Function and Molecular Ecology Significance of Two Catechol-Degrading Gene Clusters in Pseudomonas putida ND6

Many bacteria metabolize aromatic compounds via catechol as a catabolic intermediate, and possess multiple genes or clusters encoding catechol-cleavage enzymes. The presence of multiple isozyme-encoding genes is a widespread phenomenon that seems to give the carrying strains a selective advantage in the natural environment over those with only a single copy. In the naphthalene-degrading strain Pseudomonas putida ND6, catechol can be converted into intermediates of the tricarboxylic acid cycle via either the ortho- or meta-cleavage pathways. In this study, we demonstrated that the catechol ortho-cleavage pathway genes (catB and catB) on the chromosome play an important role. The cat and cat operons are co-transcribed, whereas catA and catA are under independent transcriptional regulation. We examined the binding of regulatory proteins to promoters. In the presence of cis-cis-muconate, a well-studied inducer of the cat gene cluster, CatR_I and CatR_II occupy an additional downstream site, designated as the activation binding site. Notably, CatR_I binds to both the cat and cat promoters with high affinity, while CatR_II binds weakly. This is likely caused by a T to G mutation in the G/T-N₁₁-A motif. Specifically, we found that CatR_I and CatR_II regulate catB and catB in a cooperative manner, which provides new insights into naphthalene degradation.