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open-access-imgOpen AccessCloning of the genes for and characterization of the early stages of toluene and o-xylene catabolism in Pseudomonas stutzeri OX1
Author(s)
Giuseppe Bertoni,
Fabrizio Bolognese,
Elena Galli,
Pierluigi Barbieri
Publication year1996
Publication title
applied and environmental microbiology
Resource typeJournals
PublisherAmerican Society for Microbiology
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.
Subject(s)bacteria , biochemistry , biology , chemistry , gene , genetics , haeiii , microbiology and biotechnology , organic chemistry , polymerase chain reaction , pseudomonas putida , pseudomonas stutzeri , restriction fragment length polymorphism , toluene , xylene
Language(s)English
SCImago Journal Rank1.552
H-Index324
ISSN0099-2240
DOI10.1128/aem.62.10.3704-3711.1996

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