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PLASMID-ENCODED PHTHALATE CATABOLIC PATHWAY IN ARTHROBACTER KEYSERI 12B: BIOTRANSFORMATIONS OF 2-SUBSTITUTED BENZOATES AND THEIR USE IN CLONING AND CHARACTERIZATION OF PHTHALATE CATABOLISM GENES AND GENE PRODUCTS
Citation:
Eaton, R W. PLASMID-ENCODED PHTHALATE CATABOLIC PATHWAY IN ARTHROBACTER KEYSERI 12B: BIOTRANSFORMATIONS OF 2-SUBSTITUTED BENZOATES AND THEIR USE IN CLONING AND CHARACTERIZATION OF PHTHALATE CATABOLISM GENES AND GENE PRODUCTS. JOURNAL OF BACTERIOLOGY 183(12):3689-3703, (2001).
Description:
Several 2-substituted benzoates (including 2-trifluoromethyl-, 2-chloro-, 2-bromo-, 2-iodo-, 2-nitro-, 2-methoxy-, and 2-acetyl-benzoates) were converted by phthalate-grown Arthrobacter keyseri 12B to the corresponding 2-substituted 3,4-dihydroxybenzoates (protocatechuates). Because these products lack a carboxyl group at the 2-position, they were not substrates for the next enzyme of the phthalate catabolic pathway, 3,4-dihydroxyphthalate 2-decarboxylase, and accumulated. When these incubations were carried out in iron-containing minimal medium, the products formed colored chelates. This chromogenic response was subsequently employed to identify recombinant E. coli strains carrying genes encoding the responsible enzymes, phthalate 3,4-dioxygenase and 3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase, from the 130 kbp plasmid pRE1 of strain 12B. Beginning with the initially cloned 8.14 kbp PstI fragment of pRE824 as probe to identify recombinant plasmids carrying overlapping fragments, a DNA segment of 33.5 kbp was cloned from pRE1 on several plasmids, mapped using restriction endonucleases, and from these, the sequence of 26,274 contiguous bp was determined. Sequenced DNA included several genetic units: tnp, pca operon, ptr operon, pehA, norA fragment, and pht operon encoding a transposon resolvase, catabolism of protocatechuate (3,4-dihydroxybenzoate), an ABC transporter, a possible phthalate ester hydrolase, a fragment of a norfloxacin resistance-like transporter, and the conversion of phthalate to protocatechuate, respectively. Activities of the eight enzymes involved in the catabolism of phthalate through protocatechuate to pyruvate and oxaloacetate were demonstrated in cells or cell extracts of recombinant E. coli strains.