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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,
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.

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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