Rhodococcus sp. strain RHA1 is a gram-positive polychlorinated biphenyl (PCB) degrader which can degrade 10 ppm of PCB48 (equivalent to Aroclor1248), including tri-, tetra-, and pentachlorobiphenyls, in a few days. We isolated the 7.6-kb EcoRI-BamHI fragment carrying the biphenyl catabolic genes of RHA1 and determined their nucleotide sequence. On the basis of deduced amino acid sequence homology, we identified six bph genes, bphA1A2A3A4, bphB, and bphC, that are responsible for the initial three steps of biphenyl degradation. The order of bph genes in RHA1 is bphA1A2A3A4-bphC-bphB. This gene order differs from that of other PCB degraders reported previously. The amino acid sequences deduced from the RHA1 bph genes have a higher degree of homology with the tod genes from Pseudomonas putida F1 (49 to 79%) than with the bph genes of Pseudomonas sp. strains KF707 and KKS102 (30 to 65%). In Escherichia coli, bphA gene activity was not observed even when expression vectors were used. The activities of bphB and bphC, however, were confirmed by observing the transformation of biphenyl to a meta-cleavage compound with the aid of benzene dioxygenase activity that complemented the bphA gene activity (S. Irie, S. Doi, T. Yorifuji, M. Takagi, and K. Yano, J. Bacteriol. 169:5174-5179, 1987). The expected products of the cloned bph genes, except bphA3, were observed in E. coli in an in vitro transcription-translation system. Insertion mutations of bphA1 and bphC of Rhodococcus sp. strain RHA1 were constructed by gene replacement with cloned gene fragments. The bphA1 and bphC insertion mutants lost the ability to grow on biphenyl, demonstrating that the cloned bph genes are essential for biphenyl catabolism in this strain.

We have characterized a biphenyl degrader, Rhodococcus sp. strain RHA1. Biphenyl-grown cells of strain RHA1 efficiently transformed 45 components in the 62 major peaks of a polychlorinated biphenyl (PCB) mixture of Kanechlors 200, 300, 400, and 500 within 3 days, which includes mono-to octachlorobiphenyls. Among the intermediate metabolites of PCB transformation, di-and trichlorobenzoic acids were identified. The gradual decrease of these chlorobenzoic acids during incubation indicated that these chlorobenzoic acids would also be degraded by this strain. The effect of the position of chlorine substitution was determined by using PCB mixtures that have chlorine substitutions mainly at either the ortho or the meta position. This strain transformed both types of congeners, and strong PCB transformation activity of RHA1 was indicated. RHA1 accumulated 4-chlorobenzoic acid temporally during the transformation of 4-chlorobiphenyl. The release of most chloride in the course of 2,2-dichlorobiphenyl degradation was observed. These results suggested that RHA1 would break down at least some PCB congeners into smaller molecules to a considerable extent.

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