Characterization of a protocatechuate catabolic gene cluster in Rhodococcus ruber OA1 involved in naphthalene degradation

In this study, we investigated a protocatechuate catabolic gene cluster involved in naphthalene degradation in Rhodococcus ruber OA1. Rhodococcus ruber OA1 was isolated from the pharmaceutical wastewater treatment plant of Xinhua Pharmaceutical Co., Ltd. (Zibo, China). Substrate utilization tests showed that OA1 utilizes naphthalene, phenol, benzoate, salicylate, and protocatechuate as the sole carbon and energy sources for growth. A degradation assay revealed that phthalate is an intermediate in naphthalene degradation and that the protocatechuate pathway plays an important role in naphthalene degradation. To determine the genetic basis and regulation of protocatechuate catabolism in OA1, a fosmid genomic library was constructed and a positive clone carrying the protocatechuate degradation gene cluster was isolated. Sequencing and a bioinformatics analysis identified the complete gene cluster, pcaJIGHBARC, responsible for protocatechuate degradation. Based on this gene cluster, the genes pcaGH (encoding the α and β subunits of protocatechuate 3,4-dioxygenase, 3,4-PCD) were coexpressed and the expressed products showed 3,4-PCD activity. This study illustrates a potential pathway of naphthalene degradation and identifies a protocatechuate pathway in Rhodococcus ruber OA1 for the first time, thus extending our understanding of polycyclic aromatic hydrocarbon degradation and the related aromatic compounds degraded in the process.