IDENTIFICATION OF AN ALTERNATIVE 2,3-DIHYDROXYBIPHENYL 1,2-DIOXYGENASE IN RHODOCOCCUS SP STRAIN RHA1 AND CLONING OF THE GENE

Gram-positive Rhodococcus sp. strain RHA1 possesses strong polychlorin ated biphenyl-degrading capabilities, An RHA1 bphC gene mutant, strain RDC1, had been previously constructed (E. Masai, A. Yamada, J. M. Hea ly, T. Hatta, K. Kimbara, M. Fukuda, and K. Yano, Appl. Environ. Micro biol. 61:2079-2085, 1995). An alternative 2,3-dihydroxybiphenyl 1,2-di oxygenase (2,3-DHBD), designated EtbC, was identified in RDC1 cells gr own on ethylbenzene. EtbC contained the broadest substrate specificity of any meta cleavage dioxygenase identified in a Rhodococcus strain t o date, including RHA1 BphC. EtbC was purified to near homogeneity fro m RDC1 cells grown on ethylbenzene, and a 58-amino-acid NH2-terminal s equence was determined, The NH2-terminal amino acid sequence was used for the identification of the etbC gene from an RDC1 chromosomal DNA 2 ,3-DHBD expression library. The etbC gene was successfully cloned, and we report here the determination of its nucleotide sequence. The subs trate specificity patterns of cell extract and native nondenaturing po lyacrylamide gel electrophoresis analysis identified the coexpression of two 2,3-DHBDs (BphC and EtbC) in RHA1 cells grown on either bipheny l or ethylbenzene. The possible implication of coexpressed BphC extrad iol dioxygenases in the strong polychlorinated-biphenyl degradation ac tivity of RHA1 nas suggested.