Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Sep;171(9):5048–5055. doi: 10.1128/jb.171.9.5048-5055.1989

Characterization of five genes in the upper-pathway operon of TOL plasmid pWW0 from Pseudomonas putida and identification of the gene products.

S Harayama 1, M Rekik 1, M Wubbolts 1, K Rose 1, R A Leppik 1, K N Timmis 1
PMCID: PMC210316  PMID: 2549010

Abstract

The upper operon of the TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes which transform toluene and xylenes to benzoate and toluates. The genetic organization of the operon was characterized by cloning of the upper operon genes into an expression vector and identification of their products in Escherichia coli maxicells. This analysis showed that the upper operon contains at least five genes in the order of xylC-xylM-xylA-xylB-xylN. Between the promoter of the operon and xylC, there is a 1.7-kilobase-long space of DNA in which no gene function was identified. In contrast, most of the DNA between xylC and xylN consists of coding sequences. The xylC gene encodes the 57-kilodalton benzaldehyde dehydrogenase. The xylM and xylA genes encode 35- and 40-kilodalton polypeptides, respectively, which were shown by genetic complementation tests to be subunits of xylene oxygenase. The structural gene for benzyl alcohol dehydrogenase, xylB, encodes a 40-kilodalton polypeptide. The last gene of this operon is xylN, which synthesizes a 52-kilodalton polypeptide of unknown function.

Full text

PDF
5048

Images in this article

5051Image
on p.5051
5053Image
on p.5053
5053Image
on p.5053

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bagdasarian M., Lurz R., Rückert B., Franklin F. C., Bagdasarian M. M., Frey J., Timmis K. N. Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene. 1981 Dec;16(1-3):237–247. doi: 10.1016/0378-1119(81)90080-9. [DOI] [PubMed] [Google Scholar]
  2. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Franklin F. C., Bagdasarian M., Bagdasarian M. M., Timmis K. N. 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 U S A. 1981 Dec;78(12):7458–7462. doi: 10.1073/pnas.78.12.7458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hansen J. B., Olsen R. H. Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMG1 and pMG5. J Bacteriol. 1978 Jul;135(1):227–238. doi: 10.1128/jb.135.1.227-238.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Harayama S., Engström P., Wolf-Watz H., Iino T., Hazelbauer G. L. Cloning of trg, a gene for a sensory transducer in Escherichia coli. J Bacteriol. 1982 Oct;152(1):372–383. doi: 10.1128/jb.152.1.372-383.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Harayama S., Lehrbach P. R., Timmis K. N. Transposon mutagenesis analysis of meta-cleavage pathway operon genes of the TOL plasmid of Pseudomonas putida mt-2. J Bacteriol. 1984 Oct;160(1):251–255. doi: 10.1128/jb.160.1.251-255.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Harayama S., Leppik R. A., Rekik M., Mermod N., Lehrbach P. R., Reineke W., Timmis K. N. Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product. J Bacteriol. 1986 Aug;167(2):455–461. doi: 10.1128/jb.167.2.455-461.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Harayama S., Oguchi T., Iino T. Does Tn10 transpose via the cointegrate molecule? Mol Gen Genet. 1984;194(3):444–450. doi: 10.1007/BF00425556. [DOI] [PubMed] [Google Scholar]
  10. Harayama S., Palva E. T., Hazelbauer G. L. Transposon-insertion mutants of Escherichia coli K12 defective in a component common to galactose and ribose chemotaxis. Mol Gen Genet. 1979 Mar 20;171(2):193–203. doi: 10.1007/BF00270005. [DOI] [PubMed] [Google Scholar]
  11. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  12. Hopper D. J., Taylor D. G. The purification and properties of p-cresol-(acceptor) oxidoreductase (hydroxylating), a flavocytochrome from Pseudomonas putida. Biochem J. 1977 Oct 1;167(1):155–162. doi: 10.1042/bj1670155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Inouye S., Ebina Y., Nakazawa A., Nakazawa T. Nucleotide sequence surrounding transcription initiation site of xylABC operon on TOL plasmid of Pseudomonas putida. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1688–1691. doi: 10.1073/pnas.81.6.1688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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. doi: 10.1128/jb.145.3.1137-1143.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Keil H., Saint C. M., Williams P. A. Gene organization of the first catabolic operon of TOL plasmid pWW53: production of indigo by the xylA gene product. J Bacteriol. 1987 Feb;169(2):764–770. doi: 10.1128/jb.169.2.764-770.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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. doi: 10.1128/jb.144.1.222-231.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nozaki M. Oxygenases and dioxygenases. Top Curr Chem. 1979;78:145–186. doi: 10.1007/BFb0048193. [DOI] [PubMed] [Google Scholar]
  18. O'Connor C. D., Timmis K. N. Highly repressible expression system for cloning genes that specify potentially toxic proteins. J Bacteriol. 1987 Oct;169(10):4457–4462. doi: 10.1128/jb.169.10.4457-4462.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Platt T. Transcription termination and the regulation of gene expression. Annu Rev Biochem. 1986;55:339–372. doi: 10.1146/annurev.bi.55.070186.002011. [DOI] [PubMed] [Google Scholar]
  20. Prentki P., Krisch H. M. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. doi: 10.1016/0378-1119(84)90059-3. [DOI] [PubMed] [Google Scholar]
  21. Remaut E., Stanssens P., Fiers W. Inducible high level synthesis of mature human fibroblast interferon in Escherichia coli. Nucleic Acids Res. 1983 Jul 25;11(14):4677–4688. doi: 10.1093/nar/11.14.4677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Remaut E., Tsao H., Fiers W. Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication. Gene. 1983 Apr;22(1):103–113. doi: 10.1016/0378-1119(83)90069-0. [DOI] [PubMed] [Google Scholar]
  23. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sancar A., Rupp W. D. Cloning of uvrA, lexC and ssb genes of Escherichia coli. Biochem Biophys Res Commun. 1979 Sep 12;90(1):123–129. doi: 10.1016/0006-291x(79)91598-5. [DOI] [PubMed] [Google Scholar]
  25. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  26. Worsey M. J., Williams P. A. 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. doi: 10.1128/jb.124.1.7-13.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES