Plasmid-Encoded Phthalate Catabolic Pathway in Arthrobacter keyseri 12B

Abstract

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 (formerly Micrococcus sp.) 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 used to identify recombinant Escherichia 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 a probe to identify recombinant plasmids carrying overlapping fragments, a DNA segment of 33.5 kbp was cloned from pRE1 on several plasmids and mapped using restriction endonucleases. From these plasmids, the sequence of 26,274 contiguous bp was determined. Sequenced DNA included several genetic units: tnpR, pcm operon, ptr genes, pehA, norA fragment, and pht operon, encoding a transposon resolvase, catabolism of protocatechuate (3,4-dihydroxybenzoate), a putative ATP-binding cassette 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.

Bacterial degradation of phthalate isomers and their esters

“…3). Based on the position of ring-hydroxylation, o-phthalate dioxygenase is either phthalate 4,5-dioxygenase, found mostly in Gram negative bacteria [26,49]; or phthalate 3,4-dioxygenase, present mostly in Gram positive bacteria [35]. However, there are some exceptions where phthalate 3,4-dioxygenase is present in Gram negative organism like Pseudomonas aeruginosa sp.…”

Protocatechuate 4,5-dioxygenase from Comamonas testosteroni T-2: biochemical and molecular properties of a new subgroup within class III of extradiol dioxygenases

“…(Noda et al 1990;Sugimoto et al 1999), Pseudomonas straminea (formerly P. ochraceae) (Maruyama et al 2004), Pseudomonas sp. K82 (Yun et al 2004), Rhodopseudomonas palustris (Larimer et al 2004) and in the Gram-positive Arthrobacter keyseri (Eaton 2001) (see below). Based on amino acid compositions, we believe that the enzyme from C. testosteroni Pt-L5 (Arciero et al 1983), from which a definitive picture of the enzyme emerged, is also very similar to PmdAB.…”

Functional Identification of OphR, an IclR Family Transcriptional Regulator Involved in the Regulation of the Phthalate Catabolic Operon in Rhodococcus sp. Strain DK17

“…In general, Gram-positive bacteria initially oxidize phthalate to 3,4-dihydro-3,4-dihydroxyphthalate, which is subsequently dehydrogenated and decarboxylated to form protocatechuate [2][3][4]. On the other hand, phthalate degradation in Gramnegative bacteria proceeds through oxygenation and dehydrogenation at carbons 4 and 5 to form 4,5-dihydroxyphthalate, followed by decarboxylation to yield protocatechuate [5].…”