Journal of Bacteriology, January 2003, p . 41-50, Vol . 185, No . 1

Characterization of the 4-Carboxy-4-Hydroxy-2-Oxoadipate Aldolase Gene and Operon Structure of the Protocatechuate 4,5-Cleavage Pathway Genes in Sphingomonas paucimobilis SYK-6

Hirofumi Hara,¹ Eiji Masai,^1* Keisuke Miyauchi,¹ Yoshihiro Katayama,² and Masao Fukuda¹

Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188,¹ and Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan²

Received 29 July 2002/ Accepted 4 October 2002

In the case of S . paucimobilis SYK-6, PCA is degraded via the PCA 4,5-cleavage pathway (Fig . 1) . This pathway was enzymatically characterized by Kersten et al . (16) and Maruyama and coworkers (18-22) . In this pathway, PCA is initially trans-formed to 4-carboxy-2-hydroxymuconate-6-semialdehyde(CHMS) by 4,5-PCD (LigAB) . CHMS is nonenzymatically converted to an intramolecular hemiacetal form and then oxidized by CHMS dehydrogenase . The resulting intermediate, 2-pyrone-4,6-dicarboxylate (PDC), is hydrolyzed by PDC hydrolase to yield the keto form and enol form of 4-oxalomesaconate (OMA), which are in equilibrium . OMA is converted to 4-carboxy-4-hydroxy-2-oxoadipate (CHA) by OMA hydratase . Finally, CHA is cleaved by CHA aldolase to produce pyruvate and oxaloacetate . Recently, we have identified and characterized all of the gene products and genes except the CHA aldolase gene in the SYK-6 PCA 4,5-cleavage pathway (11, 27, 28, 31, 44) . These genes are essential to PCA degradation, while the 3MGA degradation is suggested to go through both the PCA 4,5-cleavage pathway and an alternative ring-cleavage pathway . In this alternative pathway, 3MGA was finally converted to OMA and then entered the PCA 4,5-cleavage pathway . Thus, the PCA 4,5-cleavage pathway genes play a key role in PCA and 3MGA degradation (11) .

 
Recently, cloning of the PCA 4,5-cleavage pathway genes has been reported in Sphingomonas sp . strain LB126 (48), Arthrobacter keyseri 12B (6), Comamonas testosteroni BR6020 (36), and Pseudomonas ochraceae NGJ1 (23) . However, detailed information is not available in regard to the actual role and property of each of the corresponding gene products . In this study, we characterized the structure and functions of the CHA aldolase gene, which is involved in the final step of the PCA 4,5-cleavage pathway . We also examined the involvement of the two open reading frames (ORFs) found among the genes which encode the PCA 4,5-cleavage pathway enzymes, and the operon structure of this pathway genes was estimated .

 
Preparation of substrate. PDC and OMA were prepared as described earlier (28) . CHA was prepared by incubating 1 mmol of OMA with 500 U of purified OMA hydratase for 5 min (11) . Electrospray-ionization mass spectrometry (ESI-MS) analysis revealed that the m/z 201 showing [M-H]^- of OMA (where M is a molecular ion of OMA) was completely converted into m/z 219, indicating [M-H]^- of CHA by LigJ . Then, the reaction product of OMA catalyzed by purified LigJ was used as a substrate .

DNA manipulations and nucleotide sequencing. DNA manipulations were carried out essentially as described in references 1 and 38 . A Kilosequence kit (Takara Shuzo Co., Ltd., Kyoto, Japan) was used to construct a series of deletion derivatives, whose nucleotide sequences were determined by the dideoxy termination method with an ALFexpress DNA sequencer (Pharmacia Biotech, Milwaukee, Wis.) .

A Sanger reaction (39) was carried out by using the Thermosequenase fluorescence-labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom) . Sequence analysis and homology alignment were carried out with the GeneWorks programs (IntelliGenetics, Inc., Mountain View, Calif.) . The DDBJ database was used for searching homologous proteins .

Enzyme assay. According to the method of Maruyama (21), a coupled assay was used for CHA aldolase . The decrease in the absorbance at 340 nm derived from the oxidation of NADH (₃₄₀ = 6.6 x 10³ M^-1 cm^-1; pH 8.0) in a reaction mixture containing 200 µM CHA, 140 µM NADH, coupled enzymes (30 U of lactate dehydrogenase and malate dehydrogenase), 1 mM MgCl₂, and a suitable aliquot of LigK was measured in 0.1 M Tris-acetate buffer (pH 8.0) . The enzyme reaction was carried out at 30°C in a cuvette . One unit of enzyme activity is defined as that causing the oxidation of 2 µmol of NADH/min in this assay . Specific activity was expressed as units per milligram of protein . Oxaloacetate decarboxylase activity was determined by measuring the decrease in absorbance at 340 nm derived from the oxidation of NADH . The 1-ml reaction mixture contained 200 µM oxaloacetate, 140 µM NADH, 30 U of lactate dehydrogenase, 1 mM MgCl₂, and LigK enzyme in 0.1 M Tris-acetate buffer (pH 8.0) . One unit of enzyme activity is defined as that causing the oxidation of 1 µmol of NADH/min in this assay . Under these conditions, the spontaneous oxaloacetate decarboxylase activity was detected (0.01 U) . This spontaneous activity was subtracted from the raw data of oxaloacetate decarboxylase activity of LigK . Specific activity was expressed as units per milligram of protein . The K_m and V_max values were obtained from the Hanes-Woolf plots . The inhibition constant (K_i) for oxaloacetate was determined from the Dixon plot . These kinetic constants were expressed as means from at least three independent experiments .

Enzyme purification. Enzyme purification was performed according to the method described below by using a BioCAD700E apparatus (PerSeptive Biosystems, Framingham, Mass.) .

(i) Preparation of cell extract. Escherichia coli BL21(DE3) harboring pETK was grown in 100 ml of LB medium containing 100 mg of ampicillin/liter . Expression of ligK was induced for 4 h at 37°C by the addition of isopropyl-ß-D-thiogalactopyranoside (final concentration, 1 mM) when the turbidity of the culture at 660 nm reached 0.5 . Cells were harvested by centrifugation and resuspended in 20 mM Tris-HCl buffer (pH 8.0) (buffer A) . The cells were broken by two passages through a French pressure cell . The cell lysate was centrifuged at 15,000 x g for 15 min . Streptomycin (final concentration, 1% [wt/vol]) was added to the supernatant, which was centrifuged again at 15,000 x g for 15 min to remove nucleic acids . The supernatant was recovered and then centrifuged again at 170,000 x g for 60 min at 4°C . The crude extract was obtained after concentration by ultrafiltration using a minicon B15 (Amicon, Beverly, Mass.) .

(ii) POROS PI anion-exchange chromatography. The crude extract was applied to a POROS polyethyleneimine (PI) column (7.5 by 100 mm) (PerSeptive Biosystems) previously equilibrated with buffer A . The enzyme was eluted with 88 ml of linear gradient of 0 to 0.5 M NaCl . The CHA aldolase was eluted at approximately 0.20 M .

(iii) POROS HQ anion-exchange chromatography. The fractions containing CHA aldolase activity eluted from a PI column were pooled, desalted, and concentrated by ultrafiltration using a minicon B15 . The resulting solution was applied to a POROS quaternized polyethyleneimine (HQ) column (4.6 by 100 mm; PerSeptive Biosystems) previously equilibrated with buffer A . The enzyme was eluted with 33 ml of a linear gradient of 0 to 0.5 M NaCl . The fractions containing CHA aldolase activity that eluted at approximately 0.30 M were pooled .

(iv) POROS PE hydrophobic-interaction chromatography. The fractions containing CHA aldolase activity eluted from an HQ column were pooled, desalted, and concentrated . Ammonium sulfate was added to the enzyme solution to a final concentration of 2 M . After centrifugation at 15,000 x g for 10 min, the supernatant was recovered and applied to a POROS phenylether (PE) column (4.6 by 100 mm) (PerSeptive Biosystems) equilibrated with buffer B (buffer A containing 2 M ammonium sulfate) . The enzyme was eluted with 25 ml of a linear gradient of 2.0 to 0 M ammonium sulfate . The fractions containing CHA aldolase activity that eluted at approximately 1.3 M were pooled, desalted, and concentrated as described above . Glycerol was added to a final concentration of 10%, and the purified enzyme was stored at -80°C until use .

Analytical method. The protein concentration was determined by the method of Bradford (2) . The purity of the enzyme preparation was examined by sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis (SDS-15% PAGE) (17) . The molecular mass of the native enzyme was estimated by Superdex200 HR10/30 (Pharmacia Biotech) gel filtration column chromatography using a BioCAD700E apparatus . Elution was performed with 50 mM potassium phosphate buffer (pH 7.0) containing 0.15 M NaCl at a flow rate of 0.8 ml/min . The molecular weight was estimated on the basis of calibration curve of reference proteins .

To determine the N-terminal amino acid sequence, the cell extract of E . coli BL21(DE3) harboring pETK was subjected to SDS-15% PAGE and electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, Calif.) . The area at 27 kDa was cut out and analyzed on a PPSQ-21 protein sequencer (SHIMADZU, Kyoto, Japan) . The isoelectric point of LigK was determined by isoelectric focusing on an Ampholine PAG plate (pH 3.5 to 9.5; Pharmacia Biotech) using a model Multiphor II electrophoresis system (Pharmacia Biotech) .

The substrate and the reaction products were detected and identified by gas chromatography (GC)-MS using model 5971A with an Ultra-2 capillary column (50 m by 0.2 mm; Agilent technologies, Palo Alto, Calif.) and ESI-MS using HP1100 series LC-MSD (Agilent technologies) . The analytical conditions for GC-MS were the same as described previously (28) . In ESI-MS analysis, mass spectra were obtained by negative-mode ESI, with a needle voltage of -3.5 kV and a source temperature at 350°C . The sample was injected directly into the mass spectrometer; the water/methanol ratio was 90:10 (vol/vol), and the flow rate was 0.2 ml/min .

Identification of the reaction product. 200 µM CHA was incubated with purified LigK (0.5 µg) in 0.1 M Tris-acetate buffer (pH 8.0) containing 1 mM MgCl₂ for 1 min or 5 min, the reaction mixture was diluted to 1/10 with 10 mM Tris-acetate buffer (pH 8.0), and the portion of mixture (5 µl) was injected into the ESI-mass spectrometer .

In the case of GC-MS analysis, the reaction product was acidified and extracted with ethylacetate, and then the extract was trimethylsilylated . The resultant trimethylsilylated derivatives were analyzed .

The metabolites of vanillate and syringate by the ligK insertion mutant (DLK) were analyzed . DLK cells grown in 10 ml of LB medium were washed with 0.1 M Tris-acetate buffer (pH 8.0) . The cells were resuspended in the same buffer and incubated with 10 mM vanillate and 10 mM syringate for 12 h at 30°C . After centrifugation, the supernatant was diluted 20-fold with 10 mM Tris-acetate buffer (pH 8.0) and analyzed by ESI-MS as described above . On the other hand, the metabolites were extracted by ethylacetate, trimethylsilylated, and analyzed by GC-MS .

Disruption of orf1, ligK, ligR, and orf2. The 4.0-kb XhoI-SmaI fragment carrying ligK and orf1 was cloned into pBluescript II SK(+) to generate pXS4, and it was digested with PpuMI for ligK disruption or with SalI for orf1 disruption . The 1.2-kb PstI fragment containing the kanamycin resistance gene from pUC4K (47) was inserted into the PpuMI or SalI site of the 4.0-kb XhoI-SmaI fragment to construct pXS4K and pXS4K2, respectively . pXS4K and pXS4K2 were digested with BamHI and KpnI, and their inserts were cloned into pK19mobsacB (40) to generate pLKD and pF1D, respectively . The 1.8-kb ClaI-SmaI fragment carrying ligR was cloned into pUC19 to generate pCS18, and it was digested with Eco47III . The kanamycin resistance gene was inserted into this Eco47III site . The resultant plasmid, pCS18K, was digested with KpnI and SacI, and the insert containing the inactivated ligR gene was cloned into pK19mobSacB to generate pLRD . The 1.7-kb PstI fragment carrying orf2 was cloned into pUC19 to generate pPS17, and it was digested with SmaI . The kanamycin resistance gene was inserted into the SmaI site . The resultant plasmid, pPS17K, was digested with BamHI and KpnI, and the insert containing the inactivated orf2 gene was cloned into pK19mobsacB to generate pF2D .

Each of plasmids, pLKD, pF1D, pLRD, and pF2D was introduced into SYK-6 cells by electroporation, and the candidates for mutants were isolated as described previously (28) . To examine the disruption of each gene, Southern hybridization analysis was carried out . The total DNA of the candidates for ligK, ligR, and orf2 mutants were digested with PstI, and those for orf1 were digested with SmaI . The 1.2-kb PstI fragment carrying the kanamycin resistance gene, the 2.3-kb PstI fragment carrying ligR and ligK, the 4.0-kb XhoI-SmaI fragment carrying orf1, and the 1.7-kb PstI fragment carrying orf2 were labeled with the DIG system (Roche Diagnostics, Indianapolis, Ind.) and used as probes .

Reverse transcription (RT)-PCR. Cells of S . paucimobilis SYK-6 were grown in W minimal salt medium containing 10 mM vanillate until they reached the turbidity at 660 nm of 0.5 . Total RNA was prepared from 10 ml of culture by using RNeasy Mini columns (Qiagen Inc, Chatsworth, Calif.) . To remove any contaminating genomic DNA, the RNA samples were incubated with 1 U of RNase-free DNase (Takara Shuzo Co., Ltd.) in 40 mM Tris-HCl (pH 7.9) containing 1 U of RNase inhibitor (Takara Shuzo Co., Ltd.), 10 mM NaCl, 10 mM CaCl₂, and 6 mM MgSO₄ for 30 min at 37°C . RT-PCR was carried out with a BcaBEST RNA PCR kit (Takara Shuzo Co., Ltd.) . A cDNA library was obtained by an RT reaction using a hexanucleotide random priming mix . The cDNA was used as a template for subsequent PCRs with specific primers, which amplify the boundaries of ligK-orf1-ligI-lsdA and ligR-orf2-ligJ-ligA-ligB-ligC . The forward and reverse primers used were as follows: lsdA-forward (nucleotide positions from 1,363 to 1,383 in the 10.5-kb EcoRI fragment) and ligI-reverse (positions 1,924 to 1,944); ligI-forward (positions 2,528 to 2,548) and orf1-reverse (positions 2,999 to 3,019); orf1-forward (positions 3,489 to 3,509) and ligK-reverse (positions 3,740 to 3,760); internal ligR-forward (positions 4,613 to 4,533) and internal ligR-reverse (positions 5,215 to 5,235); ligR-forward (positions 5,770 to 5,790) and orf2-reverse (positions 6,476 to 6,496); internal orf2-forward (positions 5,843 to 5,863) and orf2-reverse; orf2-forward (positions 6,536 to 6,556) and ligJ-reverse (positions 6,943 to 6,963); ligJ-forward (positions 7,662 to 7,682) and ligA-reverse (positions 8,162 to 8,182); ligA-forward (positions 8,162 to 8,182) and ligB-reverse (positions 8,706 to 8,726); ligB-forward (positions 9,119 to 9,139) and ligC-reverse (positions 9,598 to 9,608); internal ligC-forward (positions 9,609 to 9,629) and internal ligC-reverse (positions 10,098 to 10,118) . Control samples in which reverse transcriptase was omitted in RT-PCR and in which genomic DNA was used as a template in PCRs were run in parallel with RT-PCRs .

Nucleotide sequence accession number. The nucleotide sequence reported in this paper was deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession no . AB073227 .

The PCA 4,5-cleavage pathway genes of S . paucimobilis SYK-6 consisted of the two divergently transcribed gene clusters of ligK-orf1-ligI-lsdA and ligR-orf2-ligJ-ligA-ligB-ligC (Fig . 1B) . Recently, all or partial sequences of the PCA 4,5-cleavage pathway genes have been reported in Sphingomonas sp . strain LB126 (48), A . keyseri 12B (6), C . testosteroni BR6020 (36), and P . ochraceae NGJ1 (23) . Two types of gene clusters can be clearly seen . Interestingly, the gene organization of the fluorene degrader, Sphingomonas sp . strain LB126, is essentially the same as that of SYK-6, although the identities of the corresponding genes between SYK-6 and LB126 vary from 50 to 88% . On the other hand, C . testosteroni BR6020 and A . keyseri 12B have a packed single gene cluster that seems to be an operon .

Purification of CHA aldolase. The 1.0-kb SalI-SphI fragment carrying ligK was cloned in pET21(+) to construct pETK, and ligK was expressed in E . coli BL21(DE3) under the control of the T7 promoter . Production of the 27-kDa protein was observed by SDS-PAGE (data not shown) . The size of the product is close to the molecular mass calculated from the deduced amino acid sequence of ligK . LigK was purified from a cell extract of E . coli BL21(DE3) harboring pETK by a series of column chromatography procedures with PI, HQ, and PE . LigK was purified approximately 52-fold to near homogeneity (>99%) with a recovery of 20% . N-terminal amino acid sequencing revealed that the first 15 residues, with the exception of the first methionine, Arg-Gly-Ala-Ala-Met-Gly-Val-Val-Val-Gln-Asn-Ile-Glu-Arg-Ala, corresponded to the deduced amino acid sequence of ligK .

Identification of the reaction product. To identify the reaction product of CHA catalyzed by the purified LigK, the reaction mixture was analyzed by ESI-MS . The fragment at m/z 219 in Fig . 2B was estimated to be the deprotonated molecular ion ([M-H]^-) of CHA (where M is a molecular ion) . The other peaks in the spectrum of CHA were originated from components of the reaction buffer containing LigK enzyme (Fig . 2A) . After 1 min of reaction, the intensity of the fragment at m/z 219 of CHA decreased to 42% of its initial intensity, and the generation of two fragments at m/z 87 and at m/z 131 corresponding to [M-H]^- of pyruvate and [M-H]^- of oxaloacetate, respectively, was observed (Fig . 2C) . This result indicated that LigK catalyzes the conversion of CHA to pyruvate and oxaloacetate . After 5 min of reaction, the intensity of the fragment at m/z 87 increased to 144% of that of the corresponding fragment in the 1-min reaction mixture, whereas the intensity of the fragment at m/z 131 decreased to 38% of that of the corresponding fragment in the 1-min reaction mixture (Fig . 2D) . These results strongly suggested that oxaloacetate was converted into pyruvate by LigK . The activity for ß-decarboxylation of oxaloacetate has been reported in CHA aldolase of P . ochraceae (21), 4-hydroxy-4-methyl-2-oxoglutarate aldolase of P . putida (45), and 2-keto-4-hydroxyglutarate aldolase of E . coli (29) . To examine whether LigK has this activity, oxaloacetate was incubated with LigK in the presence of lactate dehydrogenase and NADH . A decrease in absorbance at 340 nm derived from NADH was observed . It is concluded that LigK is able to decarboxylate oxaloacetate to generate pyruvate .

 
Enzyme properties. In accord with the previous study by Maruyama (21), the CHA aldolase activity was observed only when a divalent cation such as Mg²⁺ was present in the reaction mixture . We examined the effect of the various divalent cations for the enzyme activities of LigK . Addition of 1 mM Co²⁺, Zn²⁺, Ca²⁺, or Mn²⁺ resulted in 85, 65, 20, or 0% of the activity resulting from addition of 1 mM Mg²⁺, respectively . A similar metal dependency was observed in the decarboxylation of oxaloacetate by LigK . When 1 mM EDTA was added to the reaction mixture, both enzyme activities were completely lost in the presence of 1 mM metal ion . Aldolases are categorized as class I or class II based on their metal dependency . LigK was suggested to be one of the class II aldolases, which require the metal ion . Most class II aldolases show a significant rate enhancement in the presence of phosphate ion (37) . Addition of 0.5 mM phosphate ion in the LigK reaction mixture caused 3.0- and 1.7-fold activation of CHA aldolase and oxaloacetate decarboxylase activity, respectively .

Gel filtration column chromatography using the Superdex200 indicated that the molecular mass of the native LigK was 160 kDa . This result suggested that LigK is a homohexamer . The isoelectric point of LigK was determined to be 5.1 by isoelectric focusing gel electrophoresis . The optimal temperature of LigK for aldolase activity on CHA, and the decarboxylase activity on oxaloacetate were both determined to be 25°C . The optimal pH for aldolase activity and decarboxylase activity were estimated to be 8.0 and 7.0, respectively . The K_m for oxaloacetate (136 µM) is 12 times higher than that for CHA (11.2 µM) . The V_max for CHA aldol cleavage (265 U/mg) is 20 times higher than that for oxaloacetate decarboxylation (13.2 U/mg) .

We also examined the influence of sulfhydryl reagents on LigK . One microgram of purified LigK was preincubated with 1 mM sulfhydryl reagents for 10 min . HgCl₂ and N-ethylmaleimide inhibited 60 and 62% of the CHA aldolase and 92 and 88% of the oxaloacetate decarboxylase activities, respectively . These results suggested that some cysteine residues might be involved in the enzyme reaction . CHA aldolase activity was inhibited by oxaloacetate with a K_i value of 23 µM . As suggested by Maruyama (21), the amount of oxaloacetate in the cells might control the production of oxaloacetate from CHA .

CHA aldolase has been biochemically characterized only in P . ochraceae (21) and P . putida (45) . The molecular mass, subunit structure, and pI of LigK are very similar to those of aldolases of P . ochraceae and P . putida . In the P . ochraceae enzyme, the kinetics parameters are measured using the substrates d-CHA and l-CHA . The K_m and V_max values of the P . ochraceae enzyme for l-CHA were similar to those for LigK . In our experiment, CHA was prepared from OMA by using the purified OMA hydratase from E . coli carrying the SYK-6 ligJ gene . The physiological substrate for LigK might be an l-isomer . The K_m for oxaloacetate of the P . ochraceae enzyme was twofold higher than that of LigK, although the V_max values of these strains are similar . LigK has a significantly higher affinity for oxaloacetate than did the P . ochraceae enzyme .

Disruption of ligK in S . paucimobilis SYK-6. The ligK gene was disrupted to clarify the actual role of ligK in the catabolism of vanillate and syringate by SYK-6 . Gene inactivation was carried out using the ligK disruption plasmid, pLKD . The ligK insertional mutation was confirmed by Southern hybridization analysis using the 2.3-kb PstI fragment carrying ligK and the 1.2-kb PstI fragment carrying the kanamycin resistance gene as probes (data not shown) . The ligK and kanamycin resistance gene probes revealed that the ligK gene was inactivated by homologous recombination through the double crossover . This mutant strain was designated DLK and used for the following experiments . The obtained mutant strain DLK completely lost the ability to grow on both vanillate and syringate . This result is compatible with the deduced catabolic pathways of vanillate and syringate by SYK-6 shown in Fig . 1A .

To determine the accumulated products from vanillate and syringate incubated with DLK, 10 mM concentrations of vanillate and syringate were independently incubated with the LB-grown whole cells of DLK in the W minimal medium, and the metabolites were identified by GC-MS and ESI-MS . As shown in the gas chromatogram (Fig . 3A and B), vanillate and syringate detected with retention times of 21.2 and 25.2 min, respectively, disappeared completely, and the accumulation of PDC, the enol form of OMA, product I with a retention time of 30.5 min, and product II with a retention time of 26.1 min was observed in both cultures . In a previous study, we identified product I as the compound generated from OMA by addition of two atoms of hydrogen by NADPH-dependent reductase in the ligJ insertion mutant of SYK-6 (11) . The mass spectrum of product II accumulated in both cultures are identical but could not be assigned (data not shown) . On the other hand, ESI-MS analysis indicated accumulation of the products whose deprotonated molecular ions appeared at m/z 203 and at m/z 221 in the metabolite from vanillate and syringate (Fig . 3C and D) . In this analytical condition, neither PDC nor the enol form of OMA could be detected . We previously suggested that the ion at m/z 203 was a deprotonated molecular ion of product I . We therefore estimated that the ion at m/z 221 was generated from CHA accumulated by addition of two hydrogen atoms catalyzed by unidentified reductase(s) in DLK . To examine this hypothesis, CHA was incubated with the DLK crude extract prepared from cells grown in LB . ESI-MS of the reaction product after 10 min incubation showed that the peak at m/z 219 derived from CHA was converted to that at m/z 221 only in the presence of NADPH (data not shown) . These results strongly suggested that product II was produced from accumulated CHA . Our preliminary experiment indicated that LigJ activity was not inhibited by the presence of CHA, and thus the reason why a large amount of OMA (product I) and PDC were also accumulated from vanillate and syringate in DLK is unknown .

 
Disruption of ligR, orf1, and orf2 in S . paucimobilis SYK-6. To investigate whether ligR, orf1, and orf2 are involved in the catabolism of vanillate and syringate, each of these genes in SYK-6 was disrupted . Gene inactivation was carried out using the ligR, orf1, and orf2 disruption plasmids, pLRD, pF1D, and pF2D, respectively . The growth rates of the ligR disruption mutant, DLR, on both vanillate and syringate were decreased compared with those of SYK-6 (Fig . 4) . Based on this result and the fact that LigR has similarity with LysR-type transcriptional regulator, LigR may positively regulate the expression of the PCA 4,5-cleavage pathway genes, although it is not essential to growth of SYK-6 on vanillate and syringate . The orf1 insertion mutant, DF1, completely lost the ability to grow on both vanillate and syringate . This growth deficiency of DF1 on vanillate and syringate was complemented by introduction of pTS1210 carrying orf1 (pTSF1), indicating that orf1 is necessary for growth of SYK-6 on these compounds . Considering that orf1 has similarity with a putative transmembrane protein YbhH of E . coli, it is likely that orf1 encodes a transporter of vanillate and syringate . However, the actual role of orf1 is remained to clarify together with that of ligR . On the other hand, the disruption of orf2 did not affect the growth of SYK-6 on both vanillate and syringate .

 
RT-PCR analysis of PCA 4,5-cleavage pathway genes. To determine the operon structure of the genes included in the 10.5-kb EcoRI fragment, RT-PCR experiments were performed with total RNA isolated from SYK-6 grown on vanillate and primers complementary to neighboring ORFs . The amplification products of lsdA-ligI (581 bp), ligI-orf1 (491 bp), orf1-ligK (271 bp), ligJ-ligA (520 bp), and ligA-ligB (564 bp) were obtained . However, RT-PCR products using primer which span the ligR-orf2, orf2-ligJ, and ligB-ligC regions were not obtained (Fig . 5), while PCR using their primers with SYK-6 total DNA as a template gave the expected PCR products (data not shown) . In order to confirm the presence of the ligR, orf2, and ligC transcripts in the RNA samples, RT-PCR was carried out using primers to amplify inside of each ORF . RT-PCR products of ligR (622 bp) and ligC (509 bp) with the expected sizes were obtained (Fig . 5) . On the other hand, the RT-PCR product of orf2 did not appear, indicating the DNA region of orf2 was not transcribed in SYK-6 cells grown on vanillate .

 
In conclusion, the PCA 4,5-cleavage pathway genes of S . paucimobilis SYK-6 consist of four transcriptional units, including the ligK-orf1-ligI-lsdA cluster, the ligJAB cluster, and the monocistronic ligR and ligC genes (Fig . 1B) . In the case of the PCA 3,4-cleavage pathway genes, the diversity in gene organization and transcriptional regulation has been found (7, 10, 13, 14, 32, 35, 46) . In Acinetobacter sp . strain ADP1, all of the enzyme genes involved in this pathway constitute a single operon, pcaIJFBDKCHG, and their expression is regulated by the IclR-type transcriptional activator PcaU in concert with inducer PCA (10, 35, 46) . On the other hand, the enzyme genes of P . putida PRS2000 (13) and Agrobacterium tumefaciens A348 (32) consist of several transcriptional units . In A348, the pcaDCHGB and pcaIJ clusters are independently regulated by the LysR-type transcriptional activator PcaQ, which responds to both ß-carboxy-cis,cis-muconate and -carboxymuconolactone, and the IclR-type transcriptional activator PcaR, which responds to ß-ketoadipate, respectively (32) . To gain better understanding of the PCA 4,5-cleavage pathway genes, it is essential to address their transcriptional regulation . Determination of the promoter regions and the actual role of ligR are currently under way in our laboratory .

This work was supported in part by Grant-in-Aid for Encouragement of Young Scientists 11760057 from the Ministry of Education, Science, Sports, and Culture of Japan to E.M . H.H . was financially supported by research fellowship 2068 from the Japan Society for the Promotion of Science for Young Scientists .

1. Ausubel, F . M., R . Brent, R . E . Kingston, D . D . Moore, J . G . Seidman, J . A . Smith, and K . Struhl (ed.). 1990 . Current protocols in molecular biology . John Wiley & Sons, Inc., New York, N.Y.
2. Bradford, M . M. 1976 . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding . Anal . Biochem . 72:248-254.
3. Buchan, A., L . S . Collier, E . L . Neidle, and M . A . Moran. 2000 . Key aromatic-ring-cleaving enzyme, protocatechuate 3,4-dioxygenase, in the ecologically important marine Roseobacter lineage . Appl . Environ . Microbiol . 66:4662-4672.
4. Contzen, M., and A . Stolz. 2000 . Characterization of the genes for two protocatechuate 3,4-dioxygenases from the 4-sulfocatechol-degrading bacterium Agrobacterium radiobacter strain S2 . J . Bacteriol . 182:6123-6129.
5. Davison, J., F . Brunel, A . Phanopoulos, D . Prozzi, and P . Terpstra. 1992 . Cloning and sequencing of Pseudomonas genes determining sodium dodecyl sulfate biodegradation . Gene 114:19-24.
6. Eaton, R . W. 2001 . Plasmid-encoded phthalate catabolic pathway in Arthrobacter keyseri 12B . J . Bacteriol . 183:3689-3703.
7. Eulberg, D., S . Lakner, L . A . Golovleva, and M . Schlömann. 1998 . Characterization of a protocatechuate catabolic gene cluster from Rhodococcus opacus 1CP: evidence for a merged enzyme with 4-carboxymuconolactone-decarboxylating and 3-oxoadipate enol-lactone-hydrolyzing activity . J . Bacteriol . 180:1072-1081.
8. Finan, T . M., S . Weidner, K . Wong, J . Buhrmester, P . Chain, F . J . Vorhölter, I . Hernandez-Lucas, A . Becker, A . Cowie, J . Gouzy, B . Golding, and A . Pühler. 2001 . The complete sequence of the 1,683-kb pSymB megaplasmid from the N₂-fixing endosymbiont Sinorhizobium meliloti . Proc . Natl . Acad . Sci . USA 98:9889-9894.
9. Frazee, R . W., D . M . Livingston, D . C . LaPorte, and J . D . Lipscomb. 1993 . Cloning, sequencing, and expression of the Pseudomonas putida protocatechuate 3,4-dioxygenase genes . J . Bacteriol . 175:6194-6202.
10. Gerischer, U., A . Segura, and L . N . Ornston. 1998 . PcaU, a transcriptional activator of genes for protocatechuate utilization in Acinetobacter . J . Bacteriol . 180:1512-1524.
11. Hara, H., E . Masai, Y . Katayama, and M . Fukuda. 2000 . The 4-oxalomesaconate hydratase gene, involved in the protocatechuate 4,5-cleavage pathway, is essential to vanillate and syringate degradation in Sphingomonas paucimobilis SYK-6 . J . Bacteriol . 182:6950-6957.
12. Hartnett, C., E . L . Neidle, K . L . Ngai, and L . N . Ornston. 1990 . DNA sequences of genes encoding Acinetobacter calcoaceticus protocatechuate 3,4-dioxygenase: evidence indicating shuffling of genes and of DNA sequences within genes during their evolutionally divergence . J . Bacteriol . 172:956-966.
13. Harwood, C . S., and R . E . Parales. 1996 . The ß-ketoadipate pathway and the biology of self-identity . Annu . Rev . Microbiol . 50:553-590.
14. Iwagami, S . G., K . Yang, and J . Davies. 2000 . Characterization of the protocatechuic acid catabolic gene cluster from Streptomyces sp . strain 2065 . Appl . Environ . Microbiol . 66:1499-1508.
15. Katayama, Y., S . Nishikawa, M . Nakamura, K . Yano, M . Yamasaki, N . Morohoshi, and T . Haraguchi. 1987 . Cloning and expression of Pseudomonas paucimobilis SYK-6 genes involved in the degradation of vanillate and protocatechuate in P . putida . Mokuzai Gakkaisi 33:77-79.
16. Kersten, P . J., S . Dagley, J . W . Whittaker, D . M . Arciero, and J . D . Lipscomb. 1982 . 2-pyrone-4,6-dicarboxylic acid, a catabolite of gallic acids in Pseudomonas species . J . Bacteriol . 152:1154-1162.
17. Laemmli, U . K. 1970 . Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature (London) 227:680-685.
18. Maruyama, K. 1979 . Isolation and identification of the reaction product of -hydroxy--carboxymuconic--semialdehyde dehydrogenase . J . Biochem . 86:1671-1677.
19. Maruyama, K. 1983 . Purification and properties of 2-pyrone-4,6-dicarboxylate hydrolase . J . Biochem . 93:557-565.
20. Maruyama, K. 1985 . Purification and properties of -oxalomesaconate hydratase from Pseudomonas ochraceae grown with phthalate . Biochem . Biophys . Res . Commun . 128:271-277.
21. Maruyama, K. 1990 . Purification and properties of 4-hydroxy-4-methyl-2-oxoglutarate aldolase from Pseudomonas ochraceae grown on phthalate . J . Biochem . 108:327-333.
22. Maruyama, K., N . Ariga, M . Tsuda, and K . Deguchi. 1978 . Purification and properties of -hydroxy--carboxymuconic--semialdehyde dehydrogenase . J . Biochem . 83:1125-1134.
23. Maruyama, K., M . Miwa, N . Tsujii, T . Nagai, N . Tomita, T . Harada, H . Sobajima, and H . Sugisaki. 2001 . Cloning, sequencing, and expression of the gene encoding 4-hydroxy-4-methyl-2-oxoglutarate aldolase from Pseudomonas ochraceae NGJ1 . Biosci . Biotechnol . Biochem . 65:2701-2709.
24. Masai, E., Y . Katayama, S . Kawai, S . Nishikawa, M . Yamasaki, and N . Morohoshi. 1991 . Cloning and sequencing of the gene for a Pseudomonas paucimobilis enzyme that cleaves ß-aryl ether . J . Bacteriol . 173:7950-7955.
25. Masai, E., Y . Katayama, S . Kubota, S . Kawai, M . Yamasaki, and N . Morohoshi. 1993 . A bacterial enzyme degrading the model lignin compound ß-etherase is a member of the glutathione-S-transferase superfamily . FEBS Lett . 323:135-140.
26. Masai, E., Y . Katayama, S . Nishikawa, and M . Fukuda. 1999 . Characterization of Sphingomonas paucimobilis SYK-6 genes involved in degradation of lignin-related compounds . J . Ind . Microbiol . Biotechnol . 23:364-373.
27. Masai, E., K . Momose, H . Hara, S . Nishikawa, Y . Katayama, and M . Fukuda. 2000 . Genetic and biochemical characterization of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase and its role in the protocatechuate 4,5-cleavage pathway in Sphingomonas paucimobilis SYK-6 . J . Bacteriol . 182:6651-6658.
28. Masai, E., S . Shinohara, H . Hara, S . Nishikawa, Y . Katayama, and M . Fukuda. 1999 . Genetic and biochemical characterization of a 2-pyrone-4,6-dicarboxylic acid hydrolase involved in the protocatechuate 4,5-cleavage pathway of Sphingomonas paucimobilis SYK-6 . J . Bacteriol . 181:55-62.
29. Nishihara, H., and E . E . Dekker. 1972 . Purification, substrate specificity and binding, ß-decarboxylase activity, and other properties of Escherichia coli 2-keto-4-hydroxyglutarate aldolase . J . Biol . Chem . 247:5079-5087.
30. Nishikawa, S., T . Sonoki, T . Kasahara, T . Obi, S . Kubota, S . Kawai, N . Morohoshi, and Y . Katayama. 1998 . Cloning and sequencing of the Sphingomonas (Pseudomonas) paucimobilis gene essential for the O demethylation of vanillate and syringate . Appl . Environ . Microbiol . 64:836-842.
31. Noda, Y., S . Nishikawa, K . Shiozuka, H . Kadokura, H . Nakajima, K . Yoda, Y . Katayama, N . Morohoshi, T . Haraguchi, and M . Yamasaki. 1990 . Molecular cloning of the protocatechuate 4,5-dioxygenase genes of Pseudomonas paucimobilis . J . Bacteriol . 172:2704-2709.
32. Parke, D. 1997 . Acquisition, reorganization, and merger of genes: novel management of the ß-ketoadipate pathway in Agrobacterium tumefaciens . FEMS Microbiol . Lett . 146:3-12.
33. Peng, X., T . Egashira, K . Hanashiro, E . Masai, S . Nishikawa, Y . Katayama, K . Kimbara, and M . Fukuda. 1998 . Cloning of a Sphingomonas paucimobilis SYK-6 gene encoding a novel oxygenase that cleaves lignin-related biphenyl and characterization of the enzyme . Appl . Environ . Microbiol . 64:2520-2527.
34. Peng, X., E . Masai, Y . Katayama, and M . Fukuda. 1999 . Characterization of the meta-cleavage compound hydrolase gene involved in degradation of the lignin-related biphenyl structure by Sphingomonas paucimobilis SYK-6 . Appl . Environ . Microbiol . 65:2789-2793.
35. Popp, R., T . Kohl, P . Patz, G . Trautwein, and U . Gerischer. 2002 . Differential DNA biding of transcriptional regulator PcaU from Acinetobacter sp . strain ADP1 . J . Bacteriol . 184:1988-1997.
36. Providenti, M . A., J . Mampel, S . MacSween, A . M . Cook, and R . C . Wyndham. 2001 . Comamonas testosteroni BR6020 possesses a single genetic locus for extradiol cleavage of protocatechuate . Microbiology 147:2157-2167.
37. Richards, O . C., and . W . J . Rutter. 1961 . Preparation and properties of yeast aldolase . J . Biol . Chem . 236:3177-3184.
38. Sambrook, J., E . F . Fritsch, and T . Maniatis. 1989 . Molecular cloning: a laboratory manual, 2nd ed . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
39. Sanger, F., S . Nicklen, and A . R . Coulson. 1977 . DNA sequencing with chain-terminating inhibitors . Proc . Natl . Acad . Sci . USA 74:5463-5467.
40. Schäfer, A., A . Tauch, W . Jäger, J . Kalinowski, G . Thierbach, and A . Pühler. 1994 . Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum . Gene 145:69-73.
41. Schell, M . A. 1993 . Molecular biology of the LysR family of transcriptional regulators . Annu . Rev . Microbiol . 47:597-626.
42. Short, J . M., J . M . Fernandez, J . A . Sorge, and W . Huse. 1988 . ZAP: a bacteriophage expression vector with in vivo excision properties . Nucleic Acids Res . 16:7583-7600.
43. Studier, F . W., and B . A . Moffatt. 1986 . Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes . J . Mol . Biol . 189:113-130.
44. Sugimoto, K., T . Senda, H . Aoshima, E . Masai, M . Fukuda, and Y . Mitsui. 1999 . Crystal structure of an aromatic ring opening dioxygenase LigAB which is a protocatechuate 4,5-dioxygenase . Struct . Fold . Des . 15:953-965.
45. Tack, B . F., P . J . Chapman, and S . Dagley. 1972 . Purification and properties of 4-hydroxy-4-methyl-2-oxoglutarate aldolase . J . Biol . Chem . 247:6444-6449.
46. Trautwein, G., and U . Gerischer. 2001 . Effects exerted by transcriptional regulator PcaU from Acinetobacter sp . strain ADP1 . J . Bacteriol . 183:873-881.
47. Vieira, J., and J . Messing. 1982 . The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers . Gene 19:259-268.
48. Wattiau, P., L . Bastiaens, R . van Herwijnen, L . Daal, J . R . Parsons, M-E . Renard, D . Springael, and G . R . Cornelis. 2001 . Fluorene degradation by Sphingomonas sp . LB126 proceeds through protocatechuic acid: a genetic analysis . Res . Microbiol . 152:861-872.
49. Wolgel, S . A., J . E . Dege, P . E . Perkins-Olson, C . H . Jaurez-Garcia, R . L . Crawford, E . Münck, and J . D . Lipscomb. 1993 . Purification and characterization of protocatechuate 2,3-dioxygenase from Bacillus macerans: a new extradiol catecholic dioxygenase . J . Bacteriol . 175:4414-4426.
50. Yanisch-Perron, C., J . Vieira, and J . Messing. 1985 . Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors . Gene 33:103-119.
51. Zylstra, G . J., R . H . Olsen, and D . P . Ballou. 1989 . Genetic organization and sequence of the Pseudomonas cepacia genes for the alpha and beta subunits of protocatechuate 3,4-dioxygenase . J . Bacteriol . 171:5915-5921.

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