Cloning and characterization of benzoate catabolic genes in the gram-positive polychlorinated biphenyl degrader Rhodococcus sp strain RHA1

dBenzoate catabolism is thought to play a key role in aerobic bacterial deg radation of biphenyl and polychlorinated biphenyls (PCBs). Benzoate catabol ic genes were cloned from a PCB degrader, Rhodococcus sp. strain RHA1, by u sing PCR amplification and temporal temperature gradient electrophoresis se paration. A nucleotide sequence determination revealed that the deduced ami no acid sequences encoded by the RHA1 benzoate catabolic genes, benABCDK, e xhibit 33 to 65% identity with those of Acinetobacter sp. strain ADP1 The g ene organization of the RRA1 benABCDK genes differs from that of ADP1. The RHA1 benABCDK region was localized on the chromosome, in contrast to the bi phenyl catabolic genes, which are located on linear plasmids. Escherichia c oli cells containing RHA1 benABCD transformed benzoate to catechol via 2-hy dro-1,2-dihydroxybenzoate. They transformed neither 2- nor 4-chlorobenzoate s but did transform 3-chlorobenzoate. The RHA1 benA gene was inactivated by insertion of a thiostrepton resistance gene. The resultant mutant strain, RBD169, neither grew on benzoate nor transformed benzoate, and it did not t ransform 3-chlorobenzoate. It did, however, exhibit diminished growth on bi phenyl and growth repression in the presence of a high concentration of bip henyl (13 mM). These results indicate that the cloned benABCD genes could p lay an essential role not only in benzoate catabolism but also in biphenyl catabolism in RHA1 Six rhodococcal benzoate degraders were found to have ho mologs of RHA1 benABC. In contrast, two rhodococcal strains that cannot tra nsform benzoate were found not to have RHA1 benABC homologs, suggesting tha t many Rhodococcus strains contain benzoate catabolic genes similar to RHA1 benABC.