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

• Published in: Biodegradation (Dordrecht) 1996-08, Vol.7 (4), p.353-366
• Main Authors: Foght, J.M, Westlake, D.W.S
• Format: Article
• Language: English
• Subjects: aromatic hydrocarbons; biodegradation; Biodegradation, Environmental; Biological and medical sciences; bioremediation; Biotechnology; contaminants; Culture Media; deletions; Dioxygenases; Environment and pollution; Fundamental and applied biological sciences. Psychology; Gas Chromatography-Mass Spectrometry; Genes, Bacterial - physiology; Genetically engineered organisms behavior (microorganisms, plants, animals); Industrial applications and implications. Economical aspects; Multienzyme Complexes - metabolism; mutants; Mutation - physiology; Naphthalenes - metabolism; Oxygenases - metabolism; Phenotype; plasmids; Plasmids - genetics; Polycyclic Aromatic Hydrocarbons - metabolism; Pseudomonas fluorescens; Pseudomonas fluorescens - enzymology; Pseudomonas fluorescens - genetics; Pseudomonas fluorescens - metabolism; Pseudomonas putida - enzymology; Pseudomonas putida - genetics; Pseudomonas putida - metabolism; Restriction Mapping; Sequence Deletion; Substrate Specificity; Transformation, Bacterial; transposons
• ISSN: 0923-9820; 1572-9729
• DOI: 10.1007/bf00115749

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 specificity permitting the oxidation of a suite of polycyclic aromatic and heterocyclic substrates.