PCRs for amplification of E. chaffeensis
p28-Omp14 and p28-Omp19 promoters were carried out in a 25 μl reaction volume containing 0.2 μM of each primer, 250 ng of purified E. chaffeensis (Arkansas isolate) genomic DNA, 400 μM of each of the four deoxyribonucleoside triphosphates, 1.5 mM MgSO4, 1x native HiFi PCR buffer (60 mM Tris-SO4, 18 mM (NH4)2SO4), 2.5 units HiFi polymerase. After the first denaturation step of DNA at 95°C for 2 min, amplification was carried out for 45 cycles of denaturation at 95°C for 30 s, annealing at 40°C for 30 s and extension at 72°C for 50 s and a final extension at 72°C for 2 min.
Construction of transcription plasmids
The plasmid pMT504 is a G-less cassette plasmid containing two transcription templates cloned in opposite directions to aid in driving transcription from promoters introduced upstream of the G-less cassette sequences . We constructed in vitro transcription templates, pRG147 and pRG198, by cloning the promoter regions of p28-Omp14 and p28-Omp19, respectively, into the pMT504 plasmid at EcoRV site (Figure 1). The promoter sequences selected for preparing these constructs included the sequences starting from the downstream first nucleotide of the termination codon of the upstream gene and up to the transcription start sites of the genes mapped in our previous study . Plasmid pRG147 contained a 553 bp promoter region of p28-Omp14 amplified from genomic DNA using primers RRG217 and RRG695 (Table 1). Similarly, plasmid pRG198 contained a 306 bp promoter region of p28-Omp19 amplified by primers RRG185 and RRG696. All oligonucleotide primers used in this study were designed from the genome sequence data  and were synthesized at Integrated DNA Technologies, Inc. (Coralville, Iowa). Reverse primers for promoter segments included the transcription start sites of the respective promoters but excluding any guanosine residue downstream of the transcription initiation sites. This is to avoid transcription termination caused by incorporation methylated guanosine triphosphate present in the transcription reactions (outlined below under in vitro transcription). The promoter inserts were also cloned in opposite orientation (pRG147R and pRG198R) to serve as negative controls to demonstrate promoter-specific in vitro transcription.
Transcription from pRG147, pRG198 or pMT504 plasmids results in a shorter 125-nucleotide transcripts encoded by a control transcription template positioned downstream of the Chlamydia trachomatis rRNA P1 promoter. The test transcription template contains a 153-nucleotide G-less cassette segments in the opposite direction to the control transcription template. This synthetic template results in the transcription of a 162-nucleotide transcript from the transcription start site for both the p28-Omp14 and 19 gene promoters. Supercoiled plasmids for use in the in vitro transcription assays were prepared using the QIAprep Spin Miniprep kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. The DNA sequences of the promoter templates were verified by restriction enzyme and sequencing analysis.
In vitro transcription assays
In vitro transcription reactions were performed in a 10 μl final reaction volume with the following components; 50 mM Tris-acetate buffer pH 8.0 containing 50 mM potassium acetate, 8.1 mM magnesium acetate, 27 mM ammonium acetate, 80 mM NaCl, 2 mM DTT, 400 μM ATP, 400 μM UTP, 2.1 μM [α-32P]-CTP (800 Ci mmol-1 for radioisotope detection method) or 400 μM CTP (for detection and quantification by real-time reverse transcription PCR), 100 μM sodium salt of 3'-O-methylguanosine 5'-triphosphate, 18 units of RNasin, 5% glycerol, 0.13 pmol of supercoiled DNA template and 1 μl (360 ng) of heparin-agarose purified E. chaffeensis RNAP or 0.5 μl of 1:10 dilution of E. coli core enzyme (Epicenter, Madison, WI) or 0.5 μl of 1:10 dilution of E. coli σ70-saturated holoenzyme (Epicenter, Madison, WI). For enzyme salt tolerance assays, potassium acetate and NaCl concentrations were varied over a range from 0 to 600 mM and 0 to 120 mM, respectively. In transcription reactions using E. chaffeensis recombinant σ70, RNAP holoenzyme was reconstituted by adding 360 ng of recombinant protein to 0.5 μl of 1:10 diluted E. coli core enzyme. Holoenzyme formation was allowed to occur by incubating the mixture on ice for 20 min. To assess the modulatory effect on transcription, 4.0 μg of E. chaffeensis protein lysate (preparation described below) was incubated for 20 min at room temperature with the transcription reaction mixture in the absence of an RNAP to allow binding of proteins to DNA elements of promoter segments. Next, 1 μl of the purified E. chaffeensis RNAP was added to reaction mixture. In general, transcription reactions were incubated at 37°C for varying times of 7.5 min, 15 min or 30 min and the reactions were terminated by adding 7 μl of stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol). Six microliters of the sample was electrophoresed on a 6% polyacrylamide sequencing gel containing 7 M urea. The gels were dried and transcripts were visualized by exposing an X-ray film to the gels. Autoradiographs were scanned on a HP SCANJET 5550 scanner (Hewlett-Packard®).
Isolation of E. chaffeensis RNAP
The RNAP isolation method was a modified version from the heparin-agarose procedure described in [21, 27, 55]. E. chaffeensis Arkansas isolate was grown in confluent DH82 cells (malignant canine monocyte/macrophage cells) in 300 cm2 culture flasks in 1 litre MEM tissue culture medium containing 7% fetal bovine serum (Gibco BRL®) and 1.2 mM L-glutamine . DH82 cultures infected with E. chaffeensis having predominantly reticulate bodies (RB) were harvested 48 h post-infection by centrifugation at 1,000 × g for 10 min at 4°C in an Eppendorf 5810R centrifuge. (All centrifugation steps were performed using this centrifuge.) The purification steps were all performed at 4°C. The pellet was resuspended in 25 ml sucrose potassium glutamate (SPG) buffer (218 mM sucrose, 3.76 mM KH2PO4, 7.1 mM K2HPO4, 5 mM potassium glutamate, pH 7.0) and host cells were lysed in a 40 ml Wheaton homogenizer with pestle A. The lysate was centrifuged at 800 × g for 10 min in 50 ml conical tubes to pellet host cell debris. Subsequent supernatant was centrifuged at 15,000 × g for 10 min to pellet the organisms. The RB pellet was resuspended in 2 ml of freshly prepared lysis buffer [10 mM Tris-HCl (pH 8.0), 10 mM MgCl2,1 mM EDTA, 0.3 mM dithiothreitol (DTT), 7.5% glycerol (vol/vol), 50 mM NaCl, 1x Amersham protease inhibitor mixture, and 150 μg per ml of lysozyme]. Lysis was facilitated by three passages through 27.5 G needle. Sodium deoxycholate (at final concentration of 0.05%) was added to the lysate and the suspension incubated for 30 min at 4°C. The lysate was centrifuged at 10,000 × g for 10 min and the supernatant was collected and clarified by an additional centrifugation step for 5 min.
The clarified supernatant was loaded onto pre-packed heparin-agarose column (type I-S, Sigma®) previously equilibrated with buffer A [10 mM Tris HCl (pH 8.0),10 mM MgCl2,1 mM EDTA, 0.3 mM DTT, 7.5% glycerol and 50 mM NaCl]. The suspension was adsorbed for 60 min at 4°C and the column was washed by gravity with 20 ml of buffer A for complete removal of unbound proteins. The bound proteins from the column were eluted by gravity with buffer A containing 0.6 M NaCl and 0.5 ml fractions were collected. Based on previous analysis and calculation of the void volume of the column, fractions 3-6 were pooled and dialyzed overnight against storage buffer [10 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT, 50% glycerol and 100 mM NaCl] using Slide-A-Lyzer Gamma Irradiated Dialysis Cassette (Thermo Scientific, Illinois, USA). The fractions were stored at -80°C. RNAP activity of the dialyzed fraction was determined by in vitro transcription assay.
Protein concentration of the HA purified RNAP fractions and E. chaffeensis whole-protein lysates were measured with the bicinchoninic acid protein assay reagent (Thermo Scientific, Illinois, USA) with bovine serum albumin as the protein standard.
Proteins were analyzed by electrophoresis in 7.5% sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE), followed by silver staining according to the procedures provided by the manufacturer (G Biosciences, USA) or resolved proteins were transferred onto a nitrocellulose membrane, Hybond-ECL (Amersham Biosciences, Germany), for immunoblot analysis.
Western blot (immunoblot) of RNAP extracts
E. chaffeensis RNAP purified above was subjected to SDS-PAGE and the proteins were electroblotted for 2 h at 70 V to a sheet of nitrocellulose membrane. The membrane blot was blocked in a solution containing 10% nonfat dried milk (NFDM) freshly made in TTBS [0.1% Tween-20 in 100 mM Tris-HCl (pH 7.5) and 0.9% NaCl] for 1 h at room temperature with gentle agitation. The blot was rinsed three times in TTBS and then was incubated for 1 h at room temperature or overnight at 4°C with anti-E. coli σ70 antibody, 2G10 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), diluted 1: 500 in 1% NFDM in TTBS solution. The blot was washed again three times with washing solution and then incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-mouse immunoglobulin G diluted 1:5000 in 1% NFDM in TTBS solution. The blot was rinsed again three more times with TTBS to remove excess secondary antibody and detection was carried out using chemiluminescent detection reagents (Amersham ECL™, GE Healthcare).
Properties of isolated E. chaffeensis RNAP
Assays to determine the salt tolerance of the purified enzyme have been described above. Rifampin/rifampicin is a potent inhibitor of prokaryotic RNAPs, but not for eukaryotic RNAP . As E. chaffeensis RNAP was recovered from organisms grown in eukaryotic cells (DH82), it may be potentially contaminated with eukaryotic RNAP. To confirm that the transcript formation is from E. chaffeensis RNAP but not from eukaryotic RNAP, in vitro transcription assays were performed in the presence of rifampin at a concentration of 25 μg ml-1.
Functional studies with an E. coli RNAP monoclonal antibody (2G10) demonstrated that it can effectively bind to E. coli σ70 and markedly inhibit in vitro transcriptional activity by RNAPs of E. coli  and C. trachomatis . To further assess that in vitro transcriptional activity was due to E. chaffeensis purified RNAP but not from eukaryotic RNAP, we utilized the E. coli monoclonal antibody 2G10 in inhibition assays assuming that it blocks the E. chaffeensis RNAP similar to C. trachomatis RNAP. For this experiment, 4 μg of 2G10-antibody was added in transcription reactions and the production of transcripts were assessed by following the methods described above.
Overexpression and purification of E. chaffeensis RpoD (σ70)
The entire RpoD (σ70 subunit gene) protein coding sequence, identified from the E. chaffeensis Arkansas isolate genome , was amplified by PCR and cloned into the pET32 plasmid (Novagen, Madison, WI) for producing recombinant protein. The PCR was performed using pfu DNA polymerase (Promega, Madison, WI) and with the gene-specific PCR primers, RRG742 and RRG 743 (Table 1). To facilitate directional cloning, NcoI and XhoI restriction enzyme sites were engineered in the PCR product. The PCR product was subsequently cloned into pET32 plasmid at the above restriction sites after digesting both plasmid and inserts and ligating using T4 DNA ligase. Over expression of RpoD protein and its purification was carried out with methods similarly described elsewhere [20, 57]. The concentration of the purified RpoD protein was approximately 180 ng/μl, as determined by protein estimation method (described above).
Quantification of transcription
We carried out quantification of in vitro-generated RNA transcripts of p28-Omp14 and p28-Omp19 promoters by densitometry and TaqMan probe-based real-time RT-PCR. For densitometric analysis, we quantitated the signal intensity of radio actively labelled transcripts on X-ray films using ImageQuant software 5.2 (Molecular Dynamics, Inc., Sunnyvale, CA). For real-time RT-PCR analysis, primers and TaqMan probes for the 162 and 125 nucleotide (nt) G-less cassettes were designed manually and optimized using Vector NTI Advance 11 software (Invitrogen, Carlsbad, CA). The primers and probes used for these assays were listed in Table 1. The TaqMan probe for the 162 nt cassette (RRG765) and the probe for the 125 nt cassette (RRG768) have been labelled with reporter fluorescent dyes TET and ROX and quencher dyes Iowa Black FQ and Iowa Black RQ-Sp, respectively. Real-time RT-PCR was carried out using the SuperScript™ III One-Step RT-PCR reagents (Invitrogen, Carlsbad, CA). Each RT-PCR reaction contained the following: 1x reaction mix (containing 200 μM dNTPs), 5 mM MgSO4, 100 nM of each primer, 150 nM of each TaqMan probe, 1 μl of SuperScript III reverse transcriptase/Platinum Taq mix and 1 μl of in-vitro transcribed RNA sample in a 25 μl volume. Reverse transcription was carried out for 30 min at 48°C followed by a denaturation step of 2 min at 95°C. The PCR amplification was then performed for 40 cycles with each cycle at 94°C for 15 s and 60°C for 30 s. All reactions were carried out in triplicate using a Smart Cycler system (Cepheid, Sunnyvale, CA). The threshold cycle, Ct, values of the samples (containing 4.0 μg of E. chaffeensis protein lysate) were averaged from values obtained from each reaction, and the promoter activity was calculated as a relative level of expression to the reference control in a separate tube. The relative level of expression was calculated using the mathematical model of relative expression ratio in real-time PCR under constant reference gene expression : Ratio = (E
, where E represents the PCR efficiency of one cycle in the exponential phase and was calculated according to the equation: E = 10[-1/slope].
Preparation of E. chaffeensis whole-cell soluble protein lysates
E. chaffeensis organisms were cultivated in vitro in canine macrophage (DH82) cell lines at 37°C or in ISE6 tick cells as described previously [18, 56]. The protocols for E. chaffeensis cell lysate preparations were similar to previously described methods for E. chaffeensis, A. phagocytophilum and other Gram negative bacterial organisms [49, 52, 58]. Twenty five ml of about 80-100% E. chaffeensis infected cultures were harvested using glass beads. The cultures were centrifuged at 15,560 × g for 15 min to recover infected host cells and cell free E. chaffeensis organisms. To release the organisms from host cells, the pellet was resuspended in 10 ml SPK buffer (0.5 K2HPO4, 0.5 M KH2PO4, and 0.38 M sucrose) and sonicated twice for 30 sec at a setting of 6.5 in a Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA). The cell lysates were centrifuged at 400 × g for 5 min and the supernatant containing cell free E. chaffeensis was filtered through a 5 μm and 3 μm sterile isopore membrane filters (Millipore, Billerica, MA). The filtrate containing cell free organisms was centrifuged at 15,560 × g for 15 min at 4°C. The pellet containing E. chaffeensis organisms was washed twice with 1.5 ml of lysis buffer (150 mM Tris-HCl pH 8.0, 100 mM KCl, 10 mM Magnesium Acetate, 1 mM EDTA, 2 mM DTT and 10% glycerol) and the pellet was resuspended in 1 ml of lysis buffer containing protease inhibitors (Roche Diagnostic Labs, Indianapolis, IN). The cell suspension was sonicated four times at 8.5 setting, 30 sec each time to lyse E. chaffeensis organisms. The cell lysates were centrifuged at 15,560 × g for 15 min at 4°C to pellet the insoluble fraction and the supernatant containing soluble proteins of E. chaffeensis was collected into sterile micro centrifuge tubes as 25 μl aliquots containing protease inhibitor mix and stored at -80°C until use. Protein concentration of the protein lysates, prior to adding the protease inhibitor mix, was estimated as described above.
Electrophoretic mobility shift assay (EMSA)
DNA sequence segments spanning one or more putative regulatory sequences of p28-Omp14 or p28-Omp19 gene promoters were amplified from E. chaffeensis Arkansas isolate genomic DNA using sequence specific primers and 5'end biotin-labeled reverse primers (Table 1) and evaluated for their interaction with the protein lysates. EMSA experiments and detection were carried out according to established protocols [57, 58] with a radioactive nucleotide incorporated DNA probes or using the LightShift Chemiluminescent EMSA kit (Pierce Biotechnology, Rockford, Illinois, USA) according to the specifications of the manufacturer. The assay mixtures included a non-specific DNA (salmon sperm DNA or poly dI.dC at a high concentration of 240 μg/ml or 50 μg/ml, respectively) to eliminate non-specific interactions. Briefly, about 1 ng of each of the full length or biotin-labeled partial upstream sequences was used in each reaction together with 5 μg of the E. chaffeensis whole-cell protein lysate. About 50 ng of unlabeled specific probe sequences were used as competitors. Bovine serum albumin (BSA) was included in each experiment as a non-specific protein control. The protein concentration in E. chaffeensis protein lysates used in these experiments was similar to the work reported earlier [41, 49, 58].
We carried out two-tailed t-tests with equal variances for densitometry analysis and unequal variances for the real-time RT-PCR analysis to comparatively analyse the effect of addition of E. chaffeensis whole cell protein lysate on transcription of p28-Omp14 (pRG147) and p28-Omp19 (pRG198) promoters.