Journal of General Microbiology (1989), 135, 1083-1092. Printed in Great Britain 1083 Molecular Analysis of a Plasmid-encoded Phenol Hydroxylase from Pseudomonas CF6QQ By VICTORIA SHINGLER,l* F. CHRISTOPHER H. FRANKLIN,* MASATAKA TSUDA,3 DANY HOLROYD* AND MICHAEL BAG D A S A R I A N 194 Unit for Applied Cell and Molecular Biology, University of Umeii, S-90187 Umeii, Sweden *Department of Genetics, University of Birmingham, PO Box 363, Birmingham B15 2TT, UK 3Laboratory of Genetics, Department of Biology, University of Tokyo, Hongo, Tokyo 113, Japan SMichigan Biotechnology Institute, PO Box 27609, Lansing, MI 48909, USA (Received 30 September I988 ; revised 12 January 1989 ; accepted 30 January 1989) Pseudomonas strain CF600 is able to utilize phenol and 3,4-dimethylphenol as sole carbon and energy source. We demonstrate that growth on these substrates is by virtue of plasmid-encoded phenol hydroxylase and a meta-cleavage pathway. Screening of a genomic bank, with DNA from the previously cloned catechol2,3-dioxygenase gene of the TOL plasmid pWW0, was used in the identification of a clone which could complement a phenol-hydroxylase-deficient transposon insertion mutant. Deletion mapping and polypeptide production analysis identified a 1.2 kb region of DNA encoding a 39-5kDa polypeptide which mediated this complementa- tion. Enzyme activities and growth properties of Pseudomonas strains harbouring this fragment on a broad-host-range expression vector indicate that phenol hydroxylase is a multicomponent enzyme containing the 39.5 kDa polypeptide as one component. INTRODUCTION Pseudomonads and other soil micro-organisms are able to metabolize the vast majority of natural and synthetic organic compounds (Gibson, 1984). In recent years there has been considerable interest in their ability to degrade and detoxify the increasing amounts of aromatic compounds which enter the environment as a result of man's industrial and agricultural activities. Many catabolic pathways for the degradation of aromatic compounds have been elucidated in detail in terms of their biochemistry. Although these bacteria employ a range of enzymes for the initial attack of the different substrates, the catabolic pathways tend to converge on just a few key intermediates such as catechol or substituted catechols (Ornston 8z Yeh, 1982; Dagley, 1986). These key intermediates can be further metabolized by two distinct sets of enzymes : those of the ortho-cleavage pathway (/I-ketoadipate pathway) and those of the meta- cleavage pathway (a-ketoacid pathway) (for review see Dagley, 1986). With rare exceptions, hydroxylation of the benzene ring is a rate-limiting first step in aerobic pathways for the catabolism of aromatic compounds (Chakrabarty, 1982; Dagley, 1986). The specificity of enzymes that catalyse hydroxylation is one of the factors which determine the type of compounds metabolized by the cell (Dagley, 1986; Gibson, 1984; Timmis et al., 1985). Therefore, molecular genetic studies of hydroxylase genes will be of considerable importance for subsequent purification of the enzyme and for manipulation of the pathways with respect to the level of substrate utilization and/or substrate specificity. Abbreviations: C 120, catechol1,2-dioxygenase; C230, catechol2,3-dioxygenase; 3,4-dmp, 3,4-dimethylphenol; HMSD, hydroxymuconic semialdehyde dehydrogenase ; HMSH, hydroxymuconic semialdehyde hydrolase; PH, phenol hydroxylase. 0001-5160 0 1989 SGM