Scholar
  19. Krishnan S, Prabhu Y, Phale PS (2004) O-phthalic acid, a dead-end product in one of the two pathways of phenanthrene degradation in Pseudomonas. sp strain PP2. Indian J Biochem Biophys 41:227–232Google Scholar
  20. Kukor JJ, Olsen RH (1991) Genetic organization and regulation of a meta-cleavage pathway for catechols produced from catabolism of toluene, benzene, phenol, and cresols by Pseudomonas pickettii PKO1. J Bacteriol 173:4587–4594Google Scholar
  21. Kurbatov L, Albrecht D, Herrmann H, Petruschka L (2006) Analysis of the proteome of Pseudomonas putida KT2440 grown on different sources of carbon and energy. Environ Microbiol 8:466–478CrossRefGoogle Scholar
  22. Lee N, Lee JM, Min KH, Kwon DY (2003) Purification and characterization of 2,3-dihydroxybiphenyl 1,2-dioxygenase from Comamonas sp. SMN4. J Microbiol Biotechnol 13:487–494Google Scholar
  23. Loh KC, Chua SS (2002) Ortho pathway of benzoate degradation in Pseudomonas putida: induction of meta pathway at high substrate concentrations. Enzyme Microb Technol 30:620–626CrossRefGoogle Scholar
  24. Manjasetty BA, Powlowski J, Vrielink A (2003) Crvstal structure of a bifunctional aldolase-dehydrogenase: sequestering a reactive and volatile intermediate. Proc Natl Acad Sci U S A 100:6992–6997CrossRefGoogle Scholar
  25. Nakazawa T, Nakazawa A (1970) Pyrocatechase (Pseudomonas). Methods Enzymol 17A:518–522CrossRefGoogle Scholar
  26. Nozaki M (1970) Metapyrocatechase (Pseudomonas). Methods Enzymol 17A:522–525CrossRefGoogle Scholar
  27. Parales RE, Haddock JD (2004) Biocatalytic degradation of pollutants. Curr Opin Biotechnol 15:374–379CrossRefGoogle Scholar
  28. Parales RE, Harwood CS (1993) Regulation of the pcaIJ genes for aromatic acid degradation in Pseudomonas putida. J Bacteriol 175:5829–5838Google Scholar
  29. Parales RE, Bruce NC, Schmid A, Wackett LP (2002) Biodegradation, biotransformation, and biocatalysis (B3). Appl Environ Microbiol 68:4699–4709CrossRefGoogle Scholar
  30. Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels. Anal Chem 68:850–858CrossRefGoogle Scholar
  31. Shingler V, Franklin FCH, Tsuda M, Holroyd D, Bagdasarian M (1989) Molecular analysis of a plasmid-encoded phenol hydroxylase from Pseudomonas CF600. J Gen Microbiol 135:1083–1092Google Scholar
  32. Shingler V, Powlowski J, Marklund U (1992) Nucleotide-sequence and functional-analysis of the complete phenol/3,4-dimethylphenol catabolic pathway of Pseudomonas sp. strain-CF600. J Bacteriol 174:711–724Google Scholar
  33. Wackett LP (2003) Pseudomonas putida—a versatile biocatalyst. Nat Biotechnol 21:136–138CrossRefGoogle Scholar
  34. Wheelis ML, Ornston LN (1972) Genetic control of enzyme induction in the beta-ketoadipate pathway of Pseudomonas putida: deleting mapping of cat mutations. J Bacteriol 109:790–795Google Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  1. 1.Department of Chemical and Biomolecular EngineeringNational University of SingaporeSingaporeSingapore
  2. 2.Temasek Life Sciences Laboratory, 1 Research LinkNational University of SingaporeSingaporeSingapore
  3. 3.School of Life Sciences and Chemical TechnologyNgee Ann PolytechnicSingaporeSingapore

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