Bacterial strains, growth conditions, and plasmids
Escherichia coli strain DH5α [F-, deoR, thi-1, gyrA96, recA1, endA1, relA1, supE44, Δ (lacZYA-argF) U169, hsdR17 (r-, m+), φ 80d lacZΔM15, λ-] (Stratagene, La Jolla, CA) was used for standard cloning and transformation procedures. Strain XL1-Blue MRF' [F-, thi-1, gyrA96, recA1, endA1, relA1, supE44, lac, hsdR17 (r-, m+), F' [proAB, lacIqZ)M15, Tn10 [tetr]] E. coli was grown on Luria (L) agar and in L broth plus appropriate antibiotics (ampicillin, 100 μg/mL; kanamycin, 25 μg/mL; tetracycline 15 μg/mL) at 37°C for ~24 hours; if grown in broth, the cultures were aerated (225 rpm).
Bacillus anthracis Sterne toxigenic acapsulate strain 7702 (pXO1+, pXO2-) [7, 35] was grown in Brain Heart Infusion (BHI) broth, L broth, or on Campylobacter agar with 10% (v/v) defibrinated sheep blood (CBA) at 37°C for ~24 hours in 5% CO2 in air or in 10% CO2, 5% O2, 85% N2; when grown in broth, cultures were aerated (225 rpm).
Helicobacter pylori 43504 was grown from frozen stocks for 72 hours on CBA at 37°C, passaged to fresh CBA for an additional 48 hours and then transferred to 25 mL Ham's F-12 plus 2% fetal bovine serum (HyClone, Logan, UT) and incubated for 24 hours at 37°C. All conditions were microaerobic (10% CO2, 5% O2, 85% N2). The bacteria were then centrifuged for 10 min (6,000 × g) and resuspended in 600 μL 0.9% NaCl. Aliquots (100 μL) were spread onto 6 CBA plates and incubated at 37°C for 24 hours. The resultant cultures were used in the assays as described below.
Plasmid pBS [pBluescript II SK (+), Stratagene], pBS-rocF [11], and pBS-barocF (see below) were used in this study.
Molecular biology techniques
Plasmid DNA was isolated by the alkaline lysis method [36], or by using a column chromatography kit (Qiagen, Valencia, CA) for sequencing-grade plasmid. Restriction endonuclease digestions, ligations, and other enzyme reactions were conducted according to the manufacturer's instructions (Promega, Madison, WI or New England Biolabs, Beverly, MA). PCR reactions (50 μL) contained 10 to 100 ng of DNA, PCR buffer, 2.0 to 2.5 mM MgCl2, dNTPs (each nucleotide at a concentration of 0.20 to 0.25 mM), 200 pmol of each primer, and 2.5 units of thermostable DNA polymerase. E. coli was transformed by the calcium chloride method or electroporation.
Cloning of B. anthracis rocF into pBS
The B. anthracis arginase gene, rocF (1104 bp), including the 893 bp coding region, 184 bp upstream, and 27 bp downstream was PCR-amplified from the chromosome using primers DM55(5'-cgggatcc TTAATAATAATGATGGTAGTTGCTTCA-3') and DM56(5'-ccatcgat GCAACTTCTCAGTTGCTTTTCTTACAT-3') [uppercase letters: rocF gene; lowercase letters: Bam HI (underlined) on the forward primer or Cla I (underlined) on the reverse primer]. The PCR product was digested with Bam HI and Cla I and cloned into pBS digested with the same enzymes to generate pBS-barocF. The construct was confirmed by sequence analysis, restriction enzyme digestion (data not shown), and enzyme activity (see below). B. anthracis RocF, with a predicted mass of 32.7 kDa, was shown to be expressed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (data not shown).
Construction of pBS-barocF::kan
To disrupt the B. anthracis arginase gene, the plasmid pBS-kan [37] was digested with Sma I and Hinc II to generate the blunt-ended non-polar kanamycin resistance cassette (~1.3 kb), and pBS-barocF was digested with Hind III (~4.2 kb). The pBS-barocF linearized fragment was rendered blunt-ended by Klenow and ligated to the kanamycin cassette using the Roche Rapid DNA Ligation Kit (Roche Diagnostics Corporation, Indianapolis, IN). The ligation mixture was transformed into E. coli DH5α and doubly antibiotic resistant (kanamycin, ampicillin) transformants were verified by restriction digestion and arginase assay.
Construction of E. coli XL1-Blue MRF' (pQE30-barocF), overexpressing B. anthracis RocF
Plasmid pQE30 was digested with Bam HI and Kpn I, purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). The DNA was dephosphorylated using shrimp alkaline phosphatase. The B. anthracis rocF coding region was amplified from the chromosome using DM184, 5'-GG ggatccAAAAAAGAAATTTCGGTT-3' (with non-B. anthracis sequence designated as underlined capital letters and the Bam HI site in lowercase letters) and DM67, 5'-GG ggtaccCCTTTTAGTTTTTCACCGAATA-3'(with non-B. anthracis sequence designated as underlined capital letters and the Kpn I site in lowercase letters). The ~900 bp PCR product was cloned into E. coli TOP10 using the pCR2.1 vector according to the manufacturer's instructions (TOPO TA Cloning Kit, Invitrogen Life Technologies, Carlsbad, CA 92008). After digestion with Bam HI and Kpn I, the rocF gene was purified from the vector by agarose gel electrophoresis and ligated to the digested, phosphatase-treated pQE30 and transformed into XL1-Blue MRF' to yield pQE30-barocF. Transformants (ampr, tetr) were confirmed via restriction digestion analysis, PCR analysis, and arginase activity (data not shown).
Complementation of the H. pylori arginase mutant with the B. anthracis rocF gene
The B. anthracis rocF coding region was PCR-amplified from pBS-barocF using DM278 (5'-GCGCTGCAGGGATGAAAAAAGAAATTTCGG-3' and DM279 (5'-GCCCATGGCTTCTCAGTTGCTTTTCTTAC-3'). The ~900 bp product was digested with Pst I and Nco I, and cloned downstream of the strong H. pylori urease promoter in pLSU0005 to yield pW7. Plasmid pLSU0005 is a derivative of suicide plasmid pIR203C04 [18] that has an improved multi-cloning site, the ureA promoter and a chloramphenicol resistance cassette and is used for complementation in H. pylori. Plasmid W7 (5 μg) was electroporated into the rocF mutant of H. pylori 26695 [38] and two chloramphenicol resistant transformants were confirmed by PCR analysis using flanking primers (data not shown); clones 4a and 4b were subsequently used.
Purification of the Bacillus anthracis arginase
E. coli XL1-Blue MRF' (pQE30-barocF) was grown overnight, diluted 1:100 in 500 mL of L broth plus ampicillin and tetracycline and the culture was incubated at 37°C, 225 rpm for 3–5 h (OD600 nm = ~0.7). Isopropyl thio-β-D-galactopyranoside (2.5 mM, final concentration) was added and the culture incubated for an additional 3–5 h at 37°C, 225 rpm. The resulting culture was centrifuged at 5,000 rpm (Sorval, SH-3000) for 15 min, 4°C. The supernatant was discarded and the pellet was resuspended in 1/20th to 1/40th the original culture volume in Wash/Lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 15 mM imidazole, pH 8.0). The bacteria were lysed by two passages through a French Press (16,000 psi) and maintained at 4°C throughout the remainder of the purification procedure. The lysates were then centrifuged and the supernatant containing arginase activity was retained. For every 680 μL of cytosol, 320 μL of nickel-nitrilotriacetic acid/ethanol agarose resin (Ni-NTA, Qiagen) was added. This solution was gently mixed end-over-end for 1–2 h at 4°C. The cytosol/Ni-NTA agarose mixture was equally distributed among two, 8.5 by 2.0 cm polypropylene columns. The flow through was reserved in sterile polypropylene test tubes, and the columns were washed 6–8 times with ~10 mL Wash/Lysis buffer per wash. These washes were retained for SDS-PAGE analysis. The protein was eluted using Elution Buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) at 1 mL per elution, for a total of 12 elutions per column. The flow through fractions, washes, and elutions were analyzed for protein by SDS-PAGE, and the elutions were monitored for arginase activity (data not shown). All samples were stored at 4°C or at -20°C in 50% glycerol.
Preparation of arginase-containing extracts
Bacteria were harvested from solid medium using a sterile swab and resuspended in 0.9% NaCl or phosphate-buffered saline (PBS). Broth-grown cells were centrifuged at 16,000 × g for 5 minutes. The pelleted cells were resuspended in 150 μl of 0.9% NaCl or PBS. For whole cell assays, bacteria were directly assayed. For extracts, the suspensions were sonicated (25% intensity, Sonic Dismembrator Model 500, Fisher Scientific, Pittsburgh, PA) in an ice bath twice for 30 sec with a minimum 30 sec rest on ice between pulses. Following sonication, the lysate was clarified by centrifugation at 16,000 × g for 5 minutes. The resulting supernatant was retained on ice and used the same day for determination of arginase activity.
Arginase Assay
Extracts or viable bacteria were characterized using the standard arginase assay described previously [11] or with variations (described below). The sample (25 μl) was added to 25 μL of CoCl2, MnSO4, NiCl2, FeSO4, CaCl2, MgSO4, ZnCl2, or CuSO4 (final concentration of 5 mM, except as noted) or 25 μL of deionized distilled H2O (ddH2O, no metal control). An L-ornithine (0 to 3125 μM) standard curve was generated (extinction coefficient was typically 0.00045 to 0.00070 μM-1). The samples and standards were heat-activated (50–55°C, 30 min) or were maintained on ice for 30 min. Next, 200 μL of buffered 10 mM L-arginine (15 mM MES [2-(N-morpholino)ethanesulfonic acid], pH 6.0; 15 mM Tris, pH 9.0; or 15 mM Bis-Tris Propane [BTP], pH 6.3 or 9.0) was added, and the samples were incubated at 37°C for 1 hour. Enzyme rates were linear at this time point. The reaction was stopped by the addition of acidified ninhydrin (4 mg/mL). After heating for one hour at 90–95°C, the standards and samples were measured spectrophotometrically at 515 nm (Biomate 3, Thermo Spectronic, Rochester, NY) in 1.5 ml cuvettes. Representative data were normalized for protein and are presented as specific activity in pmol or nmol L-ornithine/min/mg protein ± standard deviation with a minimum of two experiments conducted in duplicate or triplicate. Some graphs were converted to log scale since the enzyme activity is much higher under certain experimental conditions. Some experiments were performed using BTP as a buffer, because it had a broad buffering capacity of pH 6.3 to 9.5, which would eliminate any potential buffer effects. It was determined that biochemical intermediates and end products, such as putrescine, spermidine, spermine and urea, did not react with ninhydrin (data not shown).
Protein determinations
Protein determinations were performed by the Bicinchoninic Acid assay (Pierce Chemical Company, Rockford, IL), following the manufacturer's 30 minute method. NaCl or PBS was used as the negative control. The results were calculated by a standard curve using bovine serum albumin.
SDS-PAGE analysis
Proteins were electrophoresed through an SDS-polyacrylamide gel (12%) by standard methods.
Statistical Analyses
An unpaired two-tailed Welch's t test was used to determine statistical relationships (GraphPad Instat 3.05). p < 0.05 was considered significant.