The bphDEF meta-cleavage pathway genes involved in biphenyl/polychlorinated biphenyl degradation are located on a linear plasmid and separated from the initial bphACB genes in Rhodococcus sp. strain RHA1

• Published in: Gene Vol. 187; no. 1; pp. 141 - 149
• Main Authors: Masai, Eiji, Sugiyama, Katsumi, Iwashita, Naoko, Shimizu, Satoru, Hauschild, James E, Hatta, Takashi, Kimbara, Kazuhide, Yano, Keiji, Fukuda, Masao
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 10.03.1997
• Subjects: 2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase; 2-Hydroxypenta-2,4-dienoate hydratase; 4-Hydroxy-2-oxovalerate aldolase; Benzene Derivatives - metabolism; Biodegradation; Biodegradation, Environmental; Biphenyl Compounds - metabolism; Blotting, Southern; Cloning, Molecular; Dioxygenases; Electrophoresis, Gel, Pulsed-Field; Electrophoresis, Polyacrylamide Gel; Escherichia coli - genetics; Gas Chromatography-Mass Spectrometry; Gene Expression Regulation, Bacterial; Genes, Bacterial; Gram-positive bacterium; Homoprotocatechuic acid; Hydro-Lyases - chemistry; Hydro-Lyases - genetics; Hydro-Lyases - metabolism; Hydrolases - chemistry; Hydrolases - genetics; Hydrolases - metabolism; Oxo-Acid-Lyases - chemistry; Oxo-Acid-Lyases - genetics; Oxo-Acid-Lyases - metabolism; Oxygenases - chemistry; Oxygenases - genetics; Oxygenases - metabolism; Plasmids; Polychlorinated Biphenyls - metabolism; Rhodococcus; Rhodococcus - enzymology; Rhodococcus - genetics; Rhodococcus - metabolism; Sequence Alignment; aa, amino acid(s); Tod, polypeptide(s) of toluene catabolic enzyme(s); tod, gene(s) encoding Tod; kb, kilobase(s) or 1000 bp; Xyl, polypeptide(s) of toluene and xylene catabolic enzyme(s); PCB, polychlorinated biphenyl; bph, gene(s) encoding Bph; SDS, sodium dodecyl sulfate; denotes plasmid-carrier state; Homoprotocatechuic acid; GC-MS, gas chromatography-mass spectrometry; etbC, 2,3-DHBP dioxygenase gene; hpc, gene(s) encoding Hpc; Gram-positive bacterium; HOPD, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate; Biodegradation; xyl, gene(s) encoding Xyl; 4-Hydroxy-2-oxovalerate aldolase; PAGE, PA-gel electrophoresis; Bph, polypeptide(s) of biphenyl/PCB catabolic enzyme(s); ORF, open reading frame; 2,3-DHBP, 2,3-dihydroxybiphenyl; dmp, gene(s) encoding Dmp; 2-Hydroxypenta-2,4-dienoate hydratase; 2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase; Hpc, polypeptide(s) of homoprotocatechuate catabolic enzyme(s); nt, nucleotide(s); PFGE, pulsed field gel electrophoresis; Dmp, polypeptide(s) of (dimethyl) phenol catabolic enzyme(s); PA, polyacrylamide; IPTG, isopropyl β- d-thiogalactopyranoside
• DOI: 10.1016/S0378-1119(96)00748-2
• ISSN: 0378-1119

The bphACB genes responsible for the initial oxidation of the aromatic ring of biphenyl/polychlorinated biphenyls (PCB) to meta-cleavage product in Rhodococcus sp. RHA1 have been characterized. We cloned the 6.1 kb EcoRI fragment containing another extradiol dioxygenase gene ( etbC) which was induced during the growth on ethylbenzene. The bphD, bphE and bphF encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase, 2-hydroxypenta-2,4-dienoate hydratase and 4-hydroxy-2-oxovalerate aldolase, respectively, were found downstream of etbC. The deduced amino acid (aa) sequence of RHA1 bphD and bphE had 27–33% and 32–38% identity, respectively, with those of the corresponding genes in Pseudomonas. BphE and BphF are closely related to the corresponding homoprotocatechuate meta-cleavage pathway enzymes of Escherichia coli C. The bphD and bphF were expressed in E. coli and the BphD activity was detected. The etbCbphDEF genes were transcribed in biphenyl and ethylbenzene growing cells. Pulsed field gel electrophoresis (PFGE) analysis indicated that RHA1 contains three large linear plasmids. Southern blot analysis indicated that the meta-cleavage pathway for biphenyl/PCB catabolism in RHA1 is directed by the 390 kb plasmid borne bphDEF genes located separately from bphACB gene cluster on the 1100 kb plasmid.