CHARACTERIZATION OF BIPHENYL CATABOLIC GENES OF GRAM-POSITIVE POLYCHLORINATED BIPHENYL DEGRADER RHODOCOCCUS SP STRAIN RHA1

Rhodococcus sp, strain RHA1 is a gram-positive polychlorinated bipheny l (PCB) degrader which can degrade 10 ppm of PCB48 (equivalent to Aroc lor1248), including tri-, tetra-, and pentachlorobiphenyls, in a few d ays. 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 i nitial three steps of biphenyl degradation. The order of bph genes in RHA1 is bphA1A2A3A4-bphC-bphB. This gene order differs from that of ot her PCB degraders reported previously. The amino acid sequences deduce d from the RHA1 bph genes have a higher degree of homology with the to d genes from Pseudomonas putida F1 (49 to 79%) than with the bph genes of Pseudomonas sp. strains KF707 and KKS102 (30 to 65%). In Escherich ia coli, bphA gene activity was not observed even when expression vect ors were used. The activities of bphB and bphC, however, were confirme d by observing the transformation of biphenyl to a meta-cleavage compo und with the aid of benzene dioxygenase activity that complemented the bphA gene activity (S. Irie, S. Doi, T. Yorifuji, M. Takagi, and K. Y ano, J. Bacteriol. 169:5174-5179, 1987). The expected products of the cloned bph genes, except bphA3, were observed in E. call in an in vitr o transcription-translation system. insertion mutations of bphA1 and b phC of Rhodococcus sp. strain RHA1 were constructed by gene replacemen t with cloned gene fragments. The bphA1 and bphC insertion mutants los t the ability to grow on biphenyl, demonstrating that the cloned bph g enes are essential for biphenyl catabolism in this strain.