Institute of Medical Microbiology, University of Regensburg, Franz-Josef-Strauß Allee 11, D-93053 Regensburg, Germany
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Abstract |
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Introduction |
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Materials and methods |
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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 MuellerHinton agar or in LuriaBertani 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 primersone corresponding to mar position 14451465 preceded by the nucleotides (nt) ATTAAGGATCC generating a 5' BamHI site (in italics), the other corresponding to position 18791859 preceded by the nucleotides ATTATAAGCTT generating a 3' EcoRI site (in italics). The PCR produced two fragments containing the complete marR sequence from position 14451879 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 12991316 and 15051488, 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 freezethaw cycle, bacteria were lysed by sonication (2 x 2.5 min, 75 W) and centrifuged for 30 min at 27 000g. The GSTMarR 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 SDSpolyacrylamide 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 SDS17.5% 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 12991505). For autoradioactive analysis, DNA fragments were labelled at the EcoRI site with [-32P]CTP using Klenow enzyme (Boehringer-Mannheim, Mannheim, Germany). TrisborateEDTA-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 PvuIHindIII DNA fragment of the plasmid pMarO was labelled at the HindIII site attached to the marO sequence at position 1299 either with [
-32P]CTP using Klenow enzyme or with [
-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).
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Results |
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Cultures of E. coli XL-EP1 (MarR) and XL-EP2 (MarR) 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
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
, respectively (Figure 1
). One litre of culture yielded c. 2 mg of protein.
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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 were resolved on SDS17.5% 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
, respectively (Figure 2b
, lanes 2 and 4, respectively).
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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 2c). However, dimerization of wild-type MarR and MarR
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 (8590% dimer), whereas in the case of MarR
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 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
(Figure 3a
). Discrete bands of lower mobility of the probes incubated with both proteins were only observed when 32P-labelled marO was used (Figure 3b
). Two bands (Figure 3a and b
, 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 3b
). However, for MarR
, 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 3a
), and with the more susceptible radioactive technique a retardation band corresponding to complex II was visible only at amounts exceeding 300 ng of MarR
compared with 150 ng of MarR (Figure 3b
).
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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 3c). 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
compared with wild-type MarR (1.5 versus 0.03 µg) was needed for partial protection (Figure 3c
). Analysis of the complementary strand gave the same result (not shown).
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Discussion |
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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 helixturnhelix 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 structurefunction relationship by a mutational analysis of MarR. They confirmed the function of the N-terminus in proteinprotein interaction, and more importantly they defined two regions with features related to a helixturnhelix 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 proteinprotein 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 reduced the formation of homodimers significantly. The ratio of cross-linked dimers to monomers decreased when the concentration of MarR
was reduced, but was not affected in the case of the wild-type protein. This finding argues for a reduced binding constant of MarR
. 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 helixturnhelix motifs responsible for DNA binding remain intact in MarR
, 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 proteinprotein 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
dimers were only detectable at high MarR
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
) 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
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 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
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 1
), indicating that either the six additional C-terminal amino acids present in MarR
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.
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Acknowledgements |
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Notes |
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References |
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Received 23 November 2000; returned 14 February 2001; revised 12 September 2001; accepted 25 September 2001