Molecular cloning and characterization of pentachlorophenol-degrading monooxygenase genes of Pseudomonas sp. from the chemostat.
Pseudomonas sp. strain IST 103 (PCP103) capable of utilizing pentachlorophenol (PCP) was determined by utilization of a carbon source and release of the hydroxylating enzyme PCP-4 monooxygenase. The metabolites were extracted from the culture medium and analyzed by high-performance liquid chromatography. The enzyme purified to apparent homogeneity from an extract of PCP-grown cells indicated that a fraction of DEAE-cellulose ion exchange chromatography of molecular size of 30,000 kDa determined by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis was responsible for dechlorination of PCP. The plasmid isolated from the bacterium was subjected to Shotgun cloning by restriction digestion by BamHI, HindIII, and SalI, ligated to pUC19 vector, and transformed into Escherichia coli XLBlue1alpha. The recombinant clones having higher potentiality to degrade PCP were selected by utilization of a carbon source and release of intermediary metabolites during degradation of PCP as the sole source of carbon and energy. The recombinant clones, which contained an insert of 3.0 kb of SalI and HindIII sites, were sequenced and compared with gene sequences deposited in GenBank by BLAST search; this indicated homology with the thdf gene of monooxygenase of thiophene and furan. Southern blot analysis performed by developing gene probes indicated the presence of the PCP monooxygenase gene in plasmids of the bacterium.
Thakur
Indu Shekhar
IS
Department of Environmental Sciences, G. B. Pant University of Agriculture and Technology, Pantnagar, Uttaranchal, 263145, India. isthakur@hotmail.com
Verma
Praveen
P
Upadhayaya
Kailash
K
D001483:Base Sequence; D001673:Biodegradation, Environmental; D015139:Blotting, Southern; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010416:Pentachlorophenol / Q000378:metabolism*; D011549:Pseudomonas; D017422:Sequence Analysis, DNA
D010416:Pentachlorophenol; D006899:Mixed Function Oxygenases; C069781:pentachlorophenol monooxygenase