A C-terminal 18 amino acid deletion in MarR in a clinical isolate of Escherichia coli reduces MarR binding properties and increases the MIC of ciprofloxacin

Frank Notka, Hans-Jörg Linde,*, Arnd Dankesreiter, Hans-Helmut Niller and Norbert Lehn

Institute of Medical Microbiology, University of Regensburg, Franz-Josef-Strauß Allee 11, D-93053 Regensburg, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
As described recently, the different degree of fluoroquinolone resistance in a pair of sequential clinical isolates of Escherichia coli was due to the increased expression of the regulatory gene marA as a consequence of an 18 amino acid C-terminal deletion in the repressor MarR (MarR{delta}). To further investigate the molecular mechanism of the loss of repressor function, we purified recombinant wild-type and mutated MarR, and tested their respective ability to form dimers and their specific DNA binding properties to the operator region marO. The dimerization capacity was analysed by non-reducing SDS–PAGE and by disuccinimidyl suberate-mediated cross-linking of the recombinant proteins. The binding of MarR was studied using the recombinant proteins and DNA probes containing the two identified binding sites in marO in the presence or absence of specific and non-specific DNA fragments. Dimerization of MarR{delta} was reduced compared with MarR: the dimer portion was 33.8% (MarR) and 12.4% (MarR{delta}) at a protein concentration of 10 ÌM. In mobility-shift assays MarR{delta} showed a highly reduced complex formation. Footprinting analysis confirmed reduced binding of MarR{delta} to its target sites, compared with MarR. The biochemical data are in full agreement with the crystal structure of MarR, which shows that the N- and C-terminal regions of MarR contribute to dimer formation. The data also indicate a major role of the MarR dimer as opposed to the monomer in DNA binding.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Fluoroquinolone resistance of clinical Escherichia coli isolates was shown to involve target mutations in topoisomerases II (GyrA/B) and IV (ParC/E), often in combination with lowered drug accumulation due to multidrug efflux pumps.1–5 Efflux via the AcrAB efflux system is positively regulated by the transcription factor MarA, which itself is under negative control by the repressor MarR. Many mutations in MarR were demonstrated to abolish repressor function in vitro (for a review see Alekshun & Levy6). We recently described an 18 amino acid C-terminal deletion in the repressor MarR (MarR{triangleup}) leading to apparent loss of repressor function in one of two sequential highly related clinical isolates of E. coli.7 In this study we investigated the binding properties of the wild-type and the mutant MarR of these two strains. We purified recombinant wild-type and the mutant MarR, and compared their capacity to form dimers and to bind their designated target DNA. Furthermore, the binding characteristics of the proteins at the operator region were studied using in vitro DNase I protection assays.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bacterial strains and general microbiological methods

From the same patient we obtained two highly related E. coli isolates (EP1 and EP2) requiring MICs of 16 and 256 mg/L of ciprofloxacin, respectively. Both isolates have identical amino acid substitutions in GyrA and ParC; however, isolate EP2 shows increased expression of marA. The loss of repressor function of MarR in EP2 is caused by the deletion of Ala-1822 in the marR gene (GenBank acces-sion number M96235). The resulting frameshift leads to a C-terminal 18 amino acid deletion in MarR and a change of Asn-126 to Thr.7 Cultures were grown on Mueller–Hinton agar or in Luria–Bertani medium supplemented with 16 or 64 mg/L ciprofloxacin at 37&°C. Ampicillin was used for selective media.

Plasmids and DNA manipulations

For the overproduction and purification of the MarR proteins from strains EP1 and EP2, glutathione S-transferase (GST)–MarR fusion proteins with a thrombin cleavage site were constructed as follows and introduced into E. coli XL-1 blue (Stratagene, La Jolla, CA, USA) resulting in strains XL-EP1 and XL-EP2, respectively. Total genomic DNA from isolates EP1 and EP2 was prepared using the QiaAmp Tissue Kit (Qiagen, Hilden, Germany) and served as template for PCR amplification (MJ Research, Watertown, MA, USA) with two oligonucleotides as primers—one corresponding to mar position 1445–1465 preceded by the nucleotides (nt) ATTAAGGATCC generating a 5' BamHI site (in italics), the other corresponding to position 1879–1859 preceded by the nucleotides ATTATAAGCTT generating a 3' EcoRI site (in italics). The PCR produced two fragments containing the complete marR sequence from position 1445–1879 including the start and stop codon but with the T at position 1821 missing in the EP2 fragment. After digestion with BamHI and EcoRI, the fragments were directionally cloned into the plasmid pGEX-KG8 downstream and in frame of the isopropyl ß-d-thiogalactopyranoside (IPTG)-inducible transcription and translation signals and sequences encoding a GST tag region and thrombin cleavage site, resulting in the plasmids pGEX-marR-EP1 and pGEX-marR-EP2, respectively. After purification, both strands were cycle-sequenced on a 310 DNA sequencer (Perkin-Elmer, Foster City, CA, USA) with plasmid-specific primers. A marO promoter fragment was PCR amplified using genomic DNA from strain EP1 as template and two oligonucleotides corresponding to positions 1299–1316 and 1505–1488, respectively, both including EcoRI restriction sites. The EcoRI-digested DNA fragment was cloned into the corresponding pUC18 site to produce plasmid pMarO. Recombinant DNA techniques, transformation and restriction enzyme digestion were carried out using standard techniques.9

Protein purification

Single colonies of the expression strains XL-EP1 and XL-EP2 were grown overnight in the presence of ampicillin. Gene expression was induced by the addition of IPTG to a final concentration of 1 mM. After 3 h at 37&°C, cells were harvested by low-speed centrifugation. Cell pellets were resuspended in a 10% volume of buffer A [20 mM Tris– HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40 (w/v), 1 mM phenylmethanesulfonylfluoride]. After a freeze–thaw cycle, bacteria were lysed by sonication (2 x 2.5 min, 75 W) and centrifuged for 30 min at 27 000g. The GST–MarR fusion proteins contained in the supernatant were purified by affinity matrix using the Bulk GST Purification Module (Amersham Pharmacia Biotech, Little Chalfont, UK) in a batch procedure according to the instructions of the manufacturer. To elute the MarR proteins from the affinity matrix, a thrombin digestion was carried out for 120 min at room temperature. As a result of the cloning procedure the recombinant proteins contained an additional glycine and serine N-terminal to the native protein after digestion with thrombin.

Cross-linking, protein precipitation and native SDS–polyacrylamide gel electrophoresis

Cross-linking reactions involved pre-incubating MarR derivatives for 20 min at 30&°C in 200 µL buffer consisting of 20 mM sodium phosphate pH 7.5, 150 mM NaCl and 1 mM dithiothreitol. The homobifunctional cross-linker disuccinimidyl suberate (DSS, spacer arm length 11.4 Å; Pierce, Rockford, UK) was dissolved in dimethyl sulfoxide, which was added at 1% of the reaction volume, to give a final DSS concentration of 240 µM. Control samples contained solvent only. Proteins were cross-linked for different time intervals, and the reaction was quenched with 40 mM Tris, and the proteins were precipitated by adding 10% trichloroacetic acid and 0.5 mg/mL sodium deoxycholate. Protein concentration was determined with the DC Protein Assay (Bio-Rad, Hercules, CA, USA) using lysozyme dilutions at defined concentrations as reference. For analysis under non-reducing conditions, 750 ng protein were dissolved in loading dye without ß-mercaptoethanol. Proteins were resolved on SDS–17.5% polyacrylamide gels. The bands were visualized by silver staining and quantified with the Molecular Analyst version 1.4 (Bio-Rad).

Electrophoretic mobility shift and MarR footprinting

Gel-mobility shifts were carried out using the EcoRI-digested mar promoter fragment from plasmid pMarO (nt 1299–1505). For autoradioactive analysis, DNA fragments were labelled at the EcoRI site with [{alpha}-32P]CTP using Klenow enzyme (Boehringer-Mannheim, Mannheim, Germany). Tris–borate–EDTA-buffered 5% and 6% native polyacrylamide gels were used for gel retardation experiments. Labelled fragments were incubated with various amounts of protein and/or competitor DNA as indicated before electrophoresis at 30 mA in 0.5x TBE. For footprint analysis, a PvuI–HindIII DNA fragment of the plasmid pMarO was labelled at the HindIII site attached to the marO sequence at position 1299 either with [{alpha}-32P]CTP using Klenow enzyme or with [{gamma}-32P]ATP using polynucleotide kinase (Boehringer-Mannheim) for the complementary strand. For DNase I digestion, the incubation mixtures contained 3 ng labelled DNA and 0.5 (absence of protein) or 1 U (presence of protein) of DNase I in a total volume of 50 µL and were incubated for 1 min at room temperature. Protein contents varied as indicated. Gels with unlabelled probes were stained with ethidium bromide. When labelled probes were used, the gels were dried and analysed by autoradiography and/or phosphoimaging (Bio-Rad).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Purification of MarR

Cultures of E. coli XL-EP1 (MarR) and XL-EP2 (MarR{triangleup}) were induced with IPTG to produce the MarR proteins fused to a thrombin recognition site and a GST tag encoded by the vector. The molecular mass observed correlated with the calculated mol. wt of 42.3 kDa for the wild-type MarR and 40.3 kDa for the MarR{triangleup} fusion protein. Crude cell extracts were purified and the GST tags were removed. The proteins were >90% pure and had the expected mol. wts of 16.2 and 14.2 kDa for MarR and MarR{triangleup}, respectively (Figure 1Go). One litre of culture yielded c. 2 mg of protein.



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Figure 1. SDS–PAGE of expressed and partially purified recombinant MarR derivatives. (a) Coomassie Blue-stained gel of MarR. (b) Coomassie Blue-stained gel of MarR{triangleup}. Lanes 1, bacterial lysates without induction; lanes 2, bacterial lysates after IPTG induction; lanes 3, partially purified GST fusion proteins; lanes 4, partially purified proteins after thrombin cleavage. The protein standard is indicated on the left.

 
Dimerization of the MarR derivatives

The migration of purified and concentrated MarR proteins as dimers and higher multimers has been observed previously.10 In order to reveal differences in the monomer/ dimer equilibrium, concentrated MarR and MarR{triangleup} were resolved on SDS–17.5% polyacrylamide gels under non-reducing conditions. At protein concentrations of 10 µM, distinct monomer and dimer bands could be visualized and quantification of the stained bands gave a dimer portion of 33.8% and 12.4% for MarR and MarR{triangleup}, respectively (Figure 2bGo, lanes 2 and 4, respectively).



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Figure 2. Cross-link analysis of recombinant MarR ({blacksquare}) and MarR{triangleup} ({square}). Recombinant proteins were incubated with 240 µM DSS cross-linker in the absence (a and c) or presence (c, polyacrylamide gel not shown) of equimolar concentrations of marO DNA [key to part (c): {blacksquare}, MarR; , MarR + DNA; , MarR{triangleup}; {square}, MarR{triangleup} + DNA]. Proteins were precipitated by trichloroacetic acid after the indicated time intervals, resolved on a polyacrylamide gel and silver stained. Samples without disuccinimidyl suberate were loaded on a non-reducing polyacrylamide gel (b). The protein bands were quantified using the Bio-Rad Molecular Analyst version 1.4 and the percentage dimer of the overall protein staining was calculated. The concentration of MarR and MarRD was 3 (a), 10 (b) and 0.75 µM (c), respectively.

 
For an advanced analysis of the dimer formation we used DSS to covalently connect MarR monomers, thereby removing the resulting dimers from the equilibrium reac-tion. At a concentration of 3 µM protein and 240 µM DSS the amount of cross-linked dimers increased in a time-dependent manner with >85% of protein in dimeric complexes after 10 min for MarR (Figure 2aGo, lanes 1–6). MarR{triangleup} was capable of dimer formation, but under conditions used for MarR, the dimer part never exceeded 58% (Figure 2aGo, lanes 7–12). When lysozyme was treated with DSS and recovered at different intervals for control, no di- or multimeric complexes were observed, indicating the specificity of the DSS-mediated cross-link of MarR and MarR{triangleup} (data not shown).

In order to investigate whether the presence of the target DNA influenced dimerization, for example in ways of facilitating close contact of monomeric MarR molecules after binding to the MarR recognition site, a marO DNA fragment was added to the incubation mix at equimolar concentration. Wild-type and mutant MarR proteins at concentrations of 750 nM were pre-incubated in the presence or absence of a marO promoter fragment (750 nM) containing the two known MarR binding sequences. The samples were cross-linked by addition of DSS in a final concentration of 240 µM. The presence of the target DNA did not significantly influence dimerization (Figure 2cGo). However, dimerization of wild-type MarR and MarR{triangleup} was different when the dimer/monomer ratio was compared at protein concentrations of 3 or 0.75 µM. Dimer formation of the wild-type protein was independent from the tested concentrations (85–90% dimer), whereas in the case of MarR{triangleup} the portion of dimers decreased with lower protein concentrations from 58% (3 µM) to 32% (0.75 µM).

Binding of wild-type and mutant MarR to the operator marO

In electrophoretic mobility-shift experiments we tested the capability of the wild-type MarR and MarR{triangleup} to bind to the DNA recognition sites in marO. Analysis of ethidium bromide-stained gels revealed two distinct shifts in the mobility for the wild-type, but not for MarR{triangleup} (Figure 3aGo). Discrete bands of lower mobility of the probes incubated with both proteins were only observed when 32P-labelled marO was used (Figure 3bGo). Two bands (Figure 3a and bGo, indicated by arrows) can most readily be interpreted as corresponding to one MarR dimer (complex I) and two MarR dimers (complex II) bound to marO. With increasing amounts of MarR, complex I decreased whereas complex II increased, indicating a saturation process of MarR binding to the DNA probe (Figure 3bGo). However, for MarR{triangleup}, shifts in mobility could not be detected at 1.8 µg of protein, compared with 225 ng of the wild-type protein in the ethidium bromide-stained gel (Figure 3aGo), and with the more susceptible radioactive technique a retardation band corresponding to complex II was visible only at amounts exceeding 300 ng of MarR{triangleup} compared with 150 ng of MarR (Figure 3bGo).



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Figure 3. Electrophoretic mobilities and DNase I footprint analysis of mar promoter complexes with MarR and MarR{triangleup}. (a) A 150 ng sample of a 207 bp wild-type mar promoter fragment (nt 1299–1505) was incubated alone (lane 1) or with 225 ng, 450 ng, 1.125 µg or 1.8 µg MarR (lanes 2–5, respectively); or with 225 ng, 450 ng, 1.125 µg or 1.8 µg MarR{triangleup} (lanes 6–9, respectively). (b) A 1 ng sample of the 32P-labelled wild-type mar promoter fragment was incubated alone (lanes 1 and 6), with 150 ng, 300 ng, 600 ng or 1.2 µg of MarR (lanes 2–5, respectively), or with 150 ng, 300 ng, 600 ng or 1.2 µg of MarR{triangleup} (lanes 7–10, respectively). The samples were then subjected to electrophoresis, and gels were stained with ethidium bromide (a), or dried, and the DNA was visualized by autoradiography (b). The two postulated MarR dimer–DNA complexes are indicated. (c) A PvuI–HindIII DNA fragment of the plasmid pMarO, 32P-labelled at the HindIII linker attached to the marO sequence at position 1299 was used as probe. A/G represents the A/G sequencing reaction lane used as a reference. In the second lane (0) no MarR derivative was added. The reactions contained 3 ng labelled marO DNA. The amounts of MarR and MarR{triangleup} in each reaction were 30 ng, 150 ng, 750 ng and 1.5 µg (lanes 3–6 and 7–10, respectively). Regions protected by MarR or MarR{triangleup} (site I, site II) and sites of induced hypersensitivity (*) are indicated. Sequences of the protected sites are given at the bottom.

 
Competition of the labelled marO with non-specific (unrelated) or specific (unlabelled marO) competitor DNA was carried out to prove the specificity of MarR binding. For both MarR derivatives 1 ng of unlabelled marO DNA was efficient in competing the labelled target, as was 500 ng of unrelated DNA (data not shown).

DNase protection assays using a 400 nt end-labelled DNA fragment containing 200 nt of marO, confirmed the specific sites I and II for MarR binding identified previously,10 except that we observed a clear hypersensitivity at the start for site I (Thr-1388) and, therefore, defined T-1390 as start of the protected site I (Figure 3cGo). Both recombinant MarR proteins protected the specific target sequences with no obvious difference in protection pattern. However, regarding the amount of protein necessary for DNase protection, at least a 50-fold excess of MarR{triangleup} compared with wild-type MarR (1.5 versus 0.03 µg) was needed for partial protection (Figure 3cGo). Analysis of the complementary strand gave the same result (not shown).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Resistance to fluoroquinolones is generally mediated by mutations in structural or regulatory genes, or a combination of both. Evidence from the analysis of clinical isolates so far indicates a major role in resistance development for the primary targets of fluoroquinolones, the topoisomerases II and IV, and stress response activators, namely MarA, SoxS, Ram and RobA.6,11–13 As reported previously, the contribution of the mar regulon to fluoroquinolone resistance in this genetically highly related pair of clinical E. coli isolates increased the MIC of ciprofloxacin from 16 to >256 mg/L.7 Considering the substantial influence of MarA expression on quinolone resistance,14 more information about the repressor dysfunction of MarR is needed to understand resistance mechanisms against quinolones and other antibiotics. The blocking of efflux pumps is recognized as a strategy of plants to overcome bacterial resistance,15 and has recently become a therapeutic concept.16

Mutations in MarR affecting the repressor function were described throughout the protein and conserved as well as non-conserved positions in the recently recognized family of MarR homologues were affected.6,17 By deletion, the N-terminus was shown to be essential for repressor activity,18 and a conserved helix–turn–helix motif in the family of MarR homologues [AspXArgX5(Leu/Ile)ThrX2Gly] with putative functions in DNA binding has been revealed by computer analysis.11,18,19 Alekshun et al.20 added substantial information to the structure–function relationship by a mutational analysis of MarR. They confirmed the function of the N-terminus in protein–protein interaction, and more importantly they defined two regions with features related to a helix–turn–helix motif with evidential function in DNA binding. The crystal structure of MarR, as published recently by Alekshun et al.,21 shows MarR as a dimer with each subunit containing a winged-helix DNA-binding motif and extensive protein–protein interaction at the N- and C-terminus. In the C-terminal region, residues Lys-140 and Pro-144 are considered especially important for dimer stabilization.

Comparing the MarR proteins derived from the pair of clinical E. coli isolates, we could demonstrate that the C-terminus is of special importance for MarR dimerization and DNA binding. Cross-linking experiments showed a high potential of wild-type MarR to form dimers, whereas the C-terminal deletion of MarR{triangleup} reduced the formation of homodimers significantly. The ratio of cross-linked dimers to monomers decreased when the concentration of MarR{triangleup} was reduced, but was not affected in the case of the wild-type protein. This finding argues for a reduced binding constant of MarR{triangleup}. The addition of marO DNA including the MarR binding sites to the cross-linking incubation mix had only minor influence on the dimerization of wild-type and mutant MarR, indicating that the role of DNA binding for dimerization can be neglected. Moreover, as the conserved helix–turn–helix motifs responsible for DNA binding remain intact in MarR{triangleup}, we speculate that dimerization is a prerequisite for efficient DNA binding, a feature that is often observed with regulatory genes. The CopR repressor,22 the lambda Cro,23 the lambda repressor,24 the tryptophan repressor25 and the Lac repressor26 are a few recent examples where the functional coupling of protein–protein association to gene regulation has been demonstrated.27 This hypothesis is strongly supported by the finding that the deletion of the 18 C-terminal amino acids of MarR reduced DNA binding activity to a much greater degree than dimerization, as demonstrated in mobility-shift and footprint analyses. In mobility-shift analyses, MarR{triangleup} dimers were only detectable at high MarR{triangleup} concentrations, and all retardation bands were disrupted easily with low amounts of competitor DNA. The difference in binding activity is also apparent from the comparison of ethidium bromide-stained agarose gels (no bands with MarR{triangleup}) with radioactive labelling of marO with 32P. Nearly abolished DNA binding of the C-terminal deleted mutant was also observed in footprint analysis. Approximately 50-fold the amount of MarR{triangleup} compared with MarR was needed to obtain comparable footprints.

Alekshun et al.20 also studied a C-terminal deletion mutant (Q121Ochre) that is six amino acids shorter than MarR{triangleup} of the present communication and contains both intact DNA binding motives. Nevertheless, this mutant protein was shown to have a 20-fold reduced DNA binding activity compared with wild-type MarR, and a weak negative complementing phenotype was observed. This phenotype was explained by a lower steady-state level of the protein as demonstrated by western blot analysis. However, MarR{triangleup} in the present study was shown to be expressed as efficiently and as stably as wild-type MarR, as demonstrated by Coomassie Blue-stained gels (Figure 1Go), indicating that either the six additional C-terminal amino acids present in MarR{triangleup} stabilize the protein, or that the lack of protein detection using western blot analysis is rather a matter of antibody recognition. Alekshun et al.20 also consider that a step subsequent to repressor binding may be defective, leading to the observed phenotype. However, with respect to our biochemical analyses, regarding dimerization and in accordance with the crystal structure, it seems conclusive that a preceding step, namely dimerization, may also be disturbed.

The finding of the 18 amino acid C-terminal deletion in a clinical isolate causing a urinary tract infection underscores the significance of this deletion for the acquisition of antibiotic resistance in patients. In full agreement with the recently resolved crystal structure,21 we demonstrated the important role of the C-terminus of the repressor protein MarR for the maintenance of the protein's regulatory function. Two distinct steps involved in the regulation activity of MarR, the dimerization and the DNA binding of MarR, were shown to be reduced to different degrees when the C-terminus of MarR was deleted. The DNA binding property seemed to be more reduced than the dimerization potential although the DNA binding domain was not affected, leading to the conclusion that MarR dimerization is a prerequisite for DNA binding.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Sequencing was carried out by Holger Melzl and Josef Köstler. Christina Paulus gave helpful advice on protein expression and cross-linking experiments.


    Notes
 
* Corresponding author. Tel: +49-941-944-6461; Fax: +49-941-944-6402; E-mail: Hans-Joerg.Linde{at}klinik.uni-regensburg.de Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Received 23 November 2000; returned 14 February 2001; revised 12 September 2001; accepted 25 September 2001