Skip to main content
SpringerLink
Log in

Transposon and spontaneous deletion mutants of plasmid-borne genes encoding polycyclic aromatic hydrocarbon degradation by a strain of Pseudomonas fluorescens

  • Articles
  • Published:
Biodegradation Aims and scope Submit manuscript

Abstract

Pseudomonas fluorescens strain LP6a, isolated from petroleum condensate-contaminated soil, utilizes the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene, anthracene and 2-methylnaphthalene as sole carbon and energy sources. The isolate also co-metabolically transforms a suite of PAHs and heterocycles including fluorene, biphenyl, acenaphthene, 1-methylnaphthalene, indole, benzothiophene, dibenzothiophene and dibenzofuran, producing a variety of oxidized metabolites. A 63 kb plasmid (pLP6a) carries genes encoding enzymes necessary for the PAH-degrading phenotype of P. fluorescens LP6a. This plasmid hybridizes to the classical naphthalene degradative plasmids NAH7 and pWW60, but has different restriction endonuclease patterns. In contrast, plasmid pLP6a failed to hybridize to plasmids isolated from several phenanthrene-utilizing strains which cannot utilize naphthalene. Plasmid pLP6a exhibits reproducible spontaneous deletions of a 38 kb region containing the degradative genes. Two gene clusters corresponding to the archetypal naphthalene degradation upper and lower pathway operons, separated by a cryptic region of 18 kb, were defined by transposon mutagenesis. Gas chromatographic-mass spectrometric analysis of metabolites accumulated by selected transposon mutants indicates that the degradative enzymes encoded by genes on pLP6a have a broad substrate specificity permitting the oxidation of a suite of polycyclic aromatic and heterocyclic substrates.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Berg CM & Berg DE (1987) Uses of transposable elements and maps of known insertions. In: Ingraham JL, Brooks Low K, Magasanic B, Schaechter M & Umbarger HE (Eds) Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology, Vol. 2 (pp 1071–1109). American Society for Microbiology Press, Washington, DC

    Google Scholar 

  • Blenkinsopp S, Sergy G, Wang Z, Fingas MF, Foght JM & Westlake DWS (1995) Oil spill bioremediation, agents: Canadian efficacy test protocols. In: Ludwigson JO (Ed) Proceedings, International Oil Spill Conference (Achieving and Maintaining Preparedness) (pp 91–96). American Petroleum Institute, Washington, DC

    Google Scholar 

  • Cane PA & Williams PA (1982) The plasmid-coded metabolism of naphthalene and 2-methylnaphthalene in Pseudomonas strains: phenotypic changes correlated with structural modification of the plasmid pWW60–1. J. Gen. Microbiol. 128: 2281–2290

    Google Scholar 

  • Carney BF & Leary JF (1989) Novel alterations in plasmid DNA associated with aromatic hydrocarbon utilization by Pseudomonas putida R5–3. Appl. Environ. Microbiol. 55: 1523–1530

    Google Scholar 

  • Cerniglia CE (1992) Biodegradation of polycyclic aromatic hydrocarbons. Biodegradation 3: 351–368

    Google Scholar 

  • Clarke PH & Laverack PD (1984) Growth characteristics of Pseudomonas strains carrying catabolic plasmids and their cured derivatives. FEMS Microbiol. Lett. 24: 109–112

    Google Scholar 

  • Denome SA, Stanley DC, Olson ES & Young KD (1993) Metabolism of dibenzothiophene and naphthalene in Pseudomonas strains: complete DNA sequence of an upper naphthalene catabolic pathway. J. Bacteriol. 175: 6890–6901

    Google Scholar 

  • Eaton RW & Chapman PJ (1992) Bacterial metabolism of naphthalene: construction and use of recombinant bacteria to study ring cleavage of 1,2-dihydroxynaphthalene and subsequent reactions. J. Bacteriol. 174: 7542–7554

    Google Scholar 

  • Edwards NT (1983) Polycyclic aromatic hydrocarbons (PAHs) in the terrestrial environment: a review. J. Environ. Qual. 12: 427–441

    Google Scholar 

  • Ensley BD, Ratzkin BJ, Osslund TD, Simon MJ, Wackett LP & Gibson DT (1983) Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo. Science 222: 167–169

    Google Scholar 

  • Evans WC, Fernley HN & Griffiths E (1965) Oxidative metabolism of phenanthrene and anthracene by soil pseudomonads. Biochem. J. 95: 819–831

    Google Scholar 

  • Fedorak PM & Westlake DWS (1981) Microbial degradation of aromatics and saturates in Prudhoe Bay crude oil as determined by glass capillary gas chromatography. Can. J. Microbiol. 27: 432–443

    Google Scholar 

  • Fedorak PM & Westlake DWS (1986) Fungal metabolism of n-alkylbenzenes. Appl. Environ. Microbiol. 51: 435–437

    Google Scholar 

  • Finnerty WR & Robinson M (1986) Microbial desulfurization of fossil fuels: a review. Biotechnol. Bioeng. Symp. 16: 205–211

    Google Scholar 

  • Foght JM & Westlake DWS (1988) Degradation of polycyclic aromatic hydrocarbons and aromatic heterocycles by a Pseudomonas species. Can. J. Microbiol. 34: 1135–1141

    Google Scholar 

  • Foght JM & Westlake DWS (1990) Expression of dibenzothiophene-degradative genes in two Pseudomonas species. Can. J. Microbiol. 36: 718–724

    Google Scholar 

  • Foght JM & Westlake DWS (1991) Cross hybridization of plasmid and genomic DNA from aromatic and polycyclic aromatic hydrocarbon degrading bacteria. Can. J. Microbiol. 37: 924–932

    Google Scholar 

  • Goyal AK & Zylstra GJ (1996) Molecular cloning of novel genes for polycyclic aromatic hydrocarbon degradation from Comamonas testosteroni GZ39. Appl. Environ. Microbiol. 62: 230–236

    Google Scholar 

  • Grosser RJ, Warshawsky D & Vestal JR (1991) Indigenous and enhanced mineralization of pyrene, benzo[a]pyrene and carbazole in soils. Appl. Environ. Microbiol. 57: 3462–3469

    Google Scholar 

  • Hunt JM (1979) Petroleum Geochemistry and Geology. San Francisco, CA, W.H. Freeman & Co.

    Google Scholar 

  • Jerina DM, Selander H, Yagi H, Wells MC, Davey JF, Mahadevan V & Gibson DT (1976) Dihydrodiols from anthracene and phenanthrene. J. Am. Chem. Soc. 98: 5988–5996

    Google Scholar 

  • Kiyohara H, Nagao K & Yana K (1982) Rapid screen for bacteria degrading water-insoluble solid hydrocarbons on agar plates. Appl. Environ. Microbiol. 43: 454–457

    Google Scholar 

  • Kiyohara H, Sugiyama M, Mondello FJ, Gibson DT & Yano K (1983) Plasmid involvement in the degradation of polycyclic aromatic hydrocarbons by a Beijerinckia species. Biochem. Biophys. Res. Commun. 111: 939–945

    Google Scholar 

  • Kiyohara H, Torigoe S, Kaida N, Asaki T, Iida T, Hayashi H & Takizawa N (1994) Cloning and characterization of a chromosomal gene cluster, pah, that encodes the upper pathway for phenanthrene and naphthalene utilization by Pseudomonas putida OUS82. J. Bacteriol. 176: 2439–2443

    Google Scholar 

  • Laborde AL & Gibson DT (1977) Metabolism of dibenzothiophene by a Beijerinkia species. Appl. Environ. Microbiol. 34: 783–790

    Google Scholar 

  • Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3: 208–218

    Google Scholar 

  • Menn F-M, Applegate BM & Sayler GS (1993) NAH plasmid-mediated catabolism of anthracene and phenanthene to naphthoic acids. J. Bacteriol. 59: 1938–1942

    Google Scholar 

  • Mueller JG, Lantz SE, Devereux R, Berg JD & Pritchard PH (1994) Studies on the microbial ecology of polycyclic aromatic hydrocarbon biodegradation. In: Hinchee RE, Leeson A, Semprini L & Ong SK (Eds) Bioremediation of Chlorinated and Polycyclic Aromatic Hydrocarbon Compounds (pp 218–230). Lewis Publishers, Boca Raton

    Google Scholar 

  • Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning: a laboratory manual. Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press

    Google Scholar 

  • Sanseverino J, Applegate BM, King JMH & Sayler GS (1993) Plasmid-mediated mineralization of naphthalene, phenanthrene and anthracene. Appl. Environ. Microbiol. 59: 1931–1937

    Google Scholar 

  • Selvaraj G & Iyer VN (1983) Suicide plasmid vehicles for insertion mutagenesis in Rhizobium meliloti and related bacteria. J. Bacteriol. 156: 1292–1300

    Google Scholar 

  • Simon MJ, Osslund TD, Saunders R, Ensley BD, Suggs S, Harcourt A, Suen W-C, Cruden DL, Gibson DT & Zylstra GJ (1993) Sequences of genes encoding naphthalene dioxygenase in Pseudomonas putida strains G7 and NCIB 9816–4. Gene 127: 31–37

    Google Scholar 

  • Smibert RM & Krieg NR (1981) General characterization. In: Gerhardt P, Murray RGE, Costilow RN, Nester EW, Wood WA, Krieg NR & Phillips GB (Eds) Manual of Methods for General Bacteriology (pp 409–443). American Society for Microbiology, Washington, DC

    Google Scholar 

  • Sutherland JB, Rafii F, Khan AA & Cerniglia CE (1995) Mechanisms of polycyclic aromatic hydrocarbon degradation. In: Young LY & Cerniglia CE (Eds) Microbial Transformation and Degradation of Toxic Organic Chemicals (pp 269–306). Wiley-Liss, Inc., New York

    Google Scholar 

  • Takizawa N, Kaida N, Torigoe S, Moritani T, Sawada T, Satoh S & Kiyohara H (1994) Identification and characterization of genes encoding polycyclic aromatic hydrocarbon dioxygenase and polycyclic aromatic hydrocarbon dihydrodiol dehydrogenase in Pseudomonas putida OUS82. J. Bacteriol. 176: 2444–2449

    Google Scholar 

  • Tsuda M & Iino T (1988) Identification and characterization of Tn4653, a transposon covering the toluene transposon Tn4651 on TOL plasmid pWW0. Mol. Gen. Genet. 213: 72–77

    Google Scholar 

  • Tsuda M & Iino T (1990) Naphthalene degrading genes on plasmid NAH7 are on a defective transposon. Mol. Gen. Genet. 233: 33–39

    Google Scholar 

  • Weissenfels WD, Beyer M & Klein J (1990) Degradation of phenanthrene, fluorene and fluoranthene by pure bacterial cultures. Appl. Microbiol. Biotechnol. 32: 479–484

    Google Scholar 

  • Williams PA, Taylor SD & Gibb LE (1988) Loss of the toluenexylene catabolic genes of TOL plasmid pWW0 during growth of Pseudomonas putida on benzoate is due to a selective growth advantage of ‘cured’ segregants. J. Gen. Microbiol. 134: 2039–2048

    Google Scholar 

  • Wyndham RC, Cashore AE, Nakatsu CH & Peel MC (1994) Catabolic transposons. Biodegradation 5: 323–342

    Google Scholar 

  • Yang Y, Chen RF & Shiaris MP (1994) Metabolism of naphthalene, fluorene and phenanthrene: preliminary characterization of a cloned gene cluster from Pseudomonas putida NCIB 9816. J. Bacteriol. 176: 2158–2164

    Google Scholar 

  • Yen K-M & Gunsalus IC (1982) Plasmid gene organization: naphthalene/salicylate oxidation. Proc. Natl. Acad. Sci. USA 79: 874–878

    Google Scholar 

  • Yen KM & Serdar CM (1988) Genetics of naphthalene catabolism in pseudomonads. Crit. Rev. Microbiol. 15: 247–268

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biological Sciences, University of Alberta, T6G 2E9, Edmonton, Alberta, Canada

    Julia M. Foght & Donald W. S. Westlake

Authors
  1. Julia M. Foght
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Donald W. S. Westlake
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Foght, J.M., Westlake, D.W.S. Transposon and spontaneous deletion mutants of plasmid-borne genes encoding polycyclic aromatic hydrocarbon degradation by a strain of Pseudomonas fluorescens . Biodegradation 7, 353–366 (1996). https://doi.org/10.1007/BF00115749

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00115749

Key words

Search

Navigation