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Proc Natl Acad Sci U S A. Dec 1981; 78(12): 7458–7462.
PMCID: PMC349287

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.

Abstract

The genetic organization of the Pseudomonas putida plasmid pWWO-161, which encodes enzymes for the degradation of toluene and related aromatic hydrocarbons, has been investigated by transposition mutagenesis and gene cloning. Catabolic genes were localized to two clusters, one for upper pathway (hydrocarbon leads to carboxylic acid) enzymes and the other for lower pathway (carboxylic acid leads to tricarboxylic acid cycle) enzymes, that are separated by a 14-kilobase DNA segment. The physical organization of the catabolic genes thus reflects their functional organization into two regulatory blocks. The pWWO-161 DNA fragments Sst I fragment C and fragment D were cloned in a broad host range vector to produce plasmid pKT530. This hybrid encodes toluate oxygenase and all meta cleavage pathway enzymes, and it enables P. putida mt-2 and Escherichia coli K-12 cells to grow on m-toluate as sole carbon source. The pKT530 plasmid also carries xylS (a gene whose product has been postulated to regulate expression of the lower pathway genes) and the control sequences of the pathway that interact with this product, because catechol 2,3-oxygenase synthesis is specifically induced by m-toluate in both P. putida and E. coli. Evidence is presented that suggests the promoter operator of the meta pathway gene functions less effectively with the RNA polymerase or xylS product of E. coli than with the enzyme or product of P. putida.

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Selected References

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  • Worsey MJ, Williams PA. Metabolism of toluene and xylenes by Pseudomonas (putida (arvilla) mt-2: evidence for a new function of the TOL plasmid. J Bacteriol. 1975 Oct;124(1):7–13. [PMC free article] [PubMed]
  • Kunz DA, Chapman PJ. Catabolism of pseudocumene and 3-ethyltoluene by Pseudomonas putida (arvilla) mt-2: evidence for new functions of the TOL (pWWO) plasmid. J Bacteriol. 1981 Apr;146(1):179–191. [PMC free article] [PubMed]
  • Worsey MJ, Franklin FC, Williams PA. Regulation of the degradative pathway enzymes coded for by the TOL plasmid (pWWO) from Pseudomonas putida mt-2. J Bacteriol. 1978 Jun;134(3):757–764. [PMC free article] [PubMed]
  • Williams PA, Murray K. Metabolism of benzoate and the methylbenzoates by Pseudomonas putida (arvilla) mt-2: evidence for the existence of a TOL plasmid. J Bacteriol. 1974 Oct;120(1):416–423. [PMC free article] [PubMed]
  • Datta N, Hedges RW, Shaw EJ, Sykes RB, Richmond MH. Properties of an R factor from Pseudomonas aeruginosa. J Bacteriol. 1971 Dec;108(3):1244–1249. [PMC free article] [PubMed]
  • Timmis KN, Cabello F, Cohen SN. Cloning and characterization of EcoRI and HindIII restriction endonuclease-generated fragments of antibiotic resistance plasmids R6-5 and R6. Mol Gen Genet. 1978 Jun 14;162(2):121–137. [PubMed]
  • Hansen JB, Olsen RH. Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMG1 and pMG5. J Bacteriol. 1978 Jul;135(1):227–238. [PMC free article] [PubMed]
  • Andrés I, Slocombe PM, Cabello F, Timmis JK, Lurz R, Burkardt HJ, Timmis KN. Plasmid replication functions. II. Cloning analysis of the repA replication region of antibiotic resistance plasmid R6-5. Mol Gen Genet. 1979 Jan 5;168(1):1–25. [PubMed]
  • Nakazawa T, Inouye S, Nakazawa A. Physical and functional mapping of RP4-TOL plasmid recombinants: analysis of insertion and deletion mutants. J Bacteriol. 1980 Oct;144(1):222–231. [PMC free article] [PubMed]
  • Downing R, Broda P. A cleavage map of the TOL plasmid of Pseudomonas putida mt-2. Mol Gen Genet. 1979;177(1):189–191. [PubMed]
  • Timmis K, Cabello F, Cohen SN. Cloning, isolation, and characterization of replication regions of complex plasmid genomes. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2242–2246. [PMC free article] [PubMed]
  • Figurski DH, Helinski DR. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. [PMC free article] [PubMed]
  • Inouye S, Nakazawa A, Nakazawa T. Molecular cloning of TOL genes xylB and xylE in Escherichia coli. J Bacteriol. 1981 Mar;145(3):1137–1143. [PMC free article] [PubMed]
  • Murray K, Duggleby CJ, Sala-Trepat JM, Williams PA. The metabolism of benzoate and methylbenzoates via the meta-cleavage pathway by Pseudomonas arvilla mt-2. Eur J Biochem. 1972 Jul 24;28(3):301–310. [PubMed]
  • Jacoby GA, Rogers JE, Jacob AE, Hedges RW. Transposition of Pseudomonas toluene-degrading genes and expression in Escherichia coli. Nature. 1978 Jul 13;274(5667):179–180. [PubMed]
  • Cohen SN. Transposable genetic elements and plasmid evolution. Nature. 1976 Oct 28;263(5580):731–738. [PubMed]

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