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.
The protein concentration was estimated following the method described by Bradford with bovine serum albumin as the standard . Proteins were separated by SDS-PAGE (10%) .
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
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.