Identification and localization of 3-phenylcatechol dioxygenase and 2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoate hydrolase genes of pseudomonas putida and expression in escherichia coli

Journal Article · Sun Apr 01 04:00:00 EDT 1990 · Applied and Environmental Microbiology; (USA)

OSTI ID:6605189

Khan, A A; Walia, S K ^[1]

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1. Oakland Univ., Rochester, MI (USA)

The bphC and bphD genes of Pseudomonas putida involved in the catabolism of polychlorinated biphenyls or biphenyl were identified, localized, and studied for expression in Escherichia coli. This was achieved by cloning a 2.4-kilobase (kb) DNA fragment of recombinant cosmid pOH101 into HindIII site of pUC plasmids downstream of a lacZ promoter and measuring the enzyme activities of 3-phenylcatechol dioxygenase (3-PDase; a product of bphC) and the meta-cleavage product 2-hydroxy-6-phenylhexa-2,4-dienoate hydrolase (a product of bphD). The amount of 3-PDase produced in E. coli was about 20 times higher than that of the enzyme produced by the parent, P. putida. Determination of expression of the bphC and bphD genes through their own promoter sequences or by using the lacZ promoter of pUC plasmids was done by cloning the DNA that encodes bphC and bphD genes in a HindIII site of a promoter selection vector (pKK232-8) upstream of the gene for chloramphenicol acetyltransferase (CAT). The recombinant plasmid (pAW787) constructed by the inserting the 2.4-kb DNA in pKK232-8 expressed both 3-PDase and CAT activities. Another hybrid construct (pAW786) in which the DNA insert was cloned in the opposite orientation lacked CAT activity but produced normal amounts of 3-PDase activity. On the basis of these results, we suggest that the bphC and bphD genes were expressed by using promoter sequences that are independent of the promoter that expresses CAT activity in E. coli. The locations of the bphC and bphD genes were determined by insertional inactivation of the open reading frames of structural genes bphC and bphD by Tn5 mutagenesis.

• OSTI ID: 6605189
• Journal Information: Applied and Environmental Microbiology; (USA), Journal Name: Applied and Environmental Microbiology; (USA) Vol. 56:4; ISSN 0099-2240; ISSN AEMID
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
550400 -- Genetics
560300 -- Chemicals Metabolism & Toxicology
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
AROMATICS
BACTERIA
CATABOLISM
CELL CONSTITUENTS
CHLORINATED AROMATIC HYDROCARBONS
CLONING
DNA
DNA HYBRIDIZATION
DNA-CLONING
ENZYMES
ESCHERICHIA COLI
GENES
GENETIC MAPPING
HALOGENATED AROMATIC HYDROCARBONS
HYBRIDIZATION
HYDROLASES
MAPPING
METABOLISM
MICROORGANISMS
MUTAGENESIS
MUTANTS
NUCLEIC ACIDS
ORGANIC CHLORINE COMPOUNDS
ORGANIC COMPOUNDS
ORGANIC HALOGEN COMPOUNDS
OXIDOREDUCTASES
OXYGENASES
PLASMIDS
PSEUDOMONAS
RECOMBINANT DNA