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CLONING AND CHARACTERIZATION OF THE PHTHALATE CATABOLISM REGION OF PRE1 OF ARTHROBACTER KEYSERI 12B
Eaton, R W. CLONING AND CHARACTERIZATION OF THE PHTHALATE CATABOLISM REGION OF PRE1 OF ARTHROBACTER KEYSERI 12B. Presented at 2001 Annual Meeting of the American Society for Microbiology, Orlando, FL, 20-24 May 2001.
o-Phthalate (benzene-1,2-dicarboxylate) is a central intermediate in the bacterial degradation of phthalate ester plasticizers as well as of a number of fused-ring polycyclic aromatic hydrocarbons found in fossil fuels. In Arthrobacter keyseri 12B, the genes encoding catabolism of phthalate are located on the 130 kbp plasmid, pRE1. The first two enzymes of the phthalate catabolic pathway, phthalate 3,4-dioxygenase and 3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase, convert the substrate analogue, 2-trifluoromethylbenzoate, to 2-trifluoromethylprotocatechuate which accumulates and forms a red chelate with iron. By taking advantage of this chromogenic biotransformation, a recombinant E. coli strain carrying the genes encoding these enzymes on a 8.14 kbp PstI fragment was identified. Beginning with the initially cloned PstI fragment 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 plasmids, the sequence of 26,274 contiguous bp was determined. Sequenced DNA included several genetic units, tnpXR, pcaDECABF and pcaR, ptrDABC, pehA, norA, and phtBAaAbAcAdCR encoding, respectively, a transposon resolvase, the catabolism of protocatechuate (3,4-dihydroxybenzoate), an ABC transporter, a possile phthalate ester hydrolase, a fragment of a norfloxacin resistance-like transporter, and the conversion of phthalate to protocatechuate. Activities of all 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.