Abstract

Polychlorinated biphenyls (PCBs) are one of the most widely distributed chlorinated pollutants in the environment. Bioremedation seems to be a promising approach for the clean-up of PCB-contaminated soils and aquatic environments. Both anaerobic and aerobic bacteria have the potential to attack PCBs. However, degradation is inefficient and/or incomplete. To develop rational strategies for improvement of PCB-degrading pathways it is necessary to acquire detailed information on the genetics, the enzymes involved and the metabolites formed. We set out to tackle this problem by isolation and characterization of the locus bph involved in PCB degradation by Pseudomomas sp. LB400, a strain particularly noteworthy for its ability to oxidize a broad spectrum of PCBs. This locus encodes seven polypeptides responsible for the four catabolic steps of the 'upper' pathway which transforms biphenyl to benzoate and 2-hydroxypenta-2,4-dienoate, three enzymes required for conversion of 2-hydroxypenta-2,4-dienoate to Krebs cycle intermediates, and a glutathione S-tranferase. Based on genetic analysis, recombinant E. coli strains encoding subsets of bph genes were constructed for a detailed analysis of pathway metabolites Biochemical analysis showed that with many ortho-chlorinated congeners attack by biphenyl-2,3 dioxygenase directly led to dihydroxybiphenyls by dehalogenation. The 'upper' pathway converted the majority of mono- and dichlorinated biphenyls into chlorobenzoates. However, more highly chlorinated PCBs were incompletely degraded in most instances. Identification of dead-end metabolites indicated that, depending on the congener, different reactions of this metabolic route limit the degradation of PCBs.