Cloning of the genes for and characterization of the early stages of toluene and o-xylene catabolism in Pseudomonas stutzeri OX1.
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.
Bertoni
G
G
Dipartimento di Genetica e di Biologia dei Microrganismi, Università degli Studi di Milano, Italy.
Bolognese
F
F
Galli
E
E
Barbieri
P
P
D001554:Benzene / Q000378:metabolism; D002396:Catechols / Q000378:metabolism; D003001:Cloning, Molecular; D003408:Cresols / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics*; D005810:Multigene Family / Q000235:genetics; D009281:Naphthalenes / Q000378:metabolism; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000235:genetics* / Q000378:metabolism*; D015183:Restriction Mapping; D020058:Styrene; D013343:Styrenes / Q000378:metabolism; D013379:Substrate Specificity; D014050:Toluene / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D014992:Xylenes / Q000378:metabolism*
D002396:Catechols; D003408:Cresols; D009281:Naphthalenes; D013343:Styrenes; D014992:Xylenes; C031721:naphthalene; D014050:Toluene; D020058:Styrene; C077977:cresol; D001554:Benzene; C026114:2-xylene