Genes and Proteins Involved in qnrS1 Induction

Bacterial strains and growth conditions.Bacterial strains in this study are summarized in Table 4. All E. coli strains were grown at 37°C in Mueller-Hinton broth (MHB; Difco) or on Mueller-Hinton agar (MHA; Difco). The Keio collection was used for the loss-of-induction screen. This collection comprises 3,985 nonessential, in-frame, single-gene deletions of E. coli K-12 strain BW25113 (36). These deletion strains, which retain a kanamycin resistance marker, were grown at 37°C in Luria-Bertani (LB; Difco) agar or MHB with 30 μg/ml kanamycin for all experiments.

Chemicals.Ampicillin, chloramphenicol, ciprofloxacin, and kanamycin were obtained from Sigma-Aldrich (St. Louis, MO).

Antimicrobial susceptibility testing.Ciprofloxacin MICs were determined on MHA by Etest (bioMérieux, Durham, NC).

Identification of putative binding sites.Proposed promoter sequences were identified using BPROM (www.softberry.com), and DnaA, Ihf, CysB, and other potential binding sites were located using Virtual Footprint with the PRODORIC library (prodoric.de) (20).

Transformation of plasmid with qnrS1 into E. coli recA mutant.A clinical plasmid, pMG306, carrying the qnrS1 gene and an insertion of a selectable chloramphenicol acetyltransferase gene (37) was extracted from E. coli J53 AziR pMG306 by a QIAprep spin miniprep kit (Qiagen Inc., Valencia, CA). The pMG306 plasmid was transformed by electroporation into E. coli DH10B, a recA mutant defective in the SOS response, selecting transformants on LB agar containing 20 μg/ml chloramphenicol.

Construction of truncated qnrS1 plasmids with various upstream sequences.The intact qnrS1 gene and 359 bp of upstream sequence from the start codon were amplified from pMG306 and cloned into pUC18 in an orientation opposite that of the lac promoter of pUC18 in order to avoid its influence on transcriptional activity of the native promoter. The plasmid generated was named pMG322. In a similar manner, three additional plasmids were constructed to contain the qnrS1 gene and 209 (pMG323), 172 (pMG327), 126 (pMG328), 92 (pMG329), and 43 (pMG330) bp of upstream sequence from the start codon of qnrS1. The recombinant plasmids were then introduced into E. coli DH10B by electroporation, selecting transformants on LB agar containing 100 μg/ml ampicillin.

Site-directed mutagenesis of qnrS1 plasmids.Three site-directed mutants of plasmid pMG322 were generated using a QuikChange II site-directed mutagenesis kit (Agilent Technologies, La Jolla, CA) according to the manufacturer's instructions. The primers were chosen for introducing 5- or 6-bp deletions at three sites and generated deletions at upstream sites −209 to −205 (pMG324), −200 to −194 (pMG325), and −194 to −189 (pMG326) (Fig. 1).

Exposure of DH10B strains to ciprofloxacin.E. coli strains carrying a qnrS1 plasmid were grown overnight at 37°C in MHB with 100 μg/ml ampicillin and suspended in fresh MHB to achieve a bacterial density of 1 × 10⁸ CFU/ml. Each bacterial suspension was then diluted 100-fold and incubated with shaking at 37°C until the cultures reached exponential growth at an optical density at 600 nm (OD₆₀₀) of 0.1 to 0.2. Ciprofloxacin was added to the aliquots of the culture to a final concentration of one-half the respective MICs. The bacterial cells were incubated for 30 min at room temperature without shaking. After centrifugation at 3,500 × g for 5 min at 4°C, the pellet was subjected to RNA extraction.

RNA extraction and quantitative real-time PCR of qnrS1.Bacterial mRNA was extracted with the RNeasy minikit (Qiagen, Valencia, CA), followed by digestion with a Turbo DNase-free kit (Ambion, Austin, TX) to remove contaminating DNA, and then was quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE). cDNA was synthesized using 100 ng of total mRNA with a Verso cDNA kit (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer's instructions. qRT-PCR for qnrS1 was carried out with the SsoFast EvaGreen supermix (Bio-Rad, Hercules, CA) and the CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA), with cycles of 20 s at 95°C, followed by 40 cycles of 3 s at 95°C and 3 s at 60°C. The qRT-PCR for lacZ was carried out after exposure to ciprofloxacin with a method identical to that for the β-galactosidase assays and then using cycles of 15 min at 95°C, followed by 40 cycles of 15 s at 95°C and 30 s at 52.5°C. All primers used in this study were designed using Primer3 (version 0.4.0) software (http://frodo.wi.mit.edu/primer3/) and synthesized at the Massachusetts General Hospital DNA Core Facility (Cambridge, MA) or Eton Bioscience (Charlestown, MA) (see Table S2 in the supplemental material). Relative expression levels were calculated using the ΔΔC_T method with expression of mdh used as an internal control, as expression of mdh did not change in response to ciprofloxacin (5). Each experiment was performed at least in triplicate, and at least two independent culture replicates were performed.

Identification of bacterial proteins binding to the qnrS1 upstream region.The identification of specific DNA sequence binding proteins was performed as previously described, with modifications (38). A 359-bp DNA fragment of sequence upstream of qnrS1 (Fig. 1) was amplified by PCR using a purified biotinylated primer (IDT, Coralville, IA), separated by agarose gel, and purified by Freeze ‘N Squeeze DNA gel extraction spin columns (Bio-Rad, Hercules, CA). Twenty μg DNA was immobilized on 2 mg of magnetic beads with covalently coupled streptavidin (Dynabeads M-280; Dynal) according to the manufacturer's instructions. DNA-linked beads were incubated with 200 μg of protein extract in 800 μl of binding buffer (10 mM HEPES, pH 8.0, 60 mM KCl, 4 mM MgCl₂, 0.1 mM EDTA, 0.25 mM dithiothreitol) containing 1 μg of poly(dI · dC), 600 ng of sheared herring sperm DNA, and 10% glycerol for 15 min at room temperature. Beads were washed once with binding buffer containing 5 ng/μl of herring sperm DNA and twice with binding buffer. Proteins were eluted in 100 μl of binding buffer containing 0.5 M NaCl and dialyzed against water for 1 h. The proteins were separated on an SDS-PAGE gel (12%), and protein bands were cut from the gel and identified by mass spectrometry (Taplin Mass Spectrometry Facility, Harvard Medical School, Boston, MA).

Plasmid construction of qnrS1 upstream region fusion to lacZYA operon in pRS415 vector.The 359-bp upstream sequence of qnrS1 (GenBank accession number JX102659) was amplified from pMG306 and cloned into pRS415 in frame to the lac promoter (39). The plasmid generated, named pMG331, was transformed by electroporation into E. coli DH5α, and transformants were selected on LB agar containing 100 μg/ml ampicillin. The plasmid was then extracted, confirmed through sequencing, and transformed by electroporation into E. coli BW25113, the background strain used in the Keio Collection. Transformants were selected on LB agar containing 100 μg/ml ampicillin.

Keio Collection batch transformation with pMG331 for screening.Samples (5 μl) from microtiter plate wells containing individual mutants of the Keio Collection were pooled (48 wells/pool) in 20 ml of LB with 30 μg/ml kanamycin and incubated overnight at 37°C. A 1:10 dilution of the overnight cultures in LB with 30 μg/ml kanamycin was grown to exponential phase, centrifuged at 4,000 rpm, washed four times with cold H₂O, and resuspended in 10% glycerol. Aliquots of 100 μl of each pool were then electroporated with pMG331, and transformants were selected on LB agar with 100 μg/ml ampicillin and 30 μg/ml kanamycin. At least 128 colonies were then patched onto LacTZ agar plates (0.01% tryptone, 0.001% yeast extract, 0.015% Bacto agar, 85 mM NaCl, 1% lactose, 0.005% 2,3,5 triphenyl tetrazolium chloride) with 100 μg/ml ampicillin, 30 μg/ml kanamycin, and 0.002 μg/ml ciprofloxacin together with three controls: parental BW25113 electroporated with pRS415, pMG331, or, as a noninducible negative control, pMG332 (a derivative of pMG331 that is missing region −200 to −188 [CCTAACCCTATC], which is critical for ciprofloxacin induction of qnrS). Of these independent patch colonies, those that resembled the pinkish-red phenotype of pRS415 missing the 12-bp critical upstream region were isolated for screening in a β-galactosidase assay.

Keio Collection β-galactosidase assay phenotype screen.The β-galactosidase assay was performed by growing duplicate 1-ml overnight cultures of prescreened candidate strains alongside the three control strains described in the previous section in a 2.2-ml polypropylene 96-deep-well plate (Thermo Scientific, Wilmington, DE) with 100 μg/ml ampicillin and 30 μg/ml kanamycin by following a modified protocol (40). The strains were then grown to an OD₆₀₀ of 0.4 to 0.5 after a 100-fold dilution into a fresh 96-deep-well plate and incubated with ¼ MIC of ciprofloxacin for 30 min at room temperature. Growth was arrested with 30 μg/ml chloramphenicol, and the density of the cultures was measured using a 96-well flat-bottom plate (USA Scientific, Ocala, FL) and read on a VMax kinetic Maxline microplate reader (Molecular Devices, Sunnyvale, CA) with an OD₆₀₀ filter. The cells were then lysed using PopCulture reagent (EMD Millipore), and a 10-fold dilution of the lysed cells was performed in buffer A (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, and 40 mM β-mercaptoethanol). Using independent culture and internal replicates in 96-well microtiter plates, the β-galactosidase reaction was initiated with 2.5 M 2-nitrophenyl β-d-galactopyranoside (ONPG) and stopped with 300 mM Na₂CO₃ at a time point that provided sufficient ONPG cleavage to read in a spectrophotometer (at 420 μm), and reaction times were recorded. β-Galactosidase activity (in Miller units) was then calculated using a standard equation (41).

Inverse PCR of candidate mutants with reduced ciprofloxacin induction of β-galactosidase.In order to determine the transposon insertion site of screened mutants identified as having altered ciprofloxacin induction, genomic DNA was extracted from the candidate Keio collection strains identified in the β-galactosidase assays using an Easy-DNA gDNA purification kit (Invitrogen, Carlsbad, CA) and suspended in water. To produce microplasmids, 1.5 μg of genomic DNA was then digested in a 10-μl reaction volume using either AgeI, BspHI, or MluI (New England BioLabs, Ipswich, MA), restriction enzymes that produced sticky ends, did not digest the kanamycin resistance determinant and were predicted to produce 1-kb average-sized fragments of the E. coli K-12 genome. The digestion was carried out for 4 h and inactivated for 20 min at the enzyme-specific temperatures. The 10-μl digestion was then ligated in a 100-μl volume at 25°C for 16 h using T4 DNA ligase (New England BioLabs, Ipswich, MA) and cleaned with a QIAquick PCR purification kit (Qiagen, Valencia, CA) in 50 μl of TE buffer. Different sets of primers within the resistance determinant that amplify outward toward the adjacent genes were synthesized (Table 2; Table S2). Inverse PCR was performed using Maxima Hot Start Taq DNA polymerase (Thermo Scientific, Wilmington, DE), a 25-fold dilution of the ligation product, and a final primer concentration of 0.75 μM on a C1000 thermal cycler (Bio-Rad, Hercules, CA) using a 94°C denaturing step for 2 min followed by 30 cycles of 94°C for 30 s, annealing for 35 s, and extending for 100 s at 72°C. Amplicons were detected through gel electrophoresis in a 1% agarose gel and submitted for sequencing to Tufts University Core Facility (Boston, MA).

Candidate gene cloning.ihfA, ihfB, cysB, and dnaA were amplified by PCR and cloned into expression vectors, either pTrcHisC with transformation into E. coli DH5α or pET28a(+) with transformation into E. coli BL21(DE3). Vectors and amplicons were doubly digested with appropriate restriction enzymes and ligated with T4 ligase. Orientation, reading frame, and integrity of cloned genes were confirmed through plasmid sequencing.

Expression and purification of histidine-tagged proteins.E. coli cells with the cloned genes of interest were grown in MHB at 37°C (OD₆₀₀ of 0.8) and then exposed to 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 2 h. The cells were then centrifuged at 10,000 × g for 45 min at 4°C and resuspended in buffer TN (50 mM Tris, pH 7.5, 500 mM NaCl) at a 0.05 volume of the original culture and sonicated on ice for six 30-s cycles with 30-s intermittent rest periods. Benzonase nuclease (Sigma-Aldrich) and 1× ProteoGuard complete protease inhibitor cocktail (Clontech, Mountain View, CA) were added to the lysate and incubated on ice for 30 min, followed by centrifugation at 10,000 × g for 45 min at 4°C and filtering the supernatant through a Steriflip-GP, 0.22-μm, polyethersulfone membrane (EMD Millipore, Billerica, MA). Purification of the histidine-tagged proteins was performed by nickel affinity chromatography (GE Healthcare, Marlborough, MA), using 1-ml fractions and incremental concentrations of imidazole. The fractions were run on SDS-PAGE gels for Coomassie staining and Western blotting (Invitrogen, Carlsbad, CA). The appropriate fractions were then cleared of imidazole using PD-10 desalting columns (GE Healthcare, Marlborough, MA) and concentrated on Amicon Ultra-15 centrifugal filter units (EMD Millipore).

Purification of DnaA followed the method of Smith and Grossman (42), with some modifications. The His₆-DnaA was overexpressed in E. coli BL21 at 30°C and an OD₆₀₀ of ∼0.4, and then 0.4 mM IPTG was added and incubated for 3 h. The 500-ml sample was centrifuged at 8,000 rpm for 10 min at 4°C. The supernatant was discarded, and the pellet was resuspended with 10 ml of Talon equilibration buffer with 5 mM imidazole and stored at −80°C. The sample was thawed at room temperature, and one tablet of protease inhibitor cocktail (cOmplete ultra tablets; Sigma-Aldrich, St. Louis, MO), MgCl₂ to a final concentration of 10 mM, and 2 μl of Benzonase were added. The mixture was stirred for ∼20 min until the viscosity decreased and centrifuged at 10,000 rpm for 20 min at 4°C. The lysed supernatant was filtered and applied to the preequilibrated 5-ml Histalon (TaKaRa Bio, Mountain View, CA) column. The column was washed with 5 column volumes of equilibration buffer, washed with 10 mM imidazole wash buffer, and eluted with 150 mM imidazole elution buffer. The purified sample was desalted with a PD-10 column (GE Healthcare) and concentrated with Amicon Ultra 10K (EMD Millipore). The concentration was measured using a NanoDrop spectrophotometer (ND1000 V3.6.0).

Electrophoretic DNA mobility shift assays.Using a LightShift chemiluminescent EMSA kit (Thermo Scientific, Wilmington, DE), the binding of CysB, DnaA, and a combination of IhfA and IhfB to the labeled 359-bp region upstream of qnrS1 (GenBank accession number JX102659) was assessed. This fragment was amplified using a biotinylated 5′ primer (Integrated DNA Technologies, Coralville, IA) and diluted to ∼1 fmol per reaction. LightShift chemiluminescent EMSA kit controls were run alongside mixtures of biotinylated DNA and various protein concentrations (0.1 μg to 1 μg) on an acrylamide nondenaturing gel, transferred to a 0.45-μm nylon membrane (Thermo Scientific, Wilmington, DE), and detected on autoradiography film (GE Healthcare, Marlborough, MA). Specificity of binding was determined by addition of 100- to 200-fold excess of specific unlabeled DNA in comparison to the same excess of nonspecific salmon sperm DNA. DnaA mobility shift studies were performed similarly except for preincubation of DnaA protein with ATP, as previously described by Bonilla and Grossman (43).

Accession number(s).The intact coding region of qnrS1 and 359-bp upstream sequence was deposited in GenBank under accession number JX102659. Comparison sequences include V. splendidus WP_061032006 and nucleotides 126738 to 127097 in NZ_LNRO01000003, V. parahaemolyticus WP_029823919 and nucleotides 20247 to 20606 in NZ_MSEH01000005, V. mytili WP_041155100.1 and the reverse complement of nucleotides 864 to 1223 in NZ_JXOK01000026.1, and V. ostreicida WP_076586686 and nucleotides 416386 to 415745 in NZ_MPHM01000002.