A gene cluster encoding biphenyl-and chlorobiphenyl-degrading enzymes was cloned from a soil pseudomonad into Pseudomonas aeruginosa PAO1161. Chromosomal DNA from polychlorinated biphenyldegrading Pseudomonas pseudoalcaligenes KF707 was digested with restriction endonuclease XhoI and cloned into the unique XhoI site of broad-host-range plasmid pKF330. Of 8,000 transformants tested, only 1, containing the chimeric plasmid pMFB1, rendered the host cell able to convert biphenyls and chlorobiphenyls to ring meta cleavage compounds via dihydrodiols and dihydroxy compounds. The chimeric plasmid contained a 7.9-kilobase XhoI insert. Subcloning experiments revealed that the genes bphA (encoding biphenyl dioxygenase), bphB (encoding dihydrodiol dehydrogenase), and bphC (encoding 2,3-dihydroxybiphenyl dioxygenase) were coded for by the 7.9-kilobase fragment. The gene order was bphA-bphB-bphC. The hydrolase activity, which converted the intermediate meta cleavage compounds to the final product, chlorobenzoic acids, and was encoded by a putative bphD gene, was missing from the cloned 7.9-kilobase fragment.

The bphC gene encoding 2,3-dihydroxybiphenyl dioxygenase was cloned from biphenyl-degrading and chlorinated biphenyl-degrading Pseudomonas paucimobilis Q1, and its complete nucleotide sequence was determined. The DNA-derived protein sequence provides the primary structure of 298 amino acids. Polyclonal antibodies raised against this protein from P. paucimobilis Q1 failed to cross-react with the previously isolated 2,3-dihydroxybiphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 [Furukawa, K., & Arimura, N. (1987) J. Bacteriol. 169, 924-927. Furukawa, K., Arimura, N., & Miyazaki, T. (1987) J. Bacteriol. 169, 427-429], despite the close similarities of these proteins in terms of their native as well as subunit molecular weights, cofactor, and enzymatic activities. The sequence homology of the 2,3-dihydroxybiphenyl dioxygenase from the two different sources is examined.

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Our results suggest the administration of FG could improve the FNE in humans. The improvement may be related to an anxiolytic effect of FG which acts via GABAergic modification.

2,3-Dihydroxybiphenyl dioxygenase, which catalyzes ring metacleavage of 2,3-dihydroxybiphenyl, is encoded by the bphC gene of Pseudomonas pseudoalcaligenes KF707 (K. Furukawa and T. Miyazaki, J. Bacteriol. 166:392-398, 1986). We determined the nucleotide sequence of a DNA fragment of 2,040 base pairs which included the bphC gene. The fragment included one open reading frame of 912 base pairs to accommodate the enzyme. The predicted processed amino acid sequence of the enzyme subunit consisted of 302 residues, and its 12 NH2-terminal residues were in perfect agreement with those determined for the enzyme. Approximately 10 base pairs upstream from the initiation codon for 2,3-dihydroxybiphenyl dioxygenase, there was a base sequence complementary to the 3' end of the 16S rRNA from Pseudomonas aeruginosa. There was no promoterlike sequence in the region upstream of the bphC gene, but another long open reading frame was present. A putative bphD gene encoding a metacleavage compound-hydrolyzing enzyme was suggested in the region downstream of the bphC gene.