Characterization of a high-affinity phenol hydroxylase from Comamonas testosteroni R5 by gene cloning, and expression in Pseudomonas aeruginosa PAO1c

Comamonas testasteroni strain R5 is a phenol-degrading bacterium which expr esses a phenol-oxygenating activity that is characterized by low K-s (the a pparent half-saturation constant in Haldane's equation) and low K-SI (the a pparent inhibition constant) values. We have now cloned the gene cluster en coding a phenol hydroxylase (phcKLMNOP) and its cognate regulator (phcR) fr om Strain R5. Transformation of Pseudomonas aeruginosa PAO1c (Phenol(-) Cat echol(+)) with pROR502, a derivative of pRO1614 containing the cloned genes , confers the ability to grow on phenol as the sole carbon source. The K-s and K-SI values for the phenol-oxygenating activity of PAO1c(pROR502) are a lmost identical to those of strain R5, suggesting that the phcKLMNOP genes encode the major phenol hydroxylase in strain R5. A phylogenetic analysis s hows the phenol hydroxylase from strain R5 to be more closely related to to luene/benzene-2-monooxygenase (Tb2m) from Pseudomonas sp. JS150 than to the phenol hydroxylases from P. putida CF600 and BH, or to the phenol hydroxyl ase from Ralstonia eutropha E2. Analysis of the substrate specificity of PA O1c(pROR502) and PAO1c derivatives expressing phenol hydroxylase from P. pu tida BH or from R. eutropha E2 indicates that these phenol hydroxylases cat alyze the oxidation not only of phenol and cresols but also of toluene and benzene.