Molecular cloning and characterization of Escherichia coli K12 ygjG gene
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Putrescine is the intermediate product of arginine decarboxylase pathway in Escherichia coli which can be used as an alternative nitrogen source. Transaminase and dehydrogenase enzymes seem to be implicated in the degradative pathway of putrescine, in which this compound is converted into γ-aminobutyrate. But genes coding for these enzymes have not been identified so far.
The 1.8-kbp DNA fragment containing E. coli K12 ygjG gene with aer-ygjG intergenic region was examined. It was found that the fragment contains σ54-depended open reading frame (ORF) of 1,380 nucleotides encoding a 459-amino acid polypeptide of approximately 49.6 kDa. The cytidine (C) residue localized 10 bp downstream of the σ54 promoter sequence was identified as the first mRNA base. The UUG translation initiation codon is situated 36 nucleotides downstream of the mRNA start. The YgjG was expressed as a his6-tag fused protein and purified to homogeneity. The protein catalyzed putrescine:2-oxoglutaric acid (2-OG) aminotransferase reaction (PATase, EC 220.127.116.11). The K m values for putrescine and 2-OG were found to be 9.2 mM and 19.0 mM, respectively. The recombinant enzyme also was able to transaminate cadaverine and, in lower extent, spermidine, and gave maximum activity at pH 9.0.
Expression of E. coli K12 ygjG coding region revealed σ54-depended ORF which encodes a 459-amino acid protein with putrescine:2-OG aminotransferase activity. The enzyme also was able to transaminate cadaverine and, in lower extent, spermidine.
KeywordsPutrescine Spermidine Cadaverine Agmatine Nitrogen Limited Condition
Polyamines, such as putrescine and spermidine, are present in virtually all living cells, from bacteria to plant and human cells, with a very important though poorly understood biological role [1, 2]. They can bind to nucleic acids, stabilize membrane and stimulate activity of several enzymes [2, 3, 4, 5]. Despite the proved necessity of intracellular polyamine for optimal cellular growth, polyamine accumulation can lead to inhibition of cellular growth and protein synthesis [6, 7]. Two major metabolic routes of polyamines breakdown have been described, one for the free bases via Δ1-pyrroline (4-aminobutyraldehyde) and the other via N-acetyl derivatives. Acetylation pathway serves to prevent polyamine toxicity in prokaryotes and eukaryotes [1, 8]. Spermidine and spermine are acetylated and further oxidized by polyamine oxidase or either deacetylated or excreted from the cell. Acetyltransferase forming exclusively of N-acetylspermidine has been described in E. coli [9, 10], but neither spermidine-deacetylating activity nor N-acetylspermidine oxidase activity has been detected, suggesting that the N-acetylspermidine is excreted or kept in the inert acetylated form . In the aminotransferase pathway of polyamine degradation, amino groups are converted from polycations to electron acceptors. Polyamine aminotransferase activity was found in some microorganisms [12, 13, 14]. In the case of putrescine, the resulting Δ1-pyrroline is an intermediate product of 4-aminobutyric acid (GABA) metabolism [8, 15, 16]. The putrescine:2-oxoglutarate (2-OG) aminotransferase enzyme (PATase, EC 18.104.22.168) of E. coli was partially purified from the mutant strains [12, 17] and characterized. The synthesis of putrescine aminotransferase is controlled by catabolite repression and nitrogen availability . But the corresponding gene remains to be identified. It was speculated that E. coli K12 ygjG gene (b3073, ) specifies the ω-aminotransferase which either removes the amino groups from compounds with terminal primary amines, or adds amino groups to compounds with an aldehyde group . Putrescine and compounds metabolized to putrescine activate ygjG expression , which suggests a possible role in putrescine catabolism. To elucidate YgjG biochemical functions, we have cloned the ygjG gene, expressed, purified an active recombinant protein and investigated some molecular and biochemical properties of the YgjG enzyme.
Cloning and characterization of the 5'-flanking region of the ygjG gene
The all three recombinant strains were found to have low β-galactosidase activity (below 200 Miller unites ) in LB broth. The strain harboring pBR-lacZ-3 also revealed low levels of expression in all media, pointing that the 282-bp fragment doesn't include noticeable promoter sequence. The strain carried plasmid pBR-σ54-lacZ-2 containing practically σ54 sequence alone, showed 3 – 5 times higher lacZ expression levels than pBR-lacZ-3 in both variant of minimal media. But lacZ activity was dramatically increased in the strain harboring pBR-σ54-lacZ-1, especially under nitrogen limited conditions. The increase was about 9-fold in M9 medium with ammonium and 28-fold in M9 with proline in comparison with pBR-σ54-lacZ-2. Thus, the 56-bp region contains promoter sequence, and we concluded that σ54 promoter localized in the Bgl I-Acc III fragment is the main promoter of ygjG. The Kpn I-Bgl I fragment is strongly essential for ygjG expression in minimal media and activation of ygjG expression (up to 6-fold) under nitrogen limitation. Because enhancer proteins are absolutely necessary for σ54-dependent transcription  we reanalyzed the sequence of this fragment and found some putative binding sites for E. coli activators: two general nitrogen regulatory protein (NRI) binding sites and binding site for integration host factor (IHF) (Fig. 1B). In addition, existence of factor of inversion stimulation (FIS) binding site was suggested http://www.ncbi.nlm.nih.gov:80/. Thus the activation of ygj G expression is complicated, depends on growth conditions and needs further investigations.
The predicted AUG initiation codon for the ygjG gene of 1290 bp (denoted downstream as ORF1) which codes 429-amino acid polypeptide (SWISS-PROT Database, P42588) is situated 126 nucleotides downstream of the mRNA start. Analysis of the nucleotide sequence revealed the other ORF (denoted as ORF2) encoding 459-amino acid protein with UUG initiation codon (Fig. 1B), which is 30 amino acids upstream from an in frame ORF1 start codon. Because both putative ORFs have the potential Shine-Dalgarno (SD) sequence , we decided to express each of them as his6-tag fused recombinant protein to determine if these recombinant constructs actually yield a catalytically active enzyme.
Cloning and expression of the ORF1 and OFR2 coding proteins
The 1.29-kbp (ORF1) and 1.38-kbp (ORF2) DNA fragments of pUC18-ygjG were cloned in translation fusion with N-terminal his6-tag sequence under the control of the T7 promoter in pET15b(+) vector. The resulting plasmids, pET-Ht-ORF1 and pET-Ht-ORF2, were expressed in E. coli BL21(DE3) cells. SDS-PAGE of cellular extracts demonstrated a high level of expression of these proteins. To elucidate which of polypeptides encodes an active enzyme, crude extracts were examined for PATase activity. The essential level of glutamate formation activity was observed in crude cell extracts of the BL21(DE3) harboring pET-Ht-ORF2. It was equal to 2.16 μmol·min-1·mg-1. The BL21(DE3)(pET-Ht-ORF1) strain exhibited only traces of PATase activity. The BL21(DE3) strain transformed with pET15b(+) as a control revealed no activity. Obviously, the UUG is the real initiation codon of the ygjG encoding 459-amino acid protein with a calculated molecular mass (Mr) of 49,644 Da.
Purification and characterization of the ygjG gene recombinant product
In order to confirm the identity of ORF2 as the gene encoding PATase, we purified recombinant protein from the BL21(DE3)(pET-Ht-ORF2) strain by immobilized-metal-affinity chromatography (IMAC) to give a final preparation having a specific activity of 11.68 μmol·min-1·mg-1 with yield of 81%. The purity of Ht-YgjG recombinant protein was about 95%. SDS-PAGE showed one distinctive band with a relative Mr of about 52 kDa. This value well corresponds to the sum of mass of 49.6 kDa for ORF2 coding protein and 2.1 kDa for his6-tag leader peptide.
Some parameters of PATase reaction were determined. The enzyme was active at the alkaline pH, with the maximum activity at pH 9.0 in Tris-HCl buffer and displayed a broad temperature optimum between 20°C and 80°C with maximum activity at 60°C. The K m values for putrescine and 2-OG were determined to be 9.2 mM and 19.0 mM, respectively.
Expression of the lacZ fusions under different growth conditions
β-Galactosidase activity, MUb
M9, Nitrogen Sufficientc
M9, Nitrogen Limitedc
Substrate specificity of purified recombinant Ht-YgjG
Relative activity (%)a
Amino group donorb
Amino group acceptorb
In addition to being involved in polyamine breakdown, the reaction catalysed by PATase appears to be the fourth reaction of the arginine decarboxylase pathway of arginine degradation to succinate via putrescine and GABA in E. coli [15, 27]. We have found that E. coli K-12 ygjG gene (denoted above as ORF2) encodes for enzyme catalyzing PATase reaction. According to published data [21, 22] and our experimental data, ygj G expression is under the control of the σ54 promoter and is induced at minimal salt medium and under nitrogen starvation. Two putative NRI binding sites and IHF binding site in ygjG upstream region proposed to be involved into transcription regulation. The cytidine localized 10 bp downstream from the σ54 promoter sequence was identified as the first mRNA base. The potential SD site (GGAG) is located 6 nucleotides upstream from the UUG initiation codon of ygj G and this distance is functional for E. coli genes . The UUG is initiation codon for about 3% of E. coli proteins  but it is less effective at promoting translation  than is AUG codon. Thus the initiation of translation from UUG codon could be a way to confine the synthesis of the YgjG protein.
We have purified Ht-YgjG recombinant protein and found that pH optimum and substrate specificity of the enzyme, except for GABA, was in a good accordance with data of Kim  obtained for partially purified PATase enzyme from mutant E. coli strain, while the K m value for 2-OG of Ht-YgjG is higher. The K m values of enzymes belonging to the class III aminotransferases are significantly different, from micromolar to millimolar levels, for different substrates (see, for example, http://www.brenda.uni-koeln.de). As mentioned above, the N-terminal sequence of YgjG may be essential for substrate specificity and enzyme activity. According to our preliminary data, K m values for putrescine and 2-OG of unfused YgjG enzyme are 3–4 times lower than those for the fused Ht-YgjG protein (data not shown). Thus, the N-end modification of YgjG by his6-tag leader fusion alters kinetic constants of the native enzyme. Therefore further investigations are needed to more detail characterization of YgjG protein.
We found that E. coli K12 ygjG σ54-depended ORF encodes the 459-amino acid protein. The recombinant enzyme, purified as a fuse with his6-tag leader, possessed the putrescine:2-OG aminotransferase activity. The enzyme also was able to transaminate cadaverine, spermidine and utilized α-ketobutyrate and pyruvate as amino acceptors.
Bacterial strains and plasmids
E. coli strains TG1(K12 supE hsd Δ5 thi Δ (lac-proAB) F'[traD 36 proAB+ lacIqlacZ ΔM15]) and BL21(DE3)(B F- dcm ompT hsdS(rB- mB-) gal λ (DE3)) were used as a recipients, MG1655 (K12 λ-, F-)  was used for gene amplification. Plasmids pUC18 (Fermentas), pBRP  and pET15b(+) (Novagen) were used as a vectors for cloning.
Construction of the recombinant plasmids
The E. coli K12 ygjG gene with upstream aer-ygjG intergenic region was amplified from MG1655 genomic DNA using two oligonucleotides 5'-TTGGATCCGATTAATTTGATTTAGATCGCA-3' and 5'-TTCTGCAGCCTGCGGGCGTACGCGTCG-3' as primers. The 1,8 kb PCR product was digested by Bam HI and Pst I and cloned into pUC18 vector, yielding pUC18-ygjG plasmid. The insertion in pUC18-ygjG was sequenced for frame verification.
The 338-bp Kpn I-Acc I, 56-bp Bgl I-Acc III and 282-bp Kpn I-Bgl I fragments were excised from pUC18-ygjG, fused with lacZ reporter gene and cloned into pBRP vector (pBR322-based plasmid containing pUC18 polylinker region), yielding pBR-σ54-lacZ-1, pBR-σ54-lacZ-2 and pBR-lacZ-3 plasmids, respectively.
The ygjG ORF1 and ORF2 sequences were amplified by PCR from pUC18-ygjG using phosphorilated downstream primers 5'-CCGATATCATGAAAGCACTTAACCGAGAG-3' and 5'-CCGATATCTTGAACAGGTTACCTTCGAG-3', respectively, designed so as to provide translation fusion between his6-tag leader sequence and the gene of interest, and upstream primer 5'-TTCTGCAGCCTGCGGGCGTACGCGTCG-3'. PCR products were cloned into blunted vector pET15b(+)/NdeI-BamHI to construct plasmids pET-Ht-ORF1 and pET-Ht-ORF2, respectively, and insertions were sequenced.
Growth, induction conditions and preparation of cell-free extracts
Strains were routinely grown in LB broth or M9 medium . Nitrogen limited conditions were provided using M9 medium lacking ammonium and supplemented with 2 mM of a proline as nitrogen source. All media contained 100 μg/ml ampiciline. Strains were grown aerobically at 37°C.
For expression of lacZ fusions, E. coli TG1 harboring pBR-σ54-lacZ-1, pBR-σ54-lacZ-2 or pBR-lacZ-3 plasmids were grown overnight in M9 medium, washed by 15 mM NaCl, diluted to optical density at 600 nm (OD600) of 0.07 by LB, M9 or M9 nitrogen limited medium. The resulting cultures were grown until reaching on OD600 of about 3.0 (stationary phase).
The synthesis of his6-tag fused recombinant proteins was induced in BL21(DE3) harboring pET-Ht-ORF1 or pET-Ht-ORF2 plasmid. When the cell density in LB medium had reached OD600 of 1.0, 1 mM isopropyl-β-D-thiogalactopyranoside was added followed by 2 h incubation.
For preparation of cell-free extracts bacteria were harvested by centrifugation and washed twice with TE buffer. The cell pellets were stored frozen for several days at -70°C without significant loss of enzyme activity. Frozen cells were thawed, suspended to 0,025 g (wet weight) per ml in buffer A (20 mM potassium phosphate, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μM PLP), and disrupted by sonication followed by centrifugation to remove debris.
RNA isolation and primer extension start site analysis
The TG1 harboring pUC18-ygjG has been grown in LB, M9 media and under nitrogen limited conditions, and the total RNAs were isolated using RNAsy MiniKit (Sigma) according to the manufacturer's recommendations.
Oligonucleotide 5'-CGCTTCTCAATGAGATTCAGGGCGTGG-3' which is complementary to the region from +44 to +71 relatively to the ygj G ORF2 initiation codon was radiolabeled by T4 polynucleotide kinase (Pharmacia) with γ-[33P]ATP (6000 Ci/mmol).
Aliquots of mRNA (5 μg) were denaturated at 65°C for 10 min, incubated in reaction buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 20 mM DTT) containing 0.5 ng of the labeled oligonucleotide, rTth reverse transcriptase (5 u) (Perkin Elmer) and 1 mM deoxyribonucleotide triphosphates at 37°C for 1 h. The synthesized cDNAs were denaturated at 65°C for 10 min and analyzed by electrophoresis on 6% acrylamide gel. The same primer was used for the DNA sequence ladder run on the same gel. The quantities of synthesized cDNAs were estimated using Typhoon 9210 imager and ImageQuant version 5.2 software (Amersham, Molecular Dynamics).
Purification of Ht-YgjG
The Ht-YgjG was purified from 150 ml of BL21(DE3)(pET-Ht-ORF2) induced culture. Frozen cells (60 mg) were thawed, suspended in 75 ml of buffer B (20 mM Tris-HCl, pH 8.0, 1 mM PMSF) and disrupted by sonication followed by centrifugation to remove debris (10 000 g, 20 min, 4°C). The imidazol and NaCl were added to supernatant up to final concentrations of 50 mM and 500 mM, respectively. The supernatant was applied to a His-Trap column (1 ml) (Pharmacia) equilibrated with the same buffer. The protein was eluted with 10 ml of buffer C (20 mM Tris-HCl, pH 8.0, 400 mM imidazol, 500 mM NaCl). The Ht-YgjG containing fractions were applied on Sephadex G-25 column (Pharmacia) equilibrated with buffer D (20 mM potassium phosphate, pH 7.4, 15% glycerol, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 10 μM PLP) and eluted in the same buffer. The Ht-YgjG containing fractions were combined, aliquoted and stored at -70°C until required.
Enzyme assays and analytical methods
β-Galactosidase assay was performed in cell-free extracts of TG1 cells harboring pBR-σ54-lacZ-1, pBR-σ54-lacZ-2 and pBR-lacZ-3 plasmids according to Miller .
PATase activity was routinely assayed as the formation of L-glutamic acid from putrescine and 2-OG at 37°C. The assay mixture contained (in 0.1 ml of total volume) 100 mM Tris-HCl, pH 9.0, 25 μM PLP, 10 mM putrescine, 10 mM 2-OG. The reaction mixture was preincubated at 37°C for 5 min, and the reaction was started by the addition of cell extract or enzyme solution (5 μl). The reaction was stopped by adding 15 μl of HCl (10%). The analysis of glutamic acid formation was carried out using a Quanta 4000E Capillary Electrophoresis System (Waters) with an uncoated fused-silica capillary (75 μm inner diam. × 60 cm) at 25 kV potential. The injection was performed hydrostatically for 25 s. The separation buffer consisted of 50 mM Tris-base, 25 mM benzoic acid, pH 8.5, 0.25 mM tetradecyl-trimethyl-ammonium bromide (for indirect UV detection at 254 nm). The calibration at three concentrations of the L-glutamic acid (0.025 mM, 0.05 mM, 0.1 mM) and three concentrations of the 2-OG (0.05 mM, 0.1 mM, 0.2 mM) was carried out.
The kinetic constants of the Ht-YgjG were evaluated by varying the concentration of putrescine at a 2-OG concentration of 20 mM or by varying the concentration of 2-OG at a putrescine concentration of 15 mM. The enzyme kinetics data were fitted to Michaelis-Menten kinetics and K m values were calculated. The substrate specificity of Ht-YgjG was determined using assay as described above with different polyamines and amino acids as amino group donors and 2-OG as acceptor, or with different acceptors and putrescine as amino group donor.
We thank Prof. Yuri I Kozlov, Dr. Mikhail M Gusyatiner, Dr. Tatiana A Yampolskaya and Dr. Vladimir M Belkov for useful discussions and thoughtful criticisms of the manuscript and Anna E Novikova for help in providing capillary electrophoresis.
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