Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1996 Dec;62(12):4471–4477. doi: 10.1128/aem.62.12.4471-4477.1996

Analysis of cumene (isopropylbenzene) degradation genes from Pseudomonas fluorescens IP01.

H Habe 1, K Kasuga 1, H Nojiri 1, H Yamane 1, T Omori 1
PMCID: PMC168274  PMID: 8953719

Abstract

We obtained the DNA fragments encoding 2-hydroxy-6-oxo-7-methylocta-2,4-dienoic acid (HOMODA) hydrolase in the cumene (isopropylbenzene) degrader Pseudomonas fluorescens strain IP01 via PCR using two synthesized oligonucleotides corresponding to the conserved regions within known meta-cleavage compound hydrolases. Following colony hybridization using the amplified DNA as a probe, a 4.5-kb HindIII fragment was isolated from P. fluorescens IP01. After determining the nucleotide sequence of this fragment, three open reading frames (ORF11 [cumH], ORF12 [cumD], and ORF13) were identified. The deduced amino acid sequence of ORF12 showed homology with meta-cleavage compound hydrolases encoded by the tod, dmp, xyl, and bph operons. Although the product of ORF12 was found to exhibit HOMODA and 2-hydroxy-6-oxohepta-2,4-dienoic acid (HOHDA) hydrolase activities, it did not exhibit 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase activity. The deduced amino acid sequence of ORF11 showed 40.4% homology with the sequence of todX in Pseudomonas putida F1 (Y. Wang, M. Ralings, D. T. Gibson, D. Labbé, H. Bergeron, R. Brousseau, and P. C. K. Lau, Mol. Gen. Genet. 246:570-579, 1995). The nucleotide sequence of ORF13 and its flanking region showed strong homology (91.0%) with IS52 from Pseudomonas savastanoi (Y. Yamada, P.-D. Lee, and T. Kosuge, Proc. Natl. Acad. Sci. USA 83:8263-8267, 1982). By characterization of cumH and cumD, the entire cum gene cluster from the cumene-degrader P. fluorescens IP01 (cumA1A2A3A4BCEGFHD) has been identified.

Full Text

The Full Text of this article is available as a PDF (310.5 KB).

Selected References

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

  1. Ahmad D., Fraser J., Sylvestre M., Larose A., Khan A., Bergeron J., Juteau J. M., Sondossi M. Sequence of the bphD gene encoding 2-hydroxy-6-oxo-(phenyl/chlorophenyl)hexa-2,4-dienoic acid (HOP/cPDA) hydrolase involved in the biphenyl/polychlorinated biphenyl degradation pathway in Comamonas testosteroni: evidence suggesting involvement of Ser112 in catalytic activity. Gene. 1995 Apr 14;156(1):69–74. doi: 10.1016/0378-1119(95)00073-f. [DOI] [PubMed] [Google Scholar]
  2. Black P. N. Primary sequence of the Escherichia coli fadL gene encoding an outer membrane protein required for long-chain fatty acid transport. J Bacteriol. 1991 Jan;173(2):435–442. doi: 10.1128/jb.173.2.435-442.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Comai L., Kosuge T. Transposable element that causes mutations in a plant pathogenic Pseudomonas sp. J Bacteriol. 1983 Jun;154(3):1162–1167. doi: 10.1128/jb.154.3.1162-1167.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Eaton R. W., Timmis K. N. Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204. J Bacteriol. 1986 Oct;168(1):123–131. doi: 10.1128/jb.168.1.123-131.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ensley B. D., Ratzkin B. J., Osslund T. D., Simon M. J., Wackett L. P., Gibson D. T. Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo. Science. 1983 Oct 14;222(4620):167–169. doi: 10.1126/science.6353574. [DOI] [PubMed] [Google Scholar]
  6. Erickson B. D., Mondello F. J. Nucleotide sequencing and transcriptional mapping of the genes encoding biphenyl dioxygenase, a multicomponent polychlorinated-biphenyl-degrading enzyme in Pseudomonas strain LB400. J Bacteriol. 1992 May;174(9):2903–2912. doi: 10.1128/jb.174.9.2903-2912.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Furukawa K., Hirose J., Suyama A., Zaiki T., Hayashida S. Gene components responsible for discrete substrate specificity in the metabolism of biphenyl (bph operon) and toluene (tod operon). J Bacteriol. 1993 Aug;175(16):5224–5232. doi: 10.1128/jb.175.16.5224-5232.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gibson D. T., Koch J. R., Kallio R. E. Oxidative degradation of aromatic hydrocarbons by microorganisms. I. Enzymatic formation of catechol from benzene. Biochemistry. 1968 Jul;7(7):2653–2662. doi: 10.1021/bi00847a031. [DOI] [PubMed] [Google Scholar]
  9. Grindley N. D., Reed R. R. Transpositional recombination in prokaryotes. Annu Rev Biochem. 1985;54:863–896. doi: 10.1146/annurev.bi.54.070185.004243. [DOI] [PubMed] [Google Scholar]
  10. Harayama S., Rekik M., Wubbolts M., Rose K., Leppik R. A., Timmis K. N. Characterization of five genes in the upper-pathway operon of TOL plasmid pWW0 from Pseudomonas putida and identification of the gene products. J Bacteriol. 1989 Sep;171(9):5048–5055. doi: 10.1128/jb.171.9.5048-5055.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hayase N., Taira K., Furukawa K. Pseudomonas putida KF715 bphABCD operon encoding biphenyl and polychlorinated biphenyl degradation: cloning, analysis, and expression in soil bacteria. J Bacteriol. 1990 Feb;172(2):1160–1164. doi: 10.1128/jb.172.2.1160-1164.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Higgins D. G., Sharp P. M. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl Biosci. 1989 Apr;5(2):151–153. doi: 10.1093/bioinformatics/5.2.151. [DOI] [PubMed] [Google Scholar]
  13. Hofer B., Backhaus S., Timmis K. N. The biphenyl/polychlorinated biphenyl-degradation locus (bph) of Pseudomonas sp. LB400 encodes four additional metabolic enzymes. Gene. 1994 Jun 24;144(1):9–16. doi: 10.1016/0378-1119(94)90196-1. [DOI] [PubMed] [Google Scholar]
  14. Hofer B., Eltis L. D., Dowling D. N., Timmis K. N. Genetic analysis of a Pseudomonas locus encoding a pathway for biphenyl/polychlorinated biphenyl degradation. Gene. 1993 Aug 16;130(1):47–55. doi: 10.1016/0378-1119(93)90345-4. [DOI] [PubMed] [Google Scholar]
  15. Horn J. M., Harayama S., Timmis K. N. DNA sequence determination of the TOL plasmid (pWWO) xylGFJ genes of Pseudomonas putida: implications for the evolution of aromatic catabolism. Mol Microbiol. 1991 Oct;5(10):2459–2474. doi: 10.1111/j.1365-2958.1991.tb02091.x. [DOI] [PubMed] [Google Scholar]
  16. Kikuchi Y., Nagata Y., Hinata M., Kimbara K., Fukuda M., Yano K., Takagi M. Identification of the bphA4 gene encoding ferredoxin reductase involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp. strain KKS102. J Bacteriol. 1994 Mar;176(6):1689–1694. doi: 10.1128/jb.176.6.1689-1694.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kimbara K., Hashimoto T., Fukuda M., Koana T., Takagi M., Oishi M., Yano K. Cloning and sequencing of two tandem genes involved in degradation of 2,3-dihydroxybiphenyl to benzoic acid in the polychlorinated biphenyl-degrading soil bacterium Pseudomonas sp. strain KKS102. J Bacteriol. 1989 May;171(5):2740–2747. doi: 10.1128/jb.171.5.2740-2747.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lau P. C., Bergeron H., Labbé D., Wang Y., Brousseau R., Gibson D. T. Sequence and expression of the todGIH genes involved in the last three steps of toluene degradation by Pseudomonas putida F1. Gene. 1994 Aug 19;146(1):7–13. doi: 10.1016/0378-1119(94)90827-3. [DOI] [PubMed] [Google Scholar]
  19. Masai E., Yamada A., Healy J. M., Hatta T., Kimbara K., Fukuda M., Yano K. Characterization of biphenyl catabolic genes of gram-positive polychlorinated biphenyl degrader Rhodococcus sp. strain RHA1. Appl Environ Microbiol. 1995 Jun;61(6):2079–2085. doi: 10.1128/aem.61.6.2079-2085.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Menn F. M., Zylstra G. J., Gibson D. T. Location and sequence of the todF gene encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida F1. Gene. 1991 Jul 31;104(1):91–94. doi: 10.1016/0378-1119(91)90470-v. [DOI] [PubMed] [Google Scholar]
  21. Nordlund I., Shingler V. Nucleotide sequences of the meta-cleavage pathway enzymes 2-hydroxymuconic semialdehyde dehydrogenase and 2-hydroxymuconic semialdehyde hydrolase from Pseudomonas CF600. Biochim Biophys Acta. 1990 Jun 21;1049(2):227–230. doi: 10.1016/0167-4781(90)90046-5. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Rak B., von Reutern M. Insertion element IS5 contains a third gene. EMBO J. 1984 Apr;3(4):807–811. doi: 10.1002/j.1460-2075.1984.tb01889.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Reichlin F., Kohler H. P. Pseudomonas sp. strain HBP1 Prp degrades 2-isopropylphenol (ortho-cumenol) via meta cleavage. Appl Environ Microbiol. 1994 Dec;60(12):4587–4591. doi: 10.1128/aem.60.12.4587-4591.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Taira K., Hirose J., Hayashida S., Furukawa K. Analysis of bph operon from the polychlorinated biphenyl-degrading strain of Pseudomonas pseudoalcaligenes KF707. J Biol Chem. 1992 Mar 5;267(7):4844–4853. [PubMed] [Google Scholar]
  26. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  27. Wang Y., Rawlings M., Gibson D. T., Labbé D., Bergeron H., Brousseau R., Lau P. C. Identification of a membrane protein and a truncated LysR-type regulator associated with the toluene degradation pathway in Pseudomonas putida F1. Mol Gen Genet. 1995 Mar 10;246(5):570–579. doi: 10.1007/BF00298963. [DOI] [PubMed] [Google Scholar]
  28. Yamada T., Lee P. D., Kosuge T. Insertion sequence elements of Pseudomonas savastanoi: Nucleotide sequence and homology with Agrobacterium tumefaciens transfer DNA. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8263–8267. doi: 10.1073/pnas.83.21.8263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  30. Zylstra G. J., McCombie W. R., Gibson D. T., Finette B. A. Toluene degradation by Pseudomonas putida F1: genetic organization of the tod operon. Appl Environ Microbiol. 1988 Jun;54(6):1498–1503. doi: 10.1128/aem.54.6.1498-1503.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. van der Meer J. R., de Vos W. M., Harayama S., Zehnder A. J. Molecular mechanisms of genetic adaptation to xenobiotic compounds. Microbiol Rev. 1992 Dec;56(4):677–694. doi: 10.1128/mr.56.4.677-694.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES