Bacterial strains, plasmids, and growth conditions.
All bacterial strains and plasmids used in this study are listed in . MDR S.
Typhimurium DT104 strains were isolated from cattle in Belgium (strain 543SA98) and France (strain BN10055). Isolate 543SA98 has a frameshift mutation in ramR
resulting in an overexpression of ramA
, and isolate BN10055 has a 2-bp deletion in the putative RamR DNA-binding site located upstream of ramA
). Mutant 14028sΔramR
derived from the susceptible strain 14028s was constructed as previously described (1
). Except where indicated, the bacterial strains were grown overnight at 37°C in Luria-Bertani (LB) broth. The pET15b vector (Novagen, Merck KGaA, Darmstadt, Germany) was used to clone and express the ramR
gene. Escherichia coli
BL21(DE3)pLysS was used as the host strain to overproduce the N-terminally hexahistidine-tagged RamR protein (His6
Bacterial strains and plasmids used in this study
Identification of the ramA transcriptional start site.
RNA was extracted from a culture of the MDR S. Typhimurium strain 543SA98 grown until it reached an optical density at 600 nm (OD600) of 0.5 using the NucleoSpin RNAII kit (Macherey-Nagel, Hoerdt, France). Specific primers (Sigma-Aldrich, Saint-Quentin Fallavier, France) SP1, SP2, and SP3 () were designed to determine the 5′ end of the ramA transcript by using the second-generation 5′/3′ RACE (rapid amplification of cDNA ends) kit (Roche Diagnostics, Bâle, Switzerland) according to the manufacturer's instructions. The resulting PCR product was sequenced by Cogenics (Meylan, France).
Overproduction and purification of RamR.
Chromosomal DNA of the S. Typhimurium 14028s strain was prepared with a QIAamp DNA minikit (Qiagen, Courtaboeuf, France). The ramR gene was amplified by PCR using Dynazyme polymerase (Ozyme, Montigny-Le-Bretonneux, France) and primers ramRXhoI and ramRNdeI (Sigma-Aldrich; ). The PCR product (595 bp) was cut by XhoI and NdeI (Promega, Madison, WI) and cloned into the corresponding cloning site of pET15b. The nucleotide sequence of the resulting pET15bramR recombinant plasmid was confirmed by sequencing. The E. coli BL21(DE3)pLysS strain was transformed with pET15bramR and grown at 20°C in 2YT broth (tryptone, 16 g/liter; yeast extract, 10 g/liter; NaCl, 5 g/liter) containing ampicillin (50 mg/liter) (Fluka Sigma-Aldrich, Saint-Quentin Fallavier, France) and chloramphenicol (30 mg/liter) (Fluka Sigma-Aldrich). At an OD600 of 0.5, the production of recombinant protein was induced by the addition of 1 mM isopropyl-β-d-1-thiogalactopyranoside (Calbiochem, Merck KGaA). Cultures were incubated for 16 h, and bacterial cells were then disrupted by three freezing and thawing cycles in the presence of lysozyme. The soluble protein His6-RamR was purified using Talon metal affinity resin (Clontech Laboratories, Mountain View, CA) with a 20 mM Na2HPO4, 150 mM NaCl, 200 mM imidazole buffer and then by gel filtration on a Superdex S75 column (Pharmacia, GE Healthcare, Waukesha, WI) with a 10 mM HEPES, 150 mM NaCl, 5 mM dithiothreitol, 10% (wt/vol) glycerol buffer. The eluates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to check the purity of the His6-RamR protein.
A fragment of the ramR-ramA intergenic region including the putative binding site of RamR was amplified by PCR using the Go Taq DNA polymerase (Promega) and primers interam3 and interam4 (Sigma-Aldrich; ). The amplicons were 97 bp in length for the S. Typhimurium 14028s strain and 95 bp for the S. Typhimurium BN10055 strain. A control 92-bp amplicon of the gyrB gene was amplified from the S. Typhimurium 14028s strain using primers gyrB4 and gyrB5. The PCR products were purified using the NucleoSpin Extract II kit (Macherey-Nagel) and were digoxigenin (DIG) labeled with the DIG gel shift kit (Roche Diagnostics). The electrophoretic mobility shift assay (EMSA) reaction mixtures had a final volume of 11 μl and contained 15.5 fmol of labeled DNA and 0, 5, 10, or 15 pmol of the purified His6-RamR protein in the binding buffer (36 mM HEPES, 240 mM KCl, pH 7.6). Competition assays were done under the same conditions with 15.5 fmol of the labeled ramR-ramA 97-bp DNA amplicon and various quantities of unlabeled DNA, in the presence of 10 pmol of His6-RamR protein. After 15 min at room temperature, the samples were loaded onto 6% nondenaturing acrylamide gels, electrophoresed at 4°C in 0.5× Tris-buffered EDTA (TBE) buffer, and transferred onto a nylon membrane. The bands revealed by the DIG gel shift kit were visualized with the ChemiSmart 5000 unit and analyzed with the ChemiCapt 50001 software (Vilber Lourmat, Marne-la-Vallée, France).
Hydroxyl radical footprinting (HRF), rather than DNase I footprinting, was chosen because it is not limited by the steric hindrance associated with the DNase I and DNA-binding proteins because of the small size of the diffusing chemical nuclease (hydroxyl radicals). It can therefore provide high-resolution footprinting of DNA-protein complexes and structural detail for them (7
). The two complementary oligonucleotides corresponding to the 97-bp fragment of the ramR-ramA
intergenic region were synthesized (Sigma-Aldrich) and separately 5′ labeled with [γ-32
P]ATP (Perkin-Elmer, Villebon-sur-Yvette, France) at 30 μCi per 10-μl reaction mixture using the T4 polynucleotide kinase (New England BioLabs, Ipswich, MA). Each labeled oligonucleotide was hybridized with its unlabeled complementary oligonucleotide. For binding assays, 100 nM purified radiolabeled DNA fragments were incubated for 15 min at room temperature with 0 or 32 μM His6
-RamR protein in 20 mM HEPES-NaOH, 240 mM NaCl (pH 7.6) buffer. Hydroxyl radical attacks were processed as previously described by Tullius and Dombroski (22
) and adapted by Castaing et al. (3
). Briefly, 10 μl of binding mix was incubated for 2 min at room temperature with 3 μl of fresh and cooled solution containing 0.1% (vol/vol) H2
, 6.7 mM ascorbate, and 0.1 mM [Fe(EDTA)]2−
. Reactions were quenched by addition of 1.8 μl of stop solution containing 80 mM thiourea and 13 mM EDTA. Maxam-Gilbert chemical sequencing reactions were also performed (11
). All samples were analyzed by electrophoresis on a denaturing 7% polyacrylamide gel. Quantification of radioactive signals was performed with the Storm apparatus and the ImageQuant software (Amersham Biosciences, GE Healthcare). The DNA footprints obtained were manually fitted to that deduced from the crystal structure of a complex formed between QacR, a member of the TetR family, and its operator site (Protein Data Bank [PDB] file 1JT0) (20
Measurement of RamR interaction with the ramA operator by SPR.
Surface plasmon resonance (SPR) experiments were performed using a Biacore T100 biosensor instrument (GE Healthcare). The 5′-biotinylated wild-type 97-bp and mutated 95-bp fragments of the ramR-ramA intergenic region, as well as the 92-bp control fragment of the gyrB gene, were immobilized to a level of 300 to 450 resonance units (RU) onto a neutravidin-coated CM5 sensor chip (GE Healthcare). Binding analyses were carried out at 25°C and at a flow rate of 30 μl/min. The His6-RamR purified protein was diluted in the running buffer (10 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.05% [vol/vol] Tween 20, and 5 g/liter bovine serum albumin [BSA], pH 7.4) and injected over the sensor surface in 2 replicates for 5 min. Dissociation was recorded for 5 min. Regeneration of the surfaces was performed with 10 mM Tris, 2 M NaCl for 1 min followed by washes for 5 min. Binding curves were corrected for nonspecific background by subtracting the curves obtained with the control fragment and the running buffer alone. The calculations of kinetic or affinity constants were done with the Biacore T100 evaluation software (version 2.02) using two models, the classical single-interaction (1:1) Langmuir model or the conformational-change model. The latter is a two-state reaction model based on the formation of a complex between the analyte and the immobilized ligand followed by a conformational change stabilizing this complex. Results were evaluated with the chi-square test.
Gene expression analysis by qRT-PCR.
Bacteria were grown until mid-log phase (OD600 of 0.6) and harvested by centrifugation. Pelleted cultures were stabilized with RNAprotect bacterial reagent (Qiagen) and stored at −80°C until use. Total RNA was extracted using the RNeasy minikit (Qiagen) according to the manufacturer's recommendations. Removal of residual genomic DNA was performed using the Turbo DNA-free kit (Ambion) and checked by negative PCR amplification of a chromosomal sequence. RNA integrity was checked by electrophoresis in a 1% agarose gel. Total RNAs were reverse transcribed using random hexamers and the Superscript III first-strand synthesis system (Applied Biosystems). Primers used for quantitative reverse transcription-PCR (qRT-PCR) are listed in . Cycling conditions were as follows: 95°C for 5 min followed by 40 cycles of 95°C for 10 s and 60°C for 15 s. After each run, amplification specificity and absence of primer dimer formation were checked with a dissociation curve acquired by heating the PCR products from 60 to 95°C. Relative quantities of transcripts were determined using the standard curve method and normalized against the geometric mean of three reference genes (gmk, gyrB, and rrs). Relative expression of each gene of interest (acrB, ramA, ramR, and tolC) was calculated as the average of three independent RNA samples. A two-tailed Student t test was used to assess significance, using a P value of <0.05 as a cutoff.