CLONING, NUCLEOTIDE-SEQUENCE, AND EXPRESSION OF THE GENE ENCODING A NOVEL DIOXYGENASE INVOLVED IN METABOLISM OF CARBOXYDIPHENYL ETHERS IN PSEUDOMONAS PSEUDOALCALIGENES POB310

Pseudomonas pseudoalcaligenes strain POB310 degrades 3- and 4-carboxyd iphenyl ether. The initial reaction involves an angular dioxygenation yielding an unstable hemiacetal that spontaneously decays to phenol an d protocatechuate. We cloned a DNA fragment containing the gene encodi ng the initial dioxygenase from an unstable, self-transmissible plasmi d. Sequence analysis revealed two open reading frames encoding protein s with putative molecular masses of 46.3 and 33.6 kDa. The deduced ami no acid sequences showed homologies to oxygenase and reductase subunit s of aromatic ring-activating dioxygenases, and contained regions iden tical to consensus sequences that bind chloroplast-like and Rieske-typ e [2Fe2S] clusters, suggesting that the initial dioxygenase is a class IA aromatic ring-activating dioxygenase system. Initial dioxygenase a ctivity was induced in bacteria grown in M9 minimal medium containing 3- or 4-carboxydiphenyl ether or phenol as carbon source, indicating t hat the regulation is dependent on the phenol pathway. The maximal spe cific activity was measured at the beginning of the exponential growth phase.