Characterization of the Isophthalate Degradation Genes of Comamonas sp. Strain E6

Abstract

The isophthalate (IPA) degradation gene cluster (iphACBDR) responsible for the conversion of IPA into protocatechuate (PCA) was isolated from Comamonas sp. strain E6, which utilizes phthalate isomers as sole carbon and energy sources via the PCA 4,5-cleavage pathway. Based on amino acid sequence similarity, the iphA, iphC, iphB, iphD, and iphR genes were predicted to code for an oxygenase component of IPA dioxygenase (IPADO), a periplasmic IPA binding receptor, a 1,2-dihydroxy-3,5-cyclohexadiene-1,5-dicarboxylate (1,5-DCD) dehydrogenase, a reductase component of IPADO, and an IclR-type transcriptional regulator, respectively. The iphACBDR genes constitute a single transcriptional unit, and transcription of the iph catabolic operon was induced during growth of E6 on IPA. The iphA, iphD, and iphB genes were expressed in Escherichia coli. Crude IphA and IphD converted IPA in the presence of NADPH into a product which was transformed to PCA by IphB. These results suggested that IPADO is a two-component dioxygenase that consists of a terminal oxygenase component (IphA) and a reductase component (IphD) and that iphB encodes the 1,5-DCD dehydrogenase. Disruption of iphA and iphB resulted in complete loss of growth of E6 on IPA. Inactivation of iphD significantly affected growth on IPA, and the iphC mutant did not grow on IPA at neutral pH. These results indicated that the iphACBD genes are essential for the catabolism of IPA in E6. Disruption of iphR resulted in faster growth of E6 on IPA, suggesting that iphR encodes a repressor for the iph catabolic operon. Promoter analysis of the operon supported this notion.Phthalate isomers (o-phthalate [OPA], terephthalate [TPA], and isophthalate [IPA]) and their esters have been largely used as plasticizers. Moreover, they are considered potential starting compounds for the production of 2-pyrone-4,6-dicarboxylic acid (PDC), an intermediate metabolite in the protocatechuate (PCA) 4,5-cleavage pathway (28). PDC is a useful compound for synthesis of biodegradable and highly functional polymers, such as powerful adhesive agents (22,29,30).OPA degradation has been reported for many bacteria, including Burkholderia cepacia DBO1 (9), Mycobacterium vanbaalenii PYR-1 (43), Arthrobacter keyseri 12B (14), Terrabacter sp. DBF63 (19), Rhodococcus sp. DK17 (10), and Rhodococcus jostii RHA1 (32). Degradation of OPA is initiated by dihydroxylation of the aromatic ring by OPA 3,4-dioxygenase (10, 14), which is found mostly in Gram-positive bacteria, or by OPA 4,5-dioxygenase (9), which is found mostly in Gramnegative bacteria, which generates OPA dihydrodiols. These products are then transformed by rearomatization and decarboxylation by a dehydrogenase and a decarboxylase, respectively, yielding PCA. OPA dioxygenase from DBO1 has been purified (5). This enzyme is a multicomponent dioxygenase composed of a dioxygenase component and a reductase component. OPA dioxygenase requires Fe 2ϩ for activity and shows narrow substrate specificity with OPA. Microbial degradation of TPA has been characteriz...

γ-Resorcylate Catabolic-Pathway Genes in the Soil Actinomycete Rhodococcus jostii RHA1

“…S6 in the supplemental material). At low pH, GRA most likely exists in the form of undissociated molecules and is transported through the membrane by passive diffusion, similar to other aromatic acids (24,(26)(27)(28). This feature might underlie the growth of the tsdT mutant on GRA at low pH.…”

“…S6 in the supplemental material). Aromatic acids in their undissociated form enter cells by passive diffusion at lower pH (24,(26)(27)(28); therefore, the growth of DTT cells was examined at low pH. When the pH of the medium was adjusted to 6.0 or 5.5, the growth retardation of DTT was partially suppressed.…”

Three-Component O -Demethylase System Essential for Catabolism of a Lignin-Derived Biphenyl Compound in Sphingobium sp. Strain SYK-6

“…A mixture of purified LigXa (20 g of protein), LigXc (20 g of protein), and LigXd (20 g of protein) was incubated with 100 M DDVA in FE22 buffer (pH 6.5) in the presence and absence of NADH at 30°C. Portions of the reaction mixtures were periodically collected and analyzed by high-performance liquid chromatography (HPLC; Acquity UPLC system; Waters) coupled with an Acquity TQ detector (Waters) using a TSKgel ODS-140HTP column (2.1 by 100 mm; Tosoh) as described previously (52). The mobile phase of the HPLC system was composed of a mixture of water (90%) and acetonitrile (10%) containing formic acid (0.1%) at a flow rate of 0.5 ml/min.…”

Transcriptional Regulation of the Terephthalate Catabolism Operon in Comamonas sp. Strain E6

“…strain E6, which is able to grow on TPA, isophthalate, and o-phthalate as the sole source of carbon and energy; and these compounds are degraded via the PCA 4,5-cleavage pathway (9,30). We reported on the characterization of two almost identical TPA catabolism gene clusters (Ͼ94% identity) of E6, tphR I C I A2 I A3 I B I A1 I (GenBank accession no.…”

“…The phthalate isomers terephthalate (TPA), isophthalate, and o-phthalate are widely used as plasticizers and are degraded by microbial catabolic pathways via protocatechuate (PCA) (8,9,26,30). The degradation of PCA is catalyzed by one of the following three aromatic ring cleavage pathways: the PCA 2,3-cleavage (17), the PCA 3,4-cleavage (14), or the PCA 4,5-cleavage (19,20) pathway.…”