Cloning of a gene cluster encoding biphenyl and chlorobiphenyl degradation in Pseudomonas pseudoalcaligenes

Abstract

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.

Versatile Transcription of Biphenyl Catabolic bphOperon in Pseudomonas pseudoalcaligenes KF707

“…BphC (23DHBP dioxygenase) and BphD (HOPD hydrolase) activities were assayed as described previously (1). One unit of BphC activity was defined as the amount of enzyme to produce 1 mol of biphenyl ring meta-cleavage compound (HOPD)/min.…”

“…The biphenyl-utilizing strain P. pseudoalcaligenes KF707 was grown in basal salt medium (BSM) supplemented with 0.2% (w/v) biphenyl as a sole source of carbon as described previously (1). Biphenyl-utilizing defective derivatives of KF707 such as KF730 (bphA1::Tn5-B21), KF748 (bphB::Tn5-B21), and KF744 (bphC::Tn5-B21) were previously constructed (20), and KF7095 (orf0::Tc R ) was constructed in this study.…”

“…The bphB gene encodes a biphenyl dihydrodiol dehydrogenase. The bphC gene encodes a 2,3-dihydroxybiphenyl (23DHBP) 1 dioxygenase that is involved in the ring meta-cleavage. The bphD gene encodes a 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase to obtain benzoate and 2-hydroxypenta-2,4-dienoate.…”

Aerobic degradation of polychlorinated biphenyls by Alcaligenes sp. JB1: metabolites and enzymes

“…Catechol 2,3-dioxygenase was determined by the absorbency of the meta cleavage product at A=375-380 nm, as described by Nozaki (1970). 2,3-Dihydroxybiphenyl 1,2-dioxygenase was measured at ),=434 nm according to Furukawa & Miyazaki (1986). These activities are expressed as ~tmoles cleavage-product formed per gram of cell-protein per minute (~tmol/g pro • min).…”

Bioconversion of 2-hydroxy-6-oxo-6-(4'-chlorophenyl) hexa-2,4-dienoic acid, the meta-cleavage product of 4-chlorobiphenyl

“…However, many bacterial strains able to degrade lower chlorinated PCBs have now been described (Furukawa & Matsumura, 1976;Bedard et al, 1986;Furukawa & Miyazaki, 1986;Khan et al, 1988;Parsons et al, 1988 ;Mass6 et al, 1984;Ahmad et al, 1990). The postulated steps for the conversion of such PCBs into the corresponding chlorobenzoic acids are illustrated in Fig.…”