Cloning of bacterial genes specifying degradation of 4-chlorobiphenyl from Pseudomonas putida OU83.
Genes capable of 4-chlorobiphenyl (4-CBP) degradation were cloned from 4-CBP-degrading Pseudomonas putida OU83 by using a genomic library which was constructed in the broad-host-range cosmid vector pCP13. P. putida AC812 containing chimeric cosmid-expressing enzymes involved in the 4-CBP degradation pathway were identified by detecting 3-phenylcatechol dioxygenase activity (3-PDA). Chimeric cosmid clones pOH83, pOH84, pOH85, pOH87, and pOH88 positive for 3-PDA grew in synthetic basal medium containing 4-CBP (5 mM) as a carbon source. Restriction digestion analysis of recombinant cosmids showed DNA inserts ranging from 6 to 30 kilobase pairs. Southern hybridization data revealed that the cloned DNA inserts originated from strain OU83. Gas chromatography-mass spectrometry analysis of the metabolites of P. putida AC812(pOH88) incubated with 4-CBP and 4'-chloro-3-phenylcatechol showed the formation of 4-chlorobenzoic acid and benzoic acid. These results demonstrate that the cloned DNA fragments contain genes encoding for chlorobiphenyl dioxygenase (cbpA), dihydrodiol dehydrogenase (cbpB), 4'-chloro-3-phenylcatechol dioxygenase (cbpC), a meta-cleavage compound (a chloro derivative of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate) hydrolase (cbpD), and a new dechlorinating activity (dcpE). The location of the cbpC gene specifying 3-PDA was determined by subcloning an EcoRI DNA fragment (9.8 kilobase pairs) of pOH88 in plasmid vector pUC19. The cloned gene encoding 3-PDA was expressed in Escherichia coli HB101 and had substrate specificity only for 3-phenylcatechol and 4'-chloro-3-phenylcatechol.
Khan
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Department of Biological Sciences, Oakland University, Rochester, Michigan 48309-4401.
Walia
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D001673:Biodegradation, Environmental; D001713:Biphenyl Compounds / Q000378:metabolism*; D003001:Cloning, Molecular; D003360:Cosmids; D004269:DNA, Bacterial / Q000235:genetics*; D015247:Deoxyribonuclease HindIII; D049308:Dioxygenases; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D010105:Oxygenases / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D001713:Biphenyl Compounds; D004269:DNA, Bacterial; D010105:Oxygenases; C058196:3-phenylcatechol dioxygenase; D049308:Dioxygenases; D015247:Deoxyribonuclease HindIII; C006983:4-chlorobiphenyl