Multiplicity of aromatic ring hydroxylation dioxygenase genes in a strong PCB degrader, Rhodococcus sp. strain RHA1 demonstrated by denaturing gradient gel electrophoresis

To address the multiplicity of aromatic ring hydroxylation dioxygenases, we used PCR amplification and denaturing gradient gel electrophoresis (DGGE). The amplified DNA fragments separated into five bands, A to E. Southern hybridization analysis of RHA1 total DNA using the probes for each band showed that band C originated from a couple of homologous genes. The nucleotide sequences of the bands showed that bands A, C, and E would be parts of new dioxygenase genes in RHA1. That of band B agreed with the bphA1 gene, which was characterized previously. That of band D did not correspond to any known gene sequences. The regions including the entire open reading frames (ORFs) were cloned and sequenced. The nucleotide sequences of ORFs suggested that the genes of bands A, C, and E may respectively encode benzoate, biphenyl, and polyhydrocarbon dioxygenases. Northern hybridization indicated the induction of the gene of band A by benzoate and biphenyl, and that of the gene of band C by biphenyl and ethylbenzene, supporting the above notions. The gene of band E was not induced by any of these substrates. Thus the combination of DGGE and Southern hybridization enable us to address the multiplicity of the ring hydroxylation dioxygenase genes and to isolate some of them.