Strains and plasmids.
The strains and plasmids used in this study are listed in Table . S. paucimobilis
SYK-6 was grown on W minimal salt medium (24
) containing 10 mM syringate or Luria-Bertani (LB) medium at 30°C. Pseudomonas putida
PpY1100 was grown on LB medium. The SYK-6 mutants with insertions of desZ
(DB), and desZ
(DBZ) were grown on LB medium containing 50 mg of kanamycin/liter, 300 mg of carbenicillin/liter, and 50 mg of kanamycin and 300 mg of carbenicillin/liter, respectively.
Strains and plasmids used in this study
3MGA was synthesized from gallate methyl ester by the method of Scheline (31
). Syringate, vanillate, protocatechuate, and gallate were purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). Tetrahydrofolate was purchased from Sigma Chemical Co. (St. Louis, Mo.).
Cloning of the gene.
A partially SalI digested gene library of SYK-6 constructed with pVK100 as the vector was introduced into P. putida
PpY1100 by triparental mating (5
). The resulting transconjugants were grown on LB medium containing 50 mg of kanamycin/liter. When the turbidity of the culture at 600 nm reached 1.0, cells were harvested and washed with 50 mM Tris-HCl buffer (pH 7.5). Cells were resuspended in 1 ml of the same buffer. The 500-μl reaction mixture contained 495 μl of the cell suspension and 5 μl of 100 mM 3MGA (final concentration, 1 mM) and was incubated at 30°C for 20 h. The cells were removed by centrifugation (15,000 × g
for 5 min), and the supernatant was filtered. The amount of 3MGA in the filtrates was analyzed by a high-pressure liquid chromatography (HPLC) system (HP1100 series LC-MSD; Agilent Technologies Co., Palo Alto, Calif.) with an ODS Hypersil C-18 column (4 by 125 mm; Agilent Technologies). The mobile phase was a mixture of water (89.5%), acetonitrile (9.5%), and acetic acid (1.0%), and the flow rate was 0.5 ml/min. Compounds were detected at 275 nm, and the retention time of 3MGA was 4.6 min.
A cosmid, pVK3-1, was obtained from a transconjugant that showed 3MGA degradation activity. The 7.0-kb XhoI fragment of pVK3-1, which conferred the 3MGA degradation activity on PpY1100 was cloned into pBluescript II KS(+) to generate pBX2F. pBXSM1 was obtained as the smallest plasmid that conferred the activity on E. coli.
DNA manipulations and nucleotide sequencing.
DNA manipulations were carried out essentially as described before (1
). A series of deletion derivatives of pBXSM1 were constructed with a Kilosequence kit (Takara Shuzo Co. Ltd., Kyoto, Japan). Nucleotide sequences were determined by the dideoxy termination method (29
) with an ALFexpress DNA sequencer (Pharmacia Biotech, Milwaukee, Wis.). A Sanger reaction was performed with a Thermosequenase fluorescently labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). Analysis of the nucleotide sequence was performed with the GeneWorks program (IntelliGenetics, Inc., Mountain View, Calif.). Multiple sequence alignment was produced with the program Clustal W, and the phylogenetic tree was inferred from the alignments with the neighbor-joining method (27
). Graphics for phylogenetic trees were produced with the TreeView program (23
). The DDBJ databases were used for searching homologous proteins.
Expression of desZ in E. coli.
The coding region of desZ was amplified by PCR with Ex Taq polymerase (Takara Shuzo) with pBXSM1 as a template and the desZ-F3 primer (TGACATATGGCTGAGATCGTCC) and desZ-R3 primer (CATCAAGCTATCCTCTCACAGG). The 1.0-kb PCR product was cloned in pT7Blue and sequenced. The 1.0-kb NdeI fragment of the resulting plasmid was inserted into pET21a(+) to generate pEDZA. E. coli BL21(DE3) harboring pEDZA were grown in 5 liters of LB medium containing 100 mg of ampicillin/liter at 30°C. The expression of desZ was induced for 4 h by adding isopropyl-β-d-thiogalactopyranoside (final concentration, 1 mM) when the turbidity of the culture at 600 nm reached 0.5.
Preparation of cell extracts, protein determination, and PAGE.
Cells were harvested and resuspended in FE2 buffer consisting of 50 mM Tris-HCl buffer (pH 7.0), 10% glycerol, 0.1 mM ferrous ammonium sulfate, and 2 mM l
-cysteine hydrochloride. Cells were ruptured by passage through a French pressure cell (Aminco, Urbana, Ill.), and centrifuged at 15,000 × g
for 15 min. The supernatant was then used as a crude enzyme. The protein concentration was determined by the method of Bradford (3
). The purity of the enzyme preparation was examined by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis (SDS-PAGE).
Purification of DesZ and determination of the N-terminal amino acid sequence.
For purification of DesZ, streptomycin sulfate was added to the crude enzyme to a final concentration of 1%. The lysate was kept on ice for 10 min and centrifuged at 15,000 × g for 15 min to remove nucleic acids. The supernatant was recovered and then centrifuged again at 110,000 × g for 60 min. Enzyme purification was performed by the method described below with a BioCAD700E apparatus (PerSeptive Biosystems, Framingham, Mass.). The enzyme solution was applied to a POROS PI (polyethyleneimine) column (16 by 100 mm; PerSeptive Biosystems) previously equilibrated with FEA buffer, consisting of 50 mM Tris-HCl buffer (pH 8.0), 0.1 mM ferrous ammonium sulfate, and 2 mM l-cysteine hydrochloride containing 0.1 M NaCl. The enzyme was eluted with 402 ml of a linear gradient of 0.1 to 0.6 M NaCl. The fractions containing 3MGA dioxygenase activity, which eluted at approximately 0.39 M, were pooled, desalted, and concentrated by centrifugal filtration with a Centriplus YM-10 (Amicon, Beverly, Mass.). The resulting solution was applied to a POROS HQ (quaternized polyethyleneimine) column (4.6 by 100 mm; PerSeptive Biosystems) equilibrated with FEA buffer containing 0.2 M NaCl. The enzyme was eluted with 50 ml of a linear gradient of 0.2 to 0.7 M NaCl. The fractions containing 3MGA dioxygenase activity, which eluted at approximately 0.37 M, were pooled, desalted, and concentrated as described above.
To determine the N-terminal amino acid sequence of DesZ, purified DesZ was separated by SDS-12% PAGE and electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, Calif.). The area on the membrane containing DesZ was cut out and analyzed on a PPSQ-21 protein sequencer (Shimadzu, Kyoto, Japan).
Identification of the reaction product.
The 1-ml assay mixture contained 50 mM Tris-HCl buffer (pH 7.0), 2 mM 3MGA, and the purified DesZ (100 μg of protein). Reactions were carried out at 30°C and stopped by the addition of methanol (final concentration, 25%). Precipitated protein was removed by centrifugation (15,000 × g for 10 min), and the supernatant was diluted 1:10, acidified, and extracted with ethyl acetate. The extract was trimethylsilylated (TMS), and then the derivatives were analyzed by gas chromatography-mass spectrometry (GC-MS). The substrate and reaction products were identified by GC-MS with a model 5971A with an Ultra-2 capillary column (50 m by 0.2 mm; Agilent Technologies). The column temperature was increased initially from 100 to 150°C and then from 150 to 300°C at rates of 20 and 3°C/min, respectively. The temperatures of injection and detection were 220 and 300°C, respectively. The mobile phase was a helium gas, and the flow rate was 1.0 ml/min.
The dioxygenase activity of DesZ was assayed by measuring the substrate-dependent oxygen consumption rate. The 2-ml assay mixture contained GTA buffer consisting of 50 mM 3,3-dimethylglutarate, 50 mM Tris, and 50 mM 2-amino-2-methyl-1,3-propanediol (pH 7.0), purified DesZ (100 μg of protein), and 1 mM 3MGA, PCA, or gallate as a substrate. The reaction was carried out at 30°C, and the oxygen consumption rate was measured with an oxygen electrode (B-505; Iijima Electronics Manufacturing Co., Ltd., Aichi, Japan). One unit of enzyme activity was defined as the amount that consumed 1 μmol of O2 per min at 30°C. Specific activity was expressed as units per milligram of protein. Km and Vmax values were obtained from Hanes-Woolf plots and expressed as means of at least three independent experiments. For kinetic analysis of DesZ, the concentration of 3MGA was changed from 0.01 to 10 mM.
Construction of insertion mutants of S. paucimobilis SYK-6.
The 0.8-kb HincII-NruI fragment in the desZ gene of pBXSM1 was replaced with the 1.3-kb EcoRV fragment carrying the kanamycin resistance gene (kan) from pIK03. The 2.8-kb XbaI-BglII fragment of the resultant plasmid, pBDDZ, containing the inactivated desZ gene, was inserted into pK19mobsacB to generate pKDDZ. The 1.5-kb XbaI-SmaI fragment carrying ligAB of pSSAB was cloned into pK19mobsacB to generate pKAB. This plasmid was then digested with MluI in ligB, blunt ended with KOD DNA polymerase (Toyobo Co. Ltd., Osaka, Japan), and ligated with a blunt-ended 1.0-kb BspHI fragment containing the ampicillin resistance gene (bla) from pUC19. The resulting plasmid was designated pAAB.
pKDDZ and pAAB were introduced into SYK-6 cells by electroporation, and candidates for mutants were isolated as described previously (20
). pKDDZ was further introduced into cells of the ligB
mutant obtained (strain DB), and candidates for desZ ligB
double mutants were isolated. To examine the disruption of each gene, Southern hybridization analysis was performed. Total DNAs of the candidates for desZ
, and desZ ligB
mutants were digested with XhoI, PvuII, or XhoI and PvuII, respectively. The 1.2-kb SmaI-PvuII fragment carrying desZ
, the 1.5-kb XbaI-SmaI fragment carrying ligB
, the 1.3-kb EcoRV fragment carrying kan
, and the 1.0-kb BspHI fragment carrying bla
were labeled with the DIG system (Roche Diagnostics, Indianapolis, Ind.) and used as probes.
Analysis of insertion mutants.
Degradation of 3MGA and gallate by SYK-6 and its mutants was assayed in a 2-ml mixture containing FE buffer, consisting of 50 mM Tris-HCl buffer (pH 7.0), 10% acetone, 10% glycerol, 1 mM FeSO4, 2 mM sodium ascorbate, 1 mM substrate, and cell extract. The assay mixtures for 3MGA and gallate contained 20 and 5 mg of protein, respectively. Reactions were performed at 30°C. A portion of the reaction mixture taken at various sampling points was diluted 1:10, acidified, and extracted with ethyl acetate. The extract was trimethylsilylated and then analyzed by GC-MS as described above.
Degradation of 3MGA in the presence of tetrahydrofolate was assayed in a 2-ml assay mixture containing 100 mM Tris-HCl (pH 8.0), 1 mM 3MGA, 1 mM tetrahydrofolate, and cell extracts of SYK-6 and the desZ ligB mutant (10 mg of protein). Reactions were carried out under anaerobic conditions at 30°C in an anaerobic box (Hirasawa Works Inc., Tokyo, Japan) that contained an atmosphere of 95% N2 and 5% H2. The reaction mixture was analyzed by GC-MS as described above.
Nucleotide sequence accession number.
The nucleotide sequence reported in this paper was deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession number AB110976.