Molecular and General Genetics MGG

, Volume 221, Issue 1, pp 113–120 | Cite as

The meta cleavage operon of TOL degradative plasmid pWWO comprises 13 genes

  • Shigeaki Harayama
  • Monique Rekik


The meta-cleavage operon of TOL plasmid pWWO of Pseudomonas putida encodes a set of enzymes which transform benzoate/toluates to Krebs cycle intermediates via extradiol (meta-) cleavage of (methyl)catechol. The genetic organization of the operon was characterized by cloning of the meta-cleavage genes into an expression vector and identification of their products in Escherichia coli maxicells. This analysis showed that the meta-cleavage operon contains 13 genes whose order and products (in kilodaltons) are The xyIXYZ genes encode three subunits of toluate 1,2-dioxygenase. The xylL, xyIE, xyIG, xylF, xylJ, xylK, xylI and xylH genes encode 1,2-dihydroxy-3,5-cyclohexadiene-1-carboxylate dehydrogenase, catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, 2-hydroxymuconic semialdehyde hydrolase, 2-oxopent-4-enoate hydratase, 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase and 4-oxalocrotonate tautomerase, respectively. The functions of xyIT and xylQ are not known at present. The comparison of the coding capacity and the sizes of the products of the meta-cleavage operon genes indicated that most of the DNA between xyIX and xyIH consists of coding sequences.

Key words

TOL plasmid Catabolism Pseudomonas putida Meta-cleavage operon Maxicells 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Assinder SJ, Williams PA (1988) Comparison of the meta pathway operons on NAH plasmid pWW60-22 and TOL plasmid pWW53-4 and its evolutionary significance. J Gen Microbiol 134:2769–2778Google Scholar
  2. Axcell BC, Geary PJ (1973) The purification and some properties of the enzyme cis-1,2-dihydroxycyclohexa-3,5-diene (nicotinamide adenine dinucleotide) oxidoreductase (cis-benzene glycol dehydrogenase). Biochem J 136:927–934Google Scholar
  3. Bayly RC, Di Berardino D (1978) Purification and properties of 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase from two strains of Pseudomonas putida. J Bacteriol 134:30–37Google Scholar
  4. Belasco JG, Higgins CF (1988) Mechanisms of mRNA decay in bacteria: a prospective. Gene 72:15–23Google Scholar
  5. Collinsworth WL, Chapman PJ, Dagley S (1973) Stereospecific enzymes in the degradation of aromatic compounds by Pseudomomans putida. J Bacteriol 113:922–931Google Scholar
  6. Duggleby CJ, Williams PA (1986) Purification and some properties of the 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase (2-hydroxymuconic semialdehyde hydrolase) encoded by the TOL plasmid pWWO from Pseudomonas putida mt-2. J Gen Microbiol 132:712–726Google Scholar
  7. Franklin FCH, Bagdasarian M, Bagdasarian MM, Timmis KN (1981) Molecular and functional analysis of the TOL plasmid pWWO from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta cleavage pathway. Proc Natl Acad Sci USA 78:7458–7462Google Scholar
  8. Ghosal D, You I-S, Gunsalus IC (1987) Nucleotide sequence and expression of gene nahH of plasmid NAH7 and homology with gene xylE of TOL plasmid pWWO. Gene 55:19–28Google Scholar
  9. Harayama S, Rekik M (1989) A simple procedure for transferring genes cloned in Escherichia coli vectors into other Gram-negative bacteria: phenotypic analysis and mapping of TOL plasmid gene xylK. Gene 78:19–27Google Scholar
  10. Harayama S, Timmis KN (1989) Catabolism of aromatic hydrocarbons by Pseudomonas. In: Hopwood DA, Chater KF (ed) Genetics of bacterial diversity. Academic Press, New York, pp 151–174Google Scholar
  11. Harayama S, Engström P, Wolf-Watz H, Iino T, Hazelbauer GL (1982) Cloning of trg, a gene for a sensory transducer in Escherichia coli. J Bacteriol 152:372–383Google Scholar
  12. Harayama S, Lehrbach PR, Timmis KN (1984) Transposon mutagenesis analysis of meta-cleavage pathway operon genes of the TOL plasmid of Pseudomonas putida mt-2. J Bacteriol 160:251–255Google Scholar
  13. Harayama S, Rekik M, Timmis KN (1986a) Genetic analysis of a relaxed substrate specificity aromatic ring dioxygenase, toluate 1,2-dioxygenase, encoded by TOL plasmid pWWO of Pseudomonas putida. Mol Gen Genet 202:226–234Google Scholar
  14. Harayama S, Leppik RA, Rekik M, Mermod N, Lehrbach PR, Reineke W, Timmis KN (1986b) Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product. J Bacteriol 167:455–461Google Scholar
  15. Harayama S, Rekik M, Wasserfallen A, Bairoch A (1987a) Evolutionary relationships between catabolic pathways for aromatics: conservation of gene order and nucleotide sequences of catechol oxidation genes of pWWO and NAH7 plasmids. Mol Gen Genet 210:241–247Google Scholar
  16. Harayama S, Mermod N, Rekik M, Lehrbach PR, Timmis KN (1987b) Roles of the divergent branches of the meta-cleavage pathway in the degradation of benzoate and substituted benzoates. J Bacteriol 169:558–564Google Scholar
  17. Harayama S, Rekik M, Wubbolts M, Rose K, Leppik RA, Timmis KN (1989a) Identification of five genes and their products in the upper pathway operon of TOL plasmid pWWO from Pseudomonas putida. J Bacteriol 171:5048–5055Google Scholar
  18. Harayama S, Rekik M, Ngai K-L, Ornston LN (1989b) Physically associated enzymes produce and metabolize 2-hydroxy-2,4dienoate, a chemically unstable intermediate formed in catechol metabolism via meta-cleavage in Pseudomonas putida. J Bacteriol 171:6251–6258Google Scholar
  19. Inouye S, Nakazawa A, Nakazawa T (1981) Molecular cloning of TOL genes xylB and xylE in Escherichia coli. J Bacteriol 145:1137–1143Google Scholar
  20. Inouye S, Nakazawa A, Nakazawa T (1984) Nucleotide sequence of the promoter region of the xylDEGF operon on TOL plasmid of Pseudomonas putida. Gene 29:323–330Google Scholar
  21. Inouye S, Nakazawa A, Nakazawa T (1986) Nucleotide sequence of the regulatory gene xylS on the Pseudomonas putida TOL plasmid and identification of the protein product. Gene 44:235–242Google Scholar
  22. Inouye S, Nakazawa A, Nakazawa T (1988) Nucleotide sequence of the regulatory gene xylR of the TOL plasmid from Pseudomonas putida. Gene 66:301–306Google Scholar
  23. Irie S, Doi S, Yorifuji T, Takagi M, Yano K (1987) Nucleotide sequencing and characterization of the genes encoding benzene oxidation enzymes of Pseudomonas putida. J Bacteriol 169:5174–5179Google Scholar
  24. Keil H, Keil S, Williams PA (1987) Molecular analysis of regulatory and structural xyl genes of the TOL plasmid pWW53-4. J Gen Micobiol 133:1149–1158Google Scholar
  25. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  26. Mermod N, Lehrbach PR, Reineke W, Timmis KN (1984) Transcription of the TOL plasmid toluate catabolic pathway operon of Pseudomonas putida is determined by a pair of coordinately and positively regulated overlapping promoters. EMBO J 3:2461–2466Google Scholar
  27. Nakai C, Hori K, Kagamiyama H, Nakazawa T, Nozaki M (1983a) Purification, subunit structure, and partial amino acid sequence of metapyrocatechase. J Biol Chem 258:2916–2922Google Scholar
  28. Nakai C, Kagamiyama H, Nozaki M, Nakazawa T, Inouye S, Ebina Y, Nakazawa A (1983b) Complete nucleotide sequence of the metapyrocatechase gene on the TOL plasmid of Pseudomonas putida mt-2. J Biol Chem 258:2923–2928Google Scholar
  29. Nakazawa T, Inouye S, Nakazawa A (1980) Physical and functional mapping of RP4-TOL plasmid recombinants: analysis of insertion and deletion mutants. J Bacteriol 144:222–231Google Scholar
  30. Neidle EL, Shapiro MK, Ornston LN (1987) Cloning and expression in Escherichia coli of Acinetobacter calcoaceticus genes for benzoate degradation. J Bacteriol 169:5496–5503Google Scholar
  31. Osborne DJ, Pickup RW, Williams PA (1988) The presence of two complete homologous meta pathway operons on TOL plasmid pWW53. J Gen Microbiol 134:2965–2975Google Scholar
  32. Patel TR, Gibson DT (1974) Purification and properties of (+)-cisnaphthalene dihydrodiol dehydrogenase of Pseudomonas putida. J Bacteriol 119:879–888Google Scholar
  33. Reineke W, Knackmuss H-J (1978) Chemical structure and biodegradativity of halogenated aromatic compounds. Substituent effects on 1,2-dioxygenation of benzoic acid. Biochim Biophys Acta 542:412–423Google Scholar
  34. Reiner AM (1972) Metabolism of aromatic compounds in bacteria. Purification and properties of the catechol-forming enzyme, 3,5-cyclohexadiene-1,2-diol-l-carboxylic acid (NAD+) oxidoreductase (decarboxylating). J Biol Chem 247:4960–4965Google Scholar
  35. Shaw LE, Williams PA (1988) Physical and functional mapping of two cointegrate plasmids derived from RP4 and TOL plasmid pDK1. J Gen Microbiol 134:2463–2474Google Scholar
  36. Yamaguchi M, Fujisawa H (1978) Characterization of NADHcytochrome c reductase, a component of benzoate 1,2-dioxygenase system from Pseudomonas arvilla C-1. J Biol Chem 253:8848–8853Google Scholar
  37. Yamaguchi M, Fujisawa H (1980) Purification and characterization of an oxygenase component in benzoate 1,2-dioxygenase system from Pseudomonas arvilla C-1. J Biol Chem 255:5058–5063Google Scholar
  38. Yamaguchi M, Fujisawa H (1982) Subunit structure of oxygenase component in benzoate-1,2-dioxygenase system from Pseudomonas arvilla C-1. J Biol Chem 257:12497–12502Google Scholar

Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • Shigeaki Harayama
    • 1
  • Monique Rekik
    • 1
  1. 1.Department of Medical BiochemistryUniversity of GenevaGeneva 4Switzerland

Personalised recommendations