In order to study the toluene and o-xylene catabolic genes of Pseudomonas stutzeri OX1, a genomic library was constructed. A 28-kb EcoRI restriction endonuclease DNA fragment, cloned into the vector plasmid pLAFR1 and designated pFB3401, permitted Pseudomonas putida PaW340 to convert toluene and o-xylene into the corresponding meta-ring fission products. Physical and functional endonuclease restriction maps have been derived from the cloned DNA fragment. Further subcloning into and deletion analysis in the Escherichia coli vector pGEM-3Z allowed the genes for the conversion of toluene or o-xylene into the corresponding catechols to be mapped within a 6-kb region of the pFB3401 insert and their direction of transcription to be determined. Following exposure to toluene, E. coli cells carrying this 6-kb region produce a mixture of o-cresol, m-cresol, and p-cresol, which are further converted to 3-methylcatechol and 4-methylcatechol. Similarly, a mixture of 2,3-dimethylphenol and 3,4-dimethylphenol, further converted into dimethylcatechols, was detected after exposure to o-xylene. The enzyme involved in the first step of toluene and o-xylene degradation exhibited a broad substrate specificity, being able to oxidize also benzene, ethylbenzene, m-xylene, p-xylene, styrene, and naphthalene. Deletions of the 6-kb region which affect the ability to convert toluene or o-xylene into the corresponding methylphenols compromise also their further oxidation to methylcatechols. This suggests that a single enzyme system could be involved in both steps of the early stages of toluene and o-xylene catabolism.

Cloning of the genes for and characterization of the early stages of toluene and o-xylene catabolism in Pseudomonas stutzeri OX1 / G. Bertoni, F. Bolognese, E. Galli, P. Barbieri. - In: APPLIED AND ENVIRONMENTAL MICROBIOLOGY. - ISSN 0099-2240. - 62:10(1996 Oct), pp. 3704-3711.

Cloning of the genes for and characterization of the early stages of toluene and o-xylene catabolism in Pseudomonas stutzeri OX1

G. Bertoni;F. Bolognese;E. Galli;P. Barbieri
1996

Abstract

In order to study the toluene and o-xylene catabolic genes of Pseudomonas stutzeri OX1, a genomic library was constructed. A 28-kb EcoRI restriction endonuclease DNA fragment, cloned into the vector plasmid pLAFR1 and designated pFB3401, permitted Pseudomonas putida PaW340 to convert toluene and o-xylene into the corresponding meta-ring fission products. Physical and functional endonuclease restriction maps have been derived from the cloned DNA fragment. Further subcloning into and deletion analysis in the Escherichia coli vector pGEM-3Z allowed the genes for the conversion of toluene or o-xylene into the corresponding catechols to be mapped within a 6-kb region of the pFB3401 insert and their direction of transcription to be determined. Following exposure to toluene, E. coli cells carrying this 6-kb region produce a mixture of o-cresol, m-cresol, and p-cresol, which are further converted to 3-methylcatechol and 4-methylcatechol. Similarly, a mixture of 2,3-dimethylphenol and 3,4-dimethylphenol, further converted into dimethylcatechols, was detected after exposure to o-xylene. The enzyme involved in the first step of toluene and o-xylene degradation exhibited a broad substrate specificity, being able to oxidize also benzene, ethylbenzene, m-xylene, p-xylene, styrene, and naphthalene. Deletions of the 6-kb region which affect the ability to convert toluene or o-xylene into the corresponding methylphenols compromise also their further oxidation to methylcatechols. This suggests that a single enzyme system could be involved in both steps of the early stages of toluene and o-xylene catabolism.
Benzene; Catechols; Cloning, Molecular; Cresols; Escherichia coli; Gene Expression; Genes, Bacterial; Multigene Family; Naphthalenes; Oxidation-Reduction; Pseudomonas; Restriction Mapping; Styrene; Styrenes; Substrate Specificity; Toluene; Transcription, Genetic; Xylenes
Settore BIO/19 - Microbiologia Generale
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/662409
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