2 NEARLY IDENTICAL AROMATIC COMPOUND HYDROLASE GENES IN A STRONG POLYCHLORINATED BIPHENYL DEGRADER, RHODOCOCCUS SP. STRAIN RHA1

The two 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD) hydrolase genes, etbD 1 and etbD2, were cloned from a strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, and their nucleotide sequences were determined. The etbD2 gene was located in the vicinity of bphA ge ne homologs and encoded an enzyme whose amino-terminal sequence was ve ry similar to the amino-terminal sequence of the HOHD hydrolase which was purified from RHA1, Using the etbD2 gene fragment as a probe, we c loned the etbD1 gene encoding the purified HOHD hydrolase by colony hy bridization. Both genes encode a product having 274 amino acid residue s and containing the nucleophile motif conserved in alpha/beta hydrola se fold enzymes. The deduced amino acid sequences mere quite similar t o the amino acid sequences of the products of the single-ring aromatic hydrolase genes, such as dmpD, cumD, todF, and xylF, and not very sim ilar to the amino acid sequences of the products of bphD genes from PC B degraders, including RHA1. The two HOHD hydrolase genes and the RHA1 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HPDA) hydrolase gene, bphD , were expressed in Escherichica coli, and their relative enzymatic ac tivities were examined. The product of bphD was very specific to HPDA, and the products of etbD1 and etbD2 were specific to HOHD. ALE of the gene products exhibited poor activities against the meta-cleavage pro duct of catechol. These results agreed with the results obtained for B phD and EtbD1 hydrolases purified from RHA1. The three hydrolase genes exhibited similar induction patterns both in an RNA slot blot hybridi zation analysis and in a reporter gene assay when a promoter probe vec tor was used. They were induced by biphenyl, ethylbenzene, benzene, to luene, and ortho-xylene. Strain RCD1, are RHA1 mutant strain lacking b oth the bphD gene and the etbD2 gene, grew well on ethylbenzene. This result suggested that the etbD1 gene product is involved in the meta-c leavage metabolic pathway of ethylbenzene.