Abstract

The bphA1A2A3A4 gene cluster, encoding a biphenyl dioxygenase from Rhodococcus globerulus P6, a gram-positive microorganism able to degrade a wide spectrum of polychlorobiphenyls (PCBs), was expressed in Pseudomonas putida, thereby allowing characterization of chlorobiphenyl oxidation by this enzyme. While P6 biphenyl dioxygenase activity was observed in P. putida containing bphA1A2A3A4, no activity was detected in Escherichia coli cells containing the same gene cluster. In E. coli, transcription of genes bphB and bphC1, located downstream of bphA1A2A3A4, was shown to be driven solely by a vector promoter, which indicated that the lack of biphenyl dioxygenase activity was not due to a lack of mRNA synthesis. Radioactive labelling of bph gene products in E. coli implied inefficient translation of the bphA gene cluster or rapid degradation of the gene products. The biosynthesis of functional P6 biphenyl dioxygenase in P. putida cells containing the same plasmid construct that yielded no activity in E. coli emphasizes the importance of the host strain for heterologous expression and shows that synthesis, correct folding, and assembly of a Rhodococcus biphenyl dioxygenase can be achieved in a gram-negative organism. Dioxygenation of six mono- and dichlorinated PCB congeners by P. putida containing the P6 bphA gene cluster indicates the following ring substitution preference for this reaction (from most to least preferred): un-, meta-, para-, and ortho- substitution. No indications were found for dioxygenation of meta/para carbon pairs, or for hydroxylation of chlorinated carbons at any position of a monochlorinated ring, suggesting a strict specificity of this biphenyl dioxygenase for attack at nonhalogenated ortho/meta vicinal carbons. This contrasts the properties of an analogous enzyme from Pseudomonas sp. strain LB400, which can both dioxygenate at meta and para positions and dehalogenate substituted ortho carbons during ortho and meta dioxygenation.