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Applied and Environmental Microbiology, October 1998, p. 3626-3632, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Analysis of the Gene Cluster Encoding Toluene/o-Xylene Monooxygenase from Pseudomonas stutzeri OX1

Giovanni Bertoni,dagger Manuela Martino, Enrica Galli, and Paola Barbieri*

Dipartimento di Genetica e di Biologia dei Microrganismi, Università degli Studi di Milano, 20133 Milan, Italy

Received 15 May 1998/Accepted 7 August 1998

The toluene/o-xylene monooxygenase cloned from Pseudomonas stutzeri OX1 displays a very broad range of substrates and a very peculiar regioselectivity, because it is able to hydroxylate more than one position on the aromatic ring of several hydrocarbons and phenols. The nucleotide sequence of the gene cluster coding for this enzymatic system has been determined. The sequence analysis revealed the presence of six open reading frames (ORFs) homologous to other genes clustered in operons coding for multicomponent monooxygenases found in benzene- and toluene-degradative pathways cloned from Pseudomonas strains. Significant similarities were also found with multicomponent monooxygenase systems for phenol, methane, alkene, and dimethyl sulfide cloned from different bacterial strains. The knockout of each ORF and complementation with the wild-type allele indicated that all six ORFs are essential for the full activity of the toluene/o-xylene monooxygenase in Escherichia coli. This analysis also shows that despite its activity on both hydrocarbons and phenols, toluene/ o-xylene monooxygenase belongs to a toluene multicomponent monooxygenase subfamily rather than to the monooxygenases active on phenols.

* Corresponding author. Mailing address: Dipartimento di Genetica e di Biologia dei Microrganismi, via Celoria, 26, 20133 Milano, Italy. Phone: (392)26605227. Fax: (392)2664551. E-mail: barbieri{at}imiucca.csi.unimi.it.

dagger Present address: Centro Nacional de Biotecnología (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain.

Applied and Environmental Microbiology, October 1998, p. 3626-3632, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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