Molecular and biochemical analysis of phthalate and terephthalate degradation byRhodococcussp. strain DK17

Abstract

Alkylbenzene-degrading Rhodococcus sp. strain DK17 is able to utilize phthalate and terephthalate as growth substrates. The genes encoding the transformation of phthalate and terephthalate to protocatechuate are organized as two separate operons, located 6.7kb away from each other. Interestingly, both the phthalate and terephthalate operons are induced in response to terephthalate while expression of the terephthalate genes is undetectable in phthalate-grown cells. In addition to two known plasmids (380-kb pDK1 and 330-kb pDK2), a third megaplasmid (750-kb pDK3) was newly identified in DK17. The phthalate and terephthalate operons are duplicated and are present on both pDK2 and pDK3. RT-PCR experiments, coupled with sequence analysis, suggest that phthalate and terephthalate degradation in DK17 proceeds through oxygenation at carbons 3 and 4 and at carbons 1 and 2 to form 3,4-dihydro-3,4-dihydroxyphthalate and 1,2-dihydro-1,2-dihydroxyterephthalate, respectively. The 3,4-dihydroxyphthalate pathway was further corroborated through colorometric tests. Apparently, the two dihydrodiol metabolites are subsequently dehydrogenated and decarboxylated to form protocatechuate, which is further degraded by a protocatechuate 3,4-dioxygenase as confirmed by a ring-cleavage enzyme assay.

Bacterial degradation of phthalate isomers and their esters

“…Presence of phthalates in the medium induces proteins that are involved in its transport and degradation [16,27,40,87,104]. Longer chain phthalate esters are diffi cult to degrade [102].…”

“…Metabolism of terephthalate is initiated by double hydroxylation of the ring at positions 1 and 2, catalyzed by terephthalate 1,2-dioxygenase to yield 2-hydro-1,2-dihydroxy terephthalic acid, which is further metabolized to 3,4-dihydroxybenzoate [16,88,99,104] (Fig. 3).…”

Benzoate Catabolite Repression of the Phthalate Degradation Pathway in Rhodococcus sp. Strain DK17

“…strain DK17 was originally isolated for the ability to grow on o-xylene and has the capability to utilize such aromatic compounds as benzene, alkylbenzenes (toluene, ethylbenzene, isopropylbenzene, and n-propyl-to n-hexylbenzenes), phenol, and phthalates as sole carbon and energy sources (2,14,15,16,17). This catabolic versatility led us to investigate the adaptability of DK17 to the presence of multiple aromatic hydrocarbons as carbon and energy sources.…”

“…Subsequent cloning and sequencing of the PCR product revealed 99% identity with the nucleotide sequence of benA1 of RHA1 (18). Primer sequences for phthalate dioxygenase were 5Ј-ATGATCCCGGC GCACATC-3Ј (ophA1 forward) and 5Ј-TCATGCCAGCACC GCCCC-3Ј (ophA1 reverse) and were based on our previous work on the induction of the DK17 phthalate operon (2). The thermocycler program used for the RT-PCRs was as follows:…”

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

“…strain DK17 is also able to utilize phthalate as a growth substrate via the Gram-positive pathway [6,7]. DK17 possesses three megaplasmids (380 kb pDK1, 330 kb pDK2, and 750 kb pDK3), and the phthalate operons in DK17 are duplicated and are present on both pDK2 and pDK3 [6,8].…”