Multiplicity of 2,3-Dihydroxybiphenyl Dioxygenase Genes in the Gram-positive Polychlorinated Biphenyl Degrading BacteriumRhodococcus rhodochrousK37

Abstract

Rhodococcus rhodochrous K37, a Gram-positive bacterium grown under alkaline conditions, was isolated for its ability to metabolize PCBs. Analysis revealed that it has eight genes encoding extradiol dioxygenase, which has 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, and these genes were designated bphC1 to bphC8. According to the classification of extradiol dioxygenases [Eltis, L. D., and Bolin, J. T., J. Bacteriol., 178, 5930-5937 (1996)], BphC3 and BphC6 belong to the type II enzyme group. The other six BphCs were classified as members of the type I extradiol dioxygenase group. BphC4 and BphC8 were classified into a new subfamily of type I, family 3. Two linear plasmids, 200 kb and 270 kb in size, were found in K37, and the bphC6 and bphC8 genes were located in the 200 kb linear plasmid. Northern hybridization analysis revealed that the bphC1, bphC2, and bphC7 genes were induced in the presence of testosterone, the bphC6 gene was induced by fluorene, and the bphC8 gene was induced by biphenyl. All eight BphC products exhibited much higher substrate activity for 2,3-dihydroxybiphenyl than for catechol, 3-methylcatechol, or 4-methylcatechol.

Polychlorinated Biphenyl/Biphenyl Degrading Gene Clusters inRhodococcussp. K37, HA99, and TA431 Are Different from Well-KnownbphGene Clusters of Rhodococci

“…Furthermore, only the bphC8 K37 gene was induced by biphenyl, and the K m value of the 2,3-dihydroxybihenyl dioxygenase activity for the BphC8 K37 product was much lower for the seven other BphC K37 products. 14) In this study, we confirmed that the bphC8 K37 gene is essential for degradation of biphenyl in K37 and analyzed the bph gene cluster. We found that the order of bph genes was different from those in other Rhodococci.…”

“…This band was transferred to a polyvinylidene difluoride membrane, and its N-terminal amino acid sequence was determined to be NH 2 -MVKSLAYM-GVTSPALAE, which is the same as the deduced amino acid sequence of BphC8 K37 in strain K37. 14) Isolation and analysis of a bph gene cluster from R. rhodochrous K37 A 3.0-kb ApaI-digested fragment including bphC8 K37 has been cloned (pKAC8). 14) In addition, we cloned the regions upstream and downstream of pKAC8 and generated a physical and genetic map (Fig.…”

“…14) Isolation and analysis of a bph gene cluster from R. rhodochrous K37 A 3.0-kb ApaI-digested fragment including bphC8 K37 has been cloned (pKAC8). 14) In addition, we cloned the regions upstream and downstream of pKAC8 and generated a physical and genetic map (Fig. 2).…”

“…14) To determine whether bphA1 K37 is induced in biphenyl containing medium, Northern hybridization was performed using the PCR amplified bphA1 K37 gene as a probe. Total RNA was prepared from K37 cells after a 5-h incubation on a medium containing biphenyl, succinate, naphthalene, fluorene, benzoate, phenol, cumene, phenanthrene, testosterone, cabazole, or dibenzofuran.…”

“…Northern hybridization was done according to the previously reported method. 14) Electrophoretically purified PCR products of bphA1 K37 were used in the preparation of DIG-labeled probe.…”