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Appl Environ Microbiol. 2010 January; 76(2): 519–527.
Published online 2009 November 20. doi:  10.1128/AEM.01270-09
PMCID: PMC2805221

Characterization of the Isophthalate Degradation Genes of Comamonas sp. Strain E6[down-pointing small open triangle]

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 Gram-negative 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 Fe2+ for activity and shows narrow substrate specificity with OPA. Microbial degradation of TPA has been characterized for Delftia tsuruhatensis T7 (40), R. jostii RHA1 (21), Comamonas testosteroni T-2 (39), and Comamonas sp. E6 (37). The function of each TPA degradation gene has been characterized for Comamonas sp. E6, which utilizes OPA, TPA, and IPA as sole carbon and energy sources via the PCA 4,5-cleavage pathway. This strain degrades TPA to 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate (1,4-DCD) by using TPA 1,2-dioxygenase (TPADO), and the resulting 1,4-DCD is converted to PCA by 1,4-DCD dehydrogenase, which dehydrogenates 1,4-DCD concomitant with decarboxylation. Reconstitution of enzyme activity from the purified proteins produced in Escherichia coli demonstrated that TPADO of E6 consists of a terminal oxygenase component (TphA2A3) and a reductase component (TphA1) (16). TPADO also requires Fe2+ for activity and has narrow substrate specificity for TPA.

On the other hand, there is less information concerning the genes and enzymes involved in IPA degradation (46). In 1995, Wang et al. briefly reported isolation of the IPA degradation gene cluster, iphA2CBA1, from C. testosteroni YZW-D (48). These authors found that iphA2, iphC, iphB, and iphA1 code for an oxygenase component of IPA dioxygenase (IPADO), a transporter, a 1,2-dihydroxy-3,5-cyclohexadiene-1,5-dicarboxylate (1,5-DCD) dehydrogenase, and a reductase component of IPADO, respectively, based on sequence similarity and mutant complementation data (Fig. (Fig.1A).1A). However, the concrete role of each iph gene and the nucleotide sequences were not presented. Even though the sequence data were deposited in a database in 2005, the actual functions have not been clarified yet.

FIG. 1.
Proposed catabolic pathway for IPA in Comamonas sp. E6 and organization of the iph operon. (A) The following gene products are involved in the conversion of IPA to PCA: IphA, the oxygenase component of IPADO; IphD, the reductase component of IPADO; IphB, ...

In this study, the IPA degradation gene cluster (iphACBDR) was isolated from Comamonas sp. E6, and the enzyme activities of the gene products and the involvement of the genes in IPA catabolism were verified. To our knowledge, this is the first report to show the actual function of each iph gene.

MATERIALS AND METHODS

Strains and plasmids.

Strains and plasmids used in this study are listed in Table Table1.1. Comamonas sp. E6 was grown in three media, W minimal salt medium (33) containing 10 mM IPA, Luria-Bertani (LB) medium (36), and 0.2× LB medium (45), at 30°C. The E6 mutants were grown in LB medium or 0.2× LB medium. If necessary, 50 mg of kanamycin/liter, 50 mg of tetracycline/liter, and 30 mg of chloramphenicol/liter were added to the media. E. coli strains were grown on LB medium at 37°C. For cultures of cells carrying antibiotic resistance markers, the media for E. coli transformants were supplemented with 100 mg of ampicillin/liter, 50 mg of kanamycin/liter, 12.5 mg of tetracycline/liter, and 30 mg of chloramphenicol/liter.

TABLE 1.
Strains and plasmids used in this study

Cloning of iph genes.

The IPA-F/IPA-R degenerate primer set (Table (Table2)2) was designed based on a conserved sequence in iphA2 from C. testosteroni YZW-D (accession no. AAX18934), the putative ring-hydroxylating dioxygenase oxygenase subunit gene from Bradyrhizobium japonicum USDA 110 (BAC46203), the putative 3-chlorobenzoate 3,4-dioxygenase oxygenase subunit gene from Bordetella bronchiseptica RB50 (CAE35136), the putative ring-hydroxylating dioxygenase oxygenase subunit gene from Rhodobacterales bacterium HTCC2654 (EAQ10830), and the putative OPA 4,5-dioxygenase oxygenase subunit gene (ohpA2) from Burkholderia xenovorans LB400 (ABE33619). This primer set was used to amplify the putative IPA dioxygenase gene sequence in E6. A 465-bp PCR-amplified fragment was used in colony hybridization as a probe to isolate the IPA degradation genes from the E6 gene libraries constructed in charomid 9-36 and pUC19 containing HindIII and EcoRI digests of the E6 total DNA, respectively. Colony and Southern hybridization analyses were performed using the digoxigenin (DIG) system (Roche, Mannheim, Germany).

TABLE 2.
Primers used in this study

DNA manipulations and DNA sequencing.

DNA manipulations, including total DNA isolation, construction of deletion derivatives, and DNA sequencing, were performed as described in a previous study (1). Sequence analysis was performed using the GeneWorks program (Intelligenetics, Inc., Mountain View, CA) and the MacVector program (MacVector, Inc., Cary, NC). Homology searches were performed using the DDBJ database with the BLAST program. Multiple alignment and pairwise alignment were performed with the CLUSTAL W software and the EMBOSS alignment tool (http://www.ebi.ac.uk/emboss/align), respectively. A distance matrix and phylogenetic trees were constructed by using the neighbor-joining method (35) and were visualized with the FigTree program (version 1.2.3; http://tree.bio.ed.ac.uk/software/figtree/).

RT-PCR.

Cells of Comamonas sp. E6 were grown in W minimal salt medium containing 10 mM IPA or succinate until the absorbance of the culture at 600 nm was 1.0. Total RNA was prepared from 30 ml of culture by using ISOGEN (Nippon Gene Co., Ltd., Tokyo, Japan). In order to remove any contaminating genomic DNA, the RNA samples were incubated with 1 U of RNase-free DNase I (Takara Bio Co., Ltd., Tokyo, Japan). A cDNA was obtained by a reverse transcription (RT) reaction using ReverTra Ace (Toyobo, Osaka, Japan) and random primers. The cDNA was used as a template for subsequent PCRs with specific primers (Table (Table2),2), which amplified the boundaries of iphA-iphC-iphB-iphD-iphR and internal regions of iphA and iphR. PCR samples were electrophoresed on a 1.5% agarose gel and visualized with ethidium bromide.

Expression of iphA, iphD, and iphB in E. coli and preparation of cell extracts.

The 0.2-kb fragment carrying the 5′ region of iphA was amplified by PCR using E6 total DNA as a template along with the IphA-F/IphA-R primer set (Table (Table2).2). The 0.2-kb PCR product was inserted into pT7Blue and sequenced. The 0.2-kb NdeI-HindIII fragment of the resulting plasmid and the 1.5-kb HindIII-EcoRI fragment of pKS50F were inserted into the NdeI-EcoRI sites of pET-17b to construct pTIP17A. The 1.7-kb NdeI-EcoRI fragment of pT17A was cloned into pET-17b to construct pTIP17A. The 0.8-kb DNA fragment carrying the 5′ region of iphD was amplified by PCR using pKS50F as the template and the IphD-F/IphD-R primer set. The 0.8-kb PCR product was cloned into pT7Blue and sequenced. The 0.7-kb SalI fragment of pKS50F was inserted into the same site of the resultant plasmid to generate pT13D. The 1.3-kb NdeI-HindIII fragment of pT13D was cloned into the same sites of pET-17b to construct pTIP13D. The 0.7-kb DNA fragment carrying the 5′ region of iphB was amplified by PCR using pKS50F as the template and the IphB-F/IphB-R primer set. The 0.7-kb PCR product was cloned into pT7Blue and sequenced. The 2.6-kb SphI-HindIII fragment of pKS50F was inserted into the same sites of the resultant plasmid to generate pT28BDR. The 1.2-kb NdeI-SalI fragment of pT28BDR was cloned into the same sites of pET-21a(+) to construct pTIP12B.

E. coli BL21(DE3) cells harboring pTIP17A, pTIP13D, or pTIP12B were grown in LB medium containing ampicillin at 30°C. Expression of the genes was induced for 4 h at 30°C by adding 1 mM isopropyl-β-d-thiogalactopyranoside when the absorbance at 600 nm of the culture reached 0.5. The cells were harvested by centrifugation and suspended in TG buffer (50 mM Tris-HCl [pH 7.0] and 10% glycerol). Cells suspended in the buffer were sonicated, and the cell lysate was centrifuged at 15,000 × g for 10 min at 4°C. The resulting supernatant was used as crude enzymes.

The protein concentration was determined by the Bradford method (8). The sizes of the proteins produced in E. coli were examined by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis, and the gels were stained with Coomassie brilliant blue R-250.

Enzyme assay.

The enzyme activity of IphD was evaluated by an NAD(P)H-dependent oxidoreductase assay using 2,6-dichrolophenolindophenol (DCPIP). The system used to measure activity (1 ml) contained 50 mM Tris-HCl (pH 7.0), 500 μM NAD(P)H, 88 μM DCPIP, and cell extract of E. coli BL21(DE3) harboring pTIP13D (10 μg of protein). Reduction of DCPIP was monitored by measuring the decrease in absorbance at 600 nm. The molecular extinction coefficient at this wavelength of DCPIP was 21,000 M−1 cm−1 (42). A measurement was obtained at 30°C using a spectrophotometer (model DU-7500; Beckman Coulter, Fullerton, CA). One unit of enzyme activity was defined as the amount of enzyme that reduced 1 μmol of DCPIP per min.

IPA dioxygenase (IPADO) activity was determined by measuring the decrease in the amount of IPA utilizing a high-pressure liquid chromatography (HPLC) system (Alliance 2690 separation module; Waters, Milford, MA) equipped with a photodiode array detector (966 photodiode array detector; Waters) and a TSKgel ODS-80 column (6 by 150 mm; Tosoh, Tokyo, Japan). The 1-ml assay mixture contained FE2 buffer [TG buffer containing 100 μM Fe(NH4)2(SO4)2·6H2O and 2 mM l-cysteine hydrochloride], 100 μM IPA, 1 mM NADPH, and cell extracts of E. coli BL21(DE3) harboring pTIP17A (1 mg of protein) and E. coli BL21(DE3) harboring pTIP13D (1 mg of protein). Portions of the reaction mixture were removed at various sampling points and analyzed by HPLC. The mobile phase used for the HPLC analysis was a mixture of water (74.5%), acetonitrile (24.5%), and phosphoric acid (1%) at a flow rate of 1 ml/min. IPA was detected at 210 nm, and its retention time was 6.8 min. The transformation activity of crude IPADO with respect to IPA was measured in a 10-min reaction. One unit of enzyme activity was defined as the amount of enzyme that degraded 1 μmol of IPA per min.

The substrate-dependent oxygen consumption of IPADO was determined with an oxygen electrode (B-505; Iijima Electronics Manufacturing Co., Ltd, Aichi, Japan). The 2-ml reaction mixture contained TG buffer, 100 μM IPA, 1 mM NADPH, and cell extracts of E. coli BL21(DE3) harboring pTIP17A (2 mg of protein) and E. coli BL21(DE3) harboring pTIP13D (2 mg of protein). The reaction was initiated by addition of NADPH, and measurement was carried out at 30°C.

Substrate preference of IPADO.

Crude IPADO was incubated with 100 μM IPA, OPA, TPA, benzoate (BA), m-hydroxybenzoate (MHB), and PCA under the assay conditions described above for 60 min. The reaction mixtures were analyzed by HPLC (ACQUITY UPLC system; Waters) using a TSKgel ODS-140HTP column (2.1 by 100 mm; Tosoh). The mobile phase was a mixture of water (90%) and acetonitrile (10%) containing 0.1% phosphoric acid at a flow rate of 0.3 ml/min. In the analysis of PCA, a mobile phase consisting of a mixture of water (99%) and acetonitrile (1%) containing 0.1% phosphoric acid was used. Compounds were detected at the following wavelengths: IPA, 210 nm; OPA, 198 nm; TPA, 242 nm; BA, 230 nm; MHB, 207 nm; and PCA, 205 nm. The retention times of IPA, OPA, TPA, BA, MHB, and PCA were 4.1, 3.1, 2.4, 9.0, 3.1, and 4.2 min, respectively.

Identification of the reaction product.

Crude IPADO was incubated with 100 μM IPA under the assay conditions described above for 120 min. The reaction was stopped by addition of acetonitrile (final concentration, 50%), and precipitated protein was removed by centrifugation (15,000 × g for 15 min). The supernatant was analyzed by electrospray ionization mass spectrometry (ESI-MS) with an ACQUITY TQ detector (Waters). In this analysis, mass spectra were obtained by using negative and positive modes with the following settings: capillary voltage, 3.0 kV; cone voltage, 10 to 40 V; source temperature, 120°C; desolvation temperature, 350°C; desolvation gas flow rate, 650 liter/h; and cone gas flow rate, 50 liter/h.

For gas chromatography-mass spectrometry (GC-MS) with a model 5971A apparatus with an Ultra-2 capillary column (50 m by 0.2 mm; Agilent Technologies), the reaction mixture was acidified to pH 1 by addition of HCl and extracted with ethyl acetate. The extract was trimethylsilylated, and then the derivatives were analyzed by GC-MS. The analytical conditions used have been described previously (25).

The product of the IPA degradation reaction catalyzed by IPADO and IphB was identified by HPLC (Alliance 2690 separation module). The 1-ml assay mixture contained FE2 buffer, 100 μM IPA, 1 mM NADPH, 1 mM NADP+, crude IphA (1 mg of protein), crude IphD (1 mg of protein), and crude IphB (500 μg of protein). The mixture was incubated for 180 min at 30°C, and compounds were detected at 260 nm. The reaction product was identified based on a comparison of the retention times and UV-visible spectra with those of authentic PCA.

Construction of iph mutants.

The 1.7-kb NdeI-EcoRI fragment of pTIP17A was cloned into the same sites of pT7Blue to construct pT17A. The 1.3-kb EcoRV fragment carrying the kanamycin resistance gene (kan) from pIK03 (the kanamycin cassette) was inserted into the blunt-ended BamHI site of pT17A to construct pT30DAk. A 3.0-kb SalI-EcoRI fragment of pT30DAk was inserted into the same sites of pK19mobsacB to generate pDAk. A 2.3-kb PstI fragment of pKS50F carrying iphD was cloned into the same site of pT7Blue to generate pT23D. The kanamycin cassette was inserted into the AatII site in iphD to construct pT36DDk. A 3.6-kb PstI fragment of pT36DDk was inserted into the same site of pK18mobsacB to generate pDDk. A 1.9-kb EcoRI-SalI fragment of pKS50F carrying iphB was cloned into the same sites of pK18mobsacB to generate pK19B. The kanamycin cassette was inserted into the SmaI site in iphB to generate pDBk. A 2.3-kb BamHI-SmaI fragment of pKS50F carrying iphC was blunt ended and cloned into the SmaI-EcoRV sites of pBluescript II KS(+) to generate pKS23C. The kanamycin cassette was inserted into the blunt-ended EcoRI site in iphC to construct pKS36DCk. A 3.6-kb BamHI-SalI fragment of pKS36DCk was inserted into the same sites of pK18mobsacB to generate pDCk. A 1.3-kb AatII-HindIII fragment of pKS50F carrying iphR was blunt ended and cloned into the EcoRV site of pBluescript II KS(+) to generate pKS13R. The kanamycin cassette was inserted into the Eco47III site in iphR to generate pKS26DRk. A 2.6-kb EcoRI-KpnI fragment of pKS26DRk was inserted into the same sites of pT7Blue to construct pT26DRk. A 2.6-kb EcoRI-SphI fragment of pT26DRk was cloned into the same sites of pK18mobsacB to generate pDRk.

In order to obtain the iph gene mutants, pDAk, pDDk, pDBk, pDCk, and pDRk were independently introduced into E6 cells by electroporation, and candidate mutants were isolated as described previously (37). The disruption of each gene was examined by Southern hybridization analysis. To confirm disruption of iphA, iphD, iphB, iphC, and iphR, total DNA of candidate mutants were digested with EcoRV-EcoRI, PstI, EcoRI-SalI, SphI, and EcoRI-HindIII, respectively. The 0.9-kb HindIII-XhoI fragment carrying iphA, the 2.3-kb PstI fragment carrying iphD, the 1.9-kb EcoRI-SalI fragment carrying iphB, the 1.4-kb SphI fragment carrying iphC, the 1.3-kb AatII-HindIII fragment carrying iphR, and the 1.3-kb EcoRV fragment carrying kan were labeled with the DIG system and used as probes.

Growth of mutants on IPA.

The iph mutants were pregrown in 10 ml of LB medium containing kanamycin. The cells were harvested by centrifugation at 5,000 × g for 10 min, washed with 2 ml of W medium, and suspended in 1 ml of the same medium. The cells were inoculated into W medium containing 10 mM IPA to obtain an absorbance at 600 nm of 0.05. Cell growth was periodically monitored by measuring the absorbance at 600 nm. Complementary plasmids pEJ18A carrying iphA, pEJ17B carrying iphB, pEJ24C carrying iphC, and pEJ13R carrying iphR constructed using pJB866 were introduced into cells of DEIA, DEIB, DEIC, and DEIR, respectively, by electroporation.

Measurement of the promoter activity.

The 2.0-kb SalI-HindIII fragment of pKS24 carrying the potential iph operon promoter was cloned into the promoter-probe vector pPR9TZ to obtain pZSH2. This plasmid was introduced into E6 and DEIR cells by triparental mating (13). E6 and DEIR cells harboring pZSH2 were pregrown in 0.2× LB medium containing chloramphenicol. The cells were harvested by centrifugation at 5,000 × g for 10 min, washed with W medium, and suspended in the same medium. The cells were inoculated into 0.2× LB medium to obtain an absorbance at 600 nm of 0.2. After 90 min of incubation, 5 mM IPA, OPA, TPA, or PCA was added, and the cultures were incubated for another 120 min. The cells were harvested and then resuspended in 20 mM Tris-HCl (pH 8.0). Cells suspended in the buffer were sonicated, and the cell lysate was centrifuged at 15,000 × g for 15 min at 4°C. The resulting supernatant was used as crude enzymes. Promoter activity was determined by performing a β-galactosidase assay using 4-methylumbelliferyl-β-d-galactopyranoside as a substrate. The accumulation of 4-methylumbelliferone (4MU) was monitored by using a spectrofluorometer (RF-1500; Shimadzu Corporation, Kyoto, Japan). One unit of promoter activity was defined as the amount of enzyme that produced 1 μmol of 4MU per min.

Nucleotide sequence accession number.

The nucleotide sequence reported in this paper has been deposited in the DDBJ, EMBL, and GenBank nucleotide sequence database under accession no. AB501291.

RESULTS AND DISCUSSION

Cloning and nucleotide sequences of the IPA degradation gene cluster.

The deduced amino acid sequence encoded by iphA2 of C. testosteroni YZW-D (accession no. AAX18934), which was predicted to encode the oxygenase component of IPADO (48), showed the highest levels of similarity (37 to 38% identity) with the sequences encoded by the putative oxygenase component genes of B. japonicum USDA 110 (BAC46203), B. bronchiseptica RB50 (CAE35136), Rhodobacterales bacterium HTCC2654 (EAQ10830), and B. xenovorans LB400 (ABE33619). Based on the amino acid sequence similarity of these gene products, a degenerate primer set for amplification of a DNA fragment containing a motif for a Rieske-type [2Fe-2S] iron-sulfur cluster was designed, and a 465-bp fragment was amplified from E6. The nucleotide sequence of this fragment showed 100% identity with the YZW-D iphA2 sequence. Southern hybridization analysis of the E6 total DNA using the 465-bp amplified product as a probe showed only one hybridization signal (data not shown), suggesting that one IPADO gene was present. The 5.0-kb HindIII fragment (pCH50) and the 4.0-kb EcoRI fragment (pUC40), which contained the 465-bp fragment, were isolated from E6 DNA libraries. The nucleotide sequences of these fragments showed that there were five open reading frames (ORFs) in the 6,942-bp region (Fig. (Fig.1B).1B). Each ORF showed more than 90% identity with the corresponding putative IPA degradation gene (iph gene) of YZW-D (accession no. AY923836). The E6 genes were designated iphA, iphC, iphB, iphD, and iphR. The levels of identity between the deduced amino acid sequences encoded by the E6 iph genes and the sequences encoded by representative similar genes are summarized in Table Table33.

TABLE 3.
Identification of iph gene functions

As shown in Fig. Fig.2A,2A, IphA is related to oxygenase components of the phthalate family (18), but it exhibited only 17% identity with E6 TphA2. Amino acid sequence alignments of IphA with oxygenase components of carbazole 1,9a-dioxygenase (CarAa) from Janthinobacterium sp. J3 (31) and 2-oxo-1,2-dihydroquinoline 8-monooxygenase (OxoO) from Pseudomonas putida 86 (26), which were previously determined to be α3 trimers by X-ray crystallographic analysis, revealed the conserved residues for the Rieske-type [2Fe-2S] cluster (residues 69 to 91 [IphA_E6 numbering]), nonheme iron binding motif (residues 177 to 337 [IphA_E6 numbering]), and subunit interface Asp (Asp174 [IphA_E6 numbering]), which was suggested to be involved in the electron transfer between the Rieske cluster of one subunit and the active site mononuclear iron in the neighboring subunit (15, 26, 31).

FIG. 2.
(A) Phylogenetic tree of IphA with phthalate family dioxygenases. The scale bar corresponds to a genetic distance of 0.5 substitution per position. Enzymes: IphA_E6, IPADO oxygenase component of Comamonas sp. E6 (accession no. BAH70269); IphA2_YZW-D, ...

IphD is related to reductase components of the phthalate family (Fig. (Fig.2B),2B), but it exhibited only 15% identity with E6 TphA1. IphD contains the motifs for a phthalate dioxygenase reductase (PDR)-like domain (flavin mononucleotide-binding pocket and NAD-binding pocket [cd06185]) and the plant-type [2Fe-2S] iron-sulfur cluster (cd00207).

IphB showed 34 to 70% identity with a short-chain dehydrogenase/reductase. There is no sequence similarity between IphB and E6 1,4-DCD dehydrogenase (TphB), which belongs to a PdxA (pyridoxal phosphate biosynthetic protein) superfamily (cl00873).

IphC showed 37% and 35% identity with a putative periplasmic TPA binding receptor of E6 (TphC) and BugT from Bordetella pertussis, which is a member of a large family of periplasmic solute receptors (the Bug family) of the tripartite tricarboxylate transporter (TTT) family (2).

IphR showed 23 to 53% identity with the putative IclR-type transcriptional regulators. It is known that the IclR-type transcriptional regulators act as both positive (12, 17) and negative (3, 47) regulators.

The iph gene cluster is also found in the genome of C. testosteroni KF-1 (Fig. (Fig.3).3). The organization of the iph genes is completely conserved in all three Comamonas strains. Moreover, the TPA catabolic gene cluster is also present and well conserved (Fig. (Fig.3).3). The iph gene clusters are located 0.8 and 0.6 kb upstream of the tph gene clusters of YZW-D and KF-1, respectively. On the other hand, E6 has two sets of the tph genes, but the physical relationship between these gene clusters remains to be elucidated.

FIG. 3.
Organization of the iph and tph gene clusters from C. testosteroni YZW-D, C. testosteroni KF-1, and Comamonas sp. E6. The accession numbers are as follows: putative iph and tph clusters of YZW-D, AY923836; putative iph and tph clusters of C. testosteroni ...

RT-PCR analysis of iph gene expression.

To define the operon structure of the iph genes, RT-PCR experiments were performed with total RNA isolated from E6 grown on IPA and primers complementary to neighboring ORFs (Fig. (Fig.1B).1B). The amplification products of iphA-iphC (673 bp), iphC-iphB (535 bp), iphB-iphD (594 bp), and iphD-iphR (519 bp) were obtained (Fig. (Fig.1C).1C). On the other hand, significantly smaller amounts of amplification products were observed when RNA was prepared from succinate-grown cells (Fig. (Fig.1C).1C). These results indicated that the iph genes of E6 are cotranscribed in an operon and that transcription of the iph catabolic operon was induced during growth on IPA.

Expression of iphA and iphD in E. coli.

The iphA and iphD genes were cloned into pET-17b to obtain pTIP17A and pTIP13D, respectively. SDS-PAGE analysis showed that 48- and 33-kDa proteins were present in cell extracts of E. coli BL21(DE3) harboring pTIP17A and pTIP13D, respectively (data not shown). These sizes were in agreement with the values calculated for the deduced amino acid sequences encoded by iphA (47,954 Da) and iphD (34,999 Da), respectively. But a substantial amount of IphD was produced in an insoluble fraction.

Since IphD is similar to reductase components of ring-hydroxylating dioxygenases, the DCPIP reductase assay was performed to examine its activity. The DCPIP reductase activity of crude IphD was detected only in the presence of NADH (0.9 ± 0.1 U/mg) or NADPH (5.6 ± 0.3 U/mg). These results indicated that iphD encodes a reductase and that NADPH appears to be the preferred electron donor for IphD.

In order to examine whether a mixture of crude IphA and IphD showed IPA transformation activity, 100 μM IPA was incubated with cell extracts of E. coli harboring pTIP17A and pTIP13D (1 mg of protein each) in the presence of 1 mM NADPH. IPA was completely degraded by the cell extracts within 60 min (specific activity, 3.8 ± 0.1 mU/mg), whereas a cell extract of E. coli harboring only pTIP17A or pTIP13D did not transform IPA. These results strongly suggested that IPADO consists of IphA and IphD. A reaction mixture containing IphA and IphD also showed IPA-dependent oxygen consumption (data not shown), providing evidence that the IphAD complex is an IPADO. In order to identify the product of the IPA reaction catalyzed by the crude extract containing IphA and IphD, the reaction mixture was directly analyzed by ESI-MS. However, no significant product peak was observed even though measurements of the sample were obtained in the negative and positive modes. The reaction product was therefore acidified and extracted with ethyl acetate. GC-MS analysis of the trimethylsilylated sample demonstrated that a peak with a retention time of 17.6 min was produced. This product was identified as m-hydroxybenzoate (MHB) based on a comparison of retention times and mass fragmentation patterns with those of authentic MHB (data not shown). Because in general dihydrodiols undergo dehydration in acid to produce phenols (23), MHB appeared to be produced from 1,5-DCD through dehydration concomitant with decarboxylation. However, it will be necessary to verify the reaction product by ESI-MS analysis using purified IPADO. The substrate preference of IPADO was examined using OPA, TPA, BA, MHB, and PCA as substrates. HPLC analysis showed no transformation of these compounds by IPADO, suggesting that IPADO is specific for IPA.

In conclusion, the data suggested that IPADO is a class IA dioxygenase that consists of a terminal oxygenase component (IphA) and a reductase component (IphD) (4) based on the presence of the motifs for a PDR-like domain and the plant-type [2Fe-2S] iron-sulfur cluster in IphD, as well as the fact that the IPADO activity was reconstituted from the crude IphA and IphD enzymes.

Expression of iphB in E. coli.

The iphB gene was cloned in pET-21a(+) to obtain pTIP12B. Expression of a 25-kDa protein was observed in E. coli BL21(DE3) harboring pTIP12B. This size is close to the predicted molecular mass of IphB (26,842 Da).

To examine whether iphB encodes a dehydrogenase involved in the conversion of the IPADO reaction product from IPA, which is thought to be 1,5-DCD, a cell extract of E. coli harboring pTIP12B (1 mg of protein) was incubated with 100 μM IPA, crude IphA (1 mg of protein), crude IphD (1 mg of protein), 1 mM NADPH, and 1 mM NADP+. After 180 min of incubation, a new peak with a retention time of 4.4 min was generated (data not shown). This product was identified as PCA by comparison of its retention time and UV-visible spectrum with those of authentic PCA. These results indicated that IPA was converted to PCA by two successive reactions catalyzed by IPADO and IphB. The generation of PCA supports the notion that IPADO catalyzes 3,4-dihydroxylation of IPA, yielding 1,5-DCD, and iphB encodes 1,5-DCD dehydrogenase, which dehydrogenates 1,5-DCD concomitant with decarboxylation.

Disruption of iph genes in Comamonas sp. E6.

To confirm that the iph genes are responsible for IPA degradation in Comamonas sp. E6, each of the iph genes was disrupted by gene replacement using plasmids containing iph genes inactivated by insertion of kan. The disruptions of the iph genes in the mutant candidates were confirmed by Southern hybridization analysis (data not shown). The resulting iph gene mutants were cultivated in W medium containing 10 mM IPA.

Cells of the iphA mutant (DEIA) were unable to grow on IPA (Fig. (Fig.4A).4A). To confirm that this growth deficiency was caused by disruption of iphA, pEJ18A carrying iphA was introduced into DEIA cells. The iphA gene in this plasmid was under control of the Pm promoter regulated by XylS. The DEIA cells harboring pEJ18A were able to grow on IPA in the presence of m-toluate, which is an inducer of XylS but cannot support the growth of E6. These results indicated that iphA is essential for growth of E6 on IPA.

FIG. 4.
Growth of the iph insertion mutants on IPA. The Comamonas sp. E6 iphA mutant (DEIA) (A), iphD mutant (DEID) (B), iphB mutant (DEIB) (C), iphC mutant (DEIC) (D), and iphR mutant (DEIR) (E) were incubated in W medium containing 10 mM IPA. The growth of ...

Disruption of iphD resulted in retarded growth on IPA. It took approximately 3.4 times longer for iphD mutant (DEID) cells than for E6 cells to enter the stationary phase, but this mutant was able to grow on IPA at a similar rate during the exponential growth phase (Fig. (Fig.4B).4B). Curiously, there was no difference between the growth on IPA of DEID cells that had been grown on IPA and the growth on IPA of E6 cells. Since the inactivation of iphD by insertion of kan in DEID cells grown on IPA was confirmed by Southern hybridization, mutations might have resulted in expression of an unidentified reductase gene. All these results suggested that iphD is essential for IPA degradation.

Cells of the iphB mutant (DEIB) also were not able to grow on IPA (Fig. (Fig.4C),4C), and the ability to grow on IPA was complemented by introduction of pEJ17B carrying iphB. These results indicated that iphB is essential for growth of E6 on IPA.

The iphC mutant (DEIC) was not able to grow on IPA at pH 7.3 (Fig. (Fig.4D).4D). DEIC cells harboring pEJ24C carrying iphC grew on IPA, indicating that iphC is required for growth of E6 on IPA at neutral pH. The effect of pH on the growth of DEIC cells was also tested, because it is commonly believed that the more IPA is in its undissociated form at a lower pH, the more it is expected to diffuse into the cells (11). DEIC was able to grow on IPA when the pH of the medium was adjusted to less than pH 6.5 (data not shown). These results suggested that iphC is involved in IPA uptake. It is noteworthy that the putative periplasmic solute binding receptor genes are present in both IPA and TPA degradation gene clusters and essential for growth of E6 on IPA and TPA. The TTT-like system appeared to play an important role in the uptake of IPA and TPA.

The iphR mutant (DEIR) cells exhibited faster growth on IPA than the E6 cells (Fig. (Fig.4E).4E). Introduction of a plasmid carrying iphR (pEJ13R) into E6 and DEIR cells resulted in retarded growth on IPA (data not shown). These results suggested that iphR negatively regulates expression of the iph catabolic operon in E6.

In addition, the growth of all of the iph mutants on OPA and TPA was the same as the growth of the wild type (data not shown), indicating that iph genes were specific for the IPA catabolic function.

Transcriptional regulation of the iph operon.

The 2.0-kb SalI-HindIII fragment of pKS24, which carries the potential iph operon promoter, was cloned into the promoter-probe vector pPR9TZ to obtain pZSH2. The activities of the iph operon promoter were measured using E6 cells harboring pZSH2 grown in the presence or absence of IPA, OPA, TPA, and PCA. The promoter activity increased 88-fold only in response to IPA (Fig. (Fig.5).5). This result indicates that IPA or its metabolites act as inducers of the iph operon. On the other hand, DEIR cells harboring pZSH2 showed constitutive expression, supporting the notion that IphR is a transcriptional repressor of the iph operon.

FIG. 5.
Regulation of the iph promoter activity by IphR. Cells of E6 harboring pZSH2 and DEIR harboring pZSH2 were grown in 0.2× LB medium with (+) or without (−) 5 mM IPA. The data are the averages ± standard deviations of three ...

Conclusions.

This study showed the roles of iph genes and the regulation of the iph catabolic operon (iphACBDR) for the first time. IPADO is related to the phthalate family and consists of a terminal oxygenase component (IphA) and a reductase component (IphD). IphB was required for conversion of the product of IPA in a reaction catalyzed by the IphAD complex to PCA. IphC appeared to code for a periplasmic IPA binding receptor, which probably interacts with membrane components of the TTT system. The iph operon is negatively regulated by IphR, and the transcription of this operon is induced by IPA and/or its metabolite(s).

Acknowledgments

This work was supported in part by Grant-in-Aid for Scientific Research (A) 18208027 from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

We thank T. Abe for his technical advice.

Footnotes

[down-pointing small open triangle]Published ahead of print on 20 November 2009.

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