Proc. Natl. Acad. Sci. USAVol. 92, pp. 5456-5460, June 1995Biochemistry

Binding of purified multiple antibiotic-resistance repressorprotein (MarR) to mar operator sequences

(Escherichia coli/gene regulation/salicylates/DNA-protein interactions)

ROBERT G. MARTIN AND JUDAH L. ROSNERLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892

Communicated by Gunther S. Stent, University of California, Berkeley, CA, March 10, 1995

ABSTRACT Elevated expression of the marORAB multi-ple antibiotic-resistance operon enhances the resistance ofEscherichia coli to various medically significant antibiotics.Transcription of the operon is repressed in vivo by themarR-encoded protein, MarR, and derepressed by salicylateand certain antibiotics. The possibility that repression resultsfrom MarR interacting with the marO operator-promoterregion was studied in vitro using purified MarR and a DNAfragment containing marO. MarR formed at least two com-plexes with marO DNA, bound >30-fold more tightly to it thanto salmon sperm DNA, and protected two separate 21-bp siteswithin marO from digestion by DNase I. Site I abuts thedownstream side of the putative -35 transcription-startsignal and includes 4 bp of the -10 signal. Site II begins 13bp downstream of site I, ending immediately before the firstbase pair of marR. Site II, '80%o homologous to site I, is notrequired for repression since a site 11-deleted mutant(marO133) was repressed in trans by wild-type MarR. Theabsence of site II did not prevent MarR from complexing withthe site I ofmarO133. Salicylate bound to MarR (Kd 0.5 mM)and weakened the interaction of MarR with sites I and II.Thus, repression of the mar operon, which curbs the antibioticresistance of E. coli, correlates with the formation of MarR-site I complexes. Salicylate appears to induce the mar operonby binding to MarR and inhibiting complex formation,whereas tetracycline and chloramphenicol, which neither bindMarR nor inhibit complex formation, must induce by anindirect mechanism.

The emergence of multiply antibiotic-resistant bacteria threat-ens the efficacy of antibiotics in combating infectious diseases(1). Among diverse microbial antibiotic-resistance mecha-nisms, the mar regulon of Escherichia coli is unique in that itis inducible by various agents often used in clinical situations:the antibiotics chloramphenicol and tetracycline (2) and aro-matic weak acids (3) including salicylate and acetylsalicylate.These agents stimulate the transcription of the marORABoperon, thereby initiating a cascade of events leading tomultiple antibiotic and superoxide resistances (4). Specifically,expression of marA, which encodes a putative transcriptionalactivator (5-7), elevates the expression of about 10 unlinkedgenes [the mar regulon (8, 9)] that affect outer membranepermeation (3), antibiotic efflux (10), and the reducing po-tential of the cell (9).Both marR and the operator-promoter region, marO, are

implicated in the regulation of the operon. Since recessivemarR mutations render the operon constitutive and the mu-tants antibiotic resistant (6, 9), it is likely that MarR is arepressor. The marO region (Fig. 1) contains, in addition topotential transcription- and translation-initiation signals, twosets of direct repeats, DR-1 and DR-2 (6). Deletion [marO133(unpublished data)] or duplication [marO (6)] of the contig-

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uous DR-1'-DR-2' sequences result in constitutive expressionof mar. It is not known whether these mutations affect marRexpression or the site of action of MarR.

In an attempt to understand the molecular basis for theregulation of the mar operon, MarR has been purified and itsability to bind to specific marO sequences has been examined.In addition, the interactions of several inducers with MarR andtheir abilities to affect its binding to marO DNA have beenstudied.

EXPERIMENTAL PROCEDURESExpression Vector and Host. To overexpress and purify

MarR, pRGM218, a marR derivative of plasmid pET15b[whose expression of cloned sequences is inducible by isopro-pyl ,B-D-thiogalactoside (IPTG) (Novagen)], was prepared asfollows and introduced into E. coli BL21(DE3), resulting instrain N8219. Plasmid pRGM174 (unpublished data) containsthe entire mar operon from position (pos.) 1311 through 2769(numbering according to ref. 6) connected anticlockwise viaBamHI and EcoRI linkers to plasmid pTA108 at pos. 435 and236 (numbering according to ref. 11). A plasmid with a marABdeletion from pos. 2052-2323 was generated in pRGM174(unpublished results), digested with Pvu II, and religated,eliminating mar nt 2341-2769 and pTA108 nt 236-58(pRGM193). pRGM193 DNA was digested with NdeI andBamHI and the fragment, extending from pos. 1499 withinmarR to the pos. 1 BamHI site of pTA108, was cloned betweenthe NdeI and BamHI sites of pETl5b (pRGM216). PCRamplification, using pRGM193 as template and two oligonu-cleotides as primers-one corresponding to mar pos. 1672-1653, the other to pos. 1446-1466 preceded by the heptanucle-otide CCCCATA (generating a second NdeI site)-produceda fragment that after digestion with NdeI contained mar pos.1445-1499 but with pos. 1445 changed from G to A. When thisoligonucleotide was cloned into pRGM216 at the NdeI site, theresulting plasmid, pRGM218, contained the complete marRsequence (and 250 bp derived from partial marA, marB, andpTA108 plasmid sequences 3' to it) cloned in-frame down-stream of the pET15b IPTG-inducible transcription and trans-lation signals and sequences encoding a histidine-tag regionand thrombin cleavage site. The latter increased the predictedmolecular mass of MarR by 3 kDa.

Protein Purification. N8219 was grown in 2 liters of Super-broth (12) at 37°C with vigorous aeration to late logarithmicphase and induced by the addition of IPTG to 0.4 mM.[Samples taken before and after induction were pelleted andboiled in dithiothreitol/SDS prior to SDS/PAGE (13).] After3 hr, the cells were harvested by centrifugation, resuspended in25 ml of 50 mM Tris, pH 7.5/5 mM dithiothreitol/5 mMEDTA (buffer A), repelleted (wet weight, 6-8 g), and frozen.

Abbreviations: DR, direct repeat; mar, multiple antibiotic-resistanceoperon composed of genes marO, marR, marA, and marB; MarR,protein product of the marR gene; pos., position of nucleotide; IPTG,isopropyl 3-D-thiogalactoside.

5456

Proc. Natl. Acad. Sci. USA 92 (1995) 5457

Ste I Site 11marO marR

I i' ~~II.I

-35 Direct Repeat I -10Sequence Sequence

:....~

Direct Repeat 2 Direct Repeat 2'Direct Repeat 1'

FIG. 1. The marO sequences and first 7 bases of marR (pos. 1379-1451; ref. 6) are shown with the sites reported below for the binding of MarR[shaded ellipses (higher order oligomers are not excluded)]. The AGGGC at pos. 1433-1437 may serve as the ribosome binding site for marR. Theheavy dashed line indicates the 20-bp deletion in marO133. The short arrows indicate inverted repeats within sites I and II. The limits of the sitesare defined by the failure of bands to appear following DNase I digestion in the presence of MarR that do appear in its absence (see Figs. 4 and5). Other probes may show that the sites extend a few bases farther than indicated. The limits of sites I and II are not identical on the two strands.

The cells were thawed, resuspended in 20 ml of buffer A, andsonically disrupted. All further purification steps were carriedout at 4°C. After adding 2.2 g of NaCl and 2 ml of 1 M Tris (pH9.5) to the sonicate, it was centrifuged at 18,000 x g for 1 hr,and the supernatant was loaded on a 500-ml Sephadex G-200column (Pharmacia) equilibrated with 50mM Tris, pH 8.5/0.5M NaCl (buffer B). Fractions were collected and assayed fortheir protein content (BCA protein assay, Pierce). The proteinin the excluded volume was discarded and fractions from thesubsequent protein peak were combined and loaded on a 50-mlbed volume column of chelating Sepharose (Pharmacia) that hadbeen preequilibrated with 2 volumes of 0.1 M NiSO4 followed by4 column volumes ofwater and 4volumes ofbuffer B. The proteinwas eluted with 500 ml of a linear gradient of 0-0.5 M imidazolein buffer B. A minor peak of non-MarR material (based onSDS/PAGE, see below) eluted just prior to MarR at -0.2 Mimidazole. The MarR fractions were combined, brought to 5 mMin EDTA, and dialyzed for 2 hr against buffer composed of 50mM Tris, pH 8.5/1 M NaCl/5 mM EDTA followed by dialysis(generally overnight) against 50 mM Tris, pH 8.5/1 M NaCI/5mM EDTA/5 mM dithiothreitol. Several dialyses were thencarried out against the same buffer lacking EDTA. The materialwas concentrated using Amicon Centriprep 10 Concentrators(W. R. Grace, Beverly, MA), and the histidine tag was removed

1 2 3 4 5

86800 w39°47800 -_.

33300 -_

26600 --

by thrombin digestion and chelating Sepharose or SephadexG-200 chromatography (14).

Labeled marO DNA 32P-end-labeled 197-bp marO DNAfragments were prepared by PCR amplification (20 cycles)using vector pRGM174 or pJLR33 (unpublished data) DNA astemplate, primers corresponding to the BamHI linker of thevector (GATCCT) joined to mar pos. 1312-1326 (operon) andmar pos. 1502-1481 {one of which was end-labeled with[y-32P]ATP (6000 cpm, DuPont) using polynucleotide kinase}and Deep Vent polymerase. Sequencing ladders were pre-pared with the CircumVent thermal sequencing kit. Enzymeswere purchased from New England Biolabs.

Gel Electrophoresis. Tris/EDTA/acetate-buffered 4%polyacrylamide gels were used for gel retardation experiments(13). End-labeled DNA fragments were preincubated for 30min with various amounts of MarR prior to electrophoresis at100 V in 50 mM Tris, pH 8.5/0.5 M NaCl/10% glycerol/0.1%bromophenol blue. Where indicated, the gel and buffer alsocontained 5 mM salicylate, acetylsalicylate, tetracycline, orchloramphenicol. The gels were dried and analyzed by radio-autography and/or PhosphorImaging (Molecular Dynamics)Cell pellets and solutions were analyzed for protein by SDS/PAGE and stained with Coomassie brilliant blue (13).MarR Footprinting and Ligand Binding. Footprint analyses

were carried out as described (15). Ligand binding to MarRwas assayed by the method of Hummel and Dreyer (16) orAhmed et at (17) using radioactive ligands diluted to 0.1 mM:[14C]chloramphenicol (10,270 cpm/10 nmol, DuPont),[7-14C]salicylate (4030 cpm/10 nmol, DuPont), [14C]acetylsa-

MarR (24g)S.s. DNA (gg)marO DNA (gg)

+ + + + + + + + +

0.33 1 3.3 100.33 1 3.3 10

20700 -W e 4111

FIG. 2. Purification and molecular mass of MarR. Protein samplestaken during the purification were analyzed by 20% SDS/PAGE.Lanes: 1, 50 ,ul from strain N8219 before induction; 2, 25 ,ul sampledafter induction; 3, size markers (Bio-Rad); 4, 200 ,ug of crude extract;and 5, 50 ,ug of purified MarR without the histidine tag. The molecularmasses of the markers in lane 3 are indicated in Da.

S 4,ISIEW

FIG. 3. Specificity of MarR binding to marO DNA. Approximately1 ng of a 32P-labeled 197-bp DNA fragment containing the marOregion was mixed with the indicated amounts of unlabeled salmonsperm DNA (S.s.) or marO DNA and preincubated with MarR.

Biochemistry: Martin and Rosner

5458 Biochemistry: Martin and Rosner

licylate (5600 cpm/10 nmol, Sigma), and [3H]tetracycline(31,600 cpm/10 nmol, DuPont).

Expression of the mar Regulon. Assays for mar regulonexpression and induction employing a mar-deleted, inaAl::lacZ reporter strain, N7962, were performed as described (8).

RESULTSPurification of MarR. IPTG-induced cultures of strain

N8219 synthesized large amounts of a protein whose molecularmass of 19 kDa was that expected (by sequence) for MarRlinked to the histidine tag and thrombin recognition signalpeptide encoded by the vector (Fig. 2, lanes 1 and 2). Crudecell extracts were prepared (Fig. 2, lane 4) and purified tohomogeneity, and the histidine tag was removed (Fig. 2, lane5). Two liters of culture yielded about 40 mg of purified proteinwith the expected molecular mass of 16 kDa. The concentratedprotein migrated as dimers and higher multimers on Sephadexgels (data not shown).

Binding of MarR to marO DNA. Binding of MarR to marODNA was indicated by gel retardation experiments. As theratio of MarR to 32P-labeled marO DNA was increased,discrete bands of lower electrophoretic mobility were ob-served; at the highest protein concentrations, the labeledcomplexes failed to enter the gel. An apparent Kd of 10-9wascalculated. The specificity of this binding was indicated by thefinding (Fig. 3) that 0.33 ,jg of marO was more effective in

MarR (gg) 0 0.67 2 6.7

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competing with the labeled complexes than 10 ,.tg (30X more)of salmon sperm DNA.To determine whether the binding of MarR to marO DNA

is site-specific, DNase I protection (footprint) experimentswere undertaken. MarR was found to protect both strands ofmarO DNA at two sites (Fig. 4). While MarR did not seem todiscriminate between site I and site II in most experiments(e.g., Fig. 4), in some, MarR appeared to protect site IIpreferentially (Fig. 5).To determine whether site II is essential for repression by

MarR, marO133, a mutant deleted for the 20 bp immediatelypreceding marR (pos. 1425-1444) but wild-type for marRAB(unpublished data), was studied. marO133 shows high consti-tutive expression of the mar operon: it is multiply antibioticresistant and expresses a mar regulon reporter gene,inaAl::lacZ (8), at a level (1900 Miller units) 44-fold higherthan that of wild-type. However, upon introduction of aplasmid carrying only the wild-type marOR genes, the expres-sion of ,3-galactosidase was reduced >10-fold (136 Millerunits). Thus, marO133 is responsive to MarR but apparentlylacks the ability to synthesize MarR [perhaps due to the loss ofthe putative marR ribosome binding site (AGGGC) in DR-1'].In accord with this, MarR retarded labeled promoter DNAfrom marO133, though less efficiently than the correspondingwild-type promoter, and protected site I from DNase I diges-tion (data not shown). This suggests that such binding sufficesto repress the mar operon.

0 0.67 2 6.7

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..................-.X|-<-t.9s: t / T~~~~~~~~~. - :., & S .it:: .......... -S-;-TA

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FIG. 4. DNase I protection of both strands of marO DNA by MarR. The footprints on the left were prepared using marO DNA 32P-labeledat the 5' end of the BamHI linker attached to the mar sequence at pos. 1312. The sequencing ladder (ACGT) to the extreme left was derived fromthe same 32P-labeled primer. The footprints on the right were prepared using marO DNA labeled at the 5' end of the complementary DNA strandat pos. 1502 as was the sequencing ladder to the extreme right. The concentrations of MarR and times of DNase I digestion are indicated. Bracketsindicate the protected regions.

Proc. Natl. Acad. Sci. USA 92 (1995)

Proc. Natl. Acad. Sci. USA 92 (1995) 5459

0.67 2 6.7 MarR (i.g)

1 2 3 1 2 3 123 Timeol dicestionm"S_ I (iiin)

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FIG. 5. DNase I protection ofmarO DNA by MarR in the absenceor presence of 5 mM salicylate. The footprints and sequencing lanesemployed the marO labeled at pos. 1502 as in the righthand portion ofFig. 4. Sites I and II are indicated.

Binding of Inducers to MarR Protein. To examine thebinding of mar operon inducers to MarR, purified protein (2mg) was loaded on Sephadex G-75 columns equilibrated with0.1 mM radioactive inducer (ligand) and effluent fractionswere analyzed for radioactivity and protein. Comigration ofsalicylate with MarR was readily detected (Fig. 6). An approx-imate Kd of 1 mM was calculated from the measured proteinand ligand concentrations (16). A Kd of 0.5 mM was estimatedby equilibrium dialysis (data not shown). There was no bindingof salicylate to horse myoglobin or egg white lysozyme butbinding to bovine serum albumin-a known salicylate bindingprotein-was readily detected. Substrate specificity was clearlydemonstrated by the failure of MarR to bind acetylsalicylate.Mar R also did not bind to chloramphenicol or nalidixic acid.Binding by tetracycline was marginal and corresponded to a Kdof >10 mM (data not shown).The possibility that the binding of salicylate to MarR

reduces its ability to complex with marO DNA was examinedby assaying the electrophoretic mobility of labeled marO DNAas a function of MarR concentration in the presence or absenceof salicylate. A significant reduction in the retardation ofmarODNA by MarR was found in the presence of 5 mM sodiumsalicylate (compare Fig. 7 A and B, lanes 7 and 8), suggestingthat salicylate inhibits the formation of MarR-marO com-plexes. However, no effect on electrophoretic migration wasseen in analogous experiments using acetylsalicylate, tetracy-cline, or chloramphenicol (data not shown). [These latter

0.14

X 0.12

0.1

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0.06

4 0.04ICIX 0.02

0 5 10 15

Fraction number

FIG. 6. Elution profiles ofMarR (e) from a Sephadex G-75 columnequilibrated with 0.1 mM [7-14C]salicylic acid (0). The molarity ofMarR was computed by dividing the protein (mg/ml) concentration by16,000.

results appear to conflict with those reported for MarR linkedto a maltose binding protein (*).]

Footprint analyses carried out in the presence of 5 mMsalicylate showed decreased MarR protection of sites I and II(Fig. 5). In some (but not all) experiments it appeared thatsalicylate preferentially reduced the protection by MarR of siteI. MarR protection of marO DNA from DNase I digestion wasnot altered by the presence of 5 mM tetracycline or chloram-phenicol (data not shown).

DISCUSSIONThe specific binding of MarR to two sites on marO (Fig. 1)suggests a significant role for these complexes in repression byMarR. Occupancy of either site might interfere with transcrip-tion of the operon. However, analysis of the marO133 mutantshows that site II is not necessary either for MarR binding tosite I or for repression of the mar operon. Similarly, cfxBl, amar constitutive mutant deleted from the second base of DR-2(pos. 1417) well into the marR coding region, is repressible byMarR (9). By binding to site I, MarR could interfere with thebinding of RNA polymerase to the -35 and -10 sequencesnecessary for transcription. The role of site II in the absenceof site I remains to be studied.The extent of sites I and II (a21 bp or -67 A) is not readily

explicable if each site were protected by a single molecule ofMarR. (The calculated diameter of a sphere corresponding toa protein of 16 kDa is -30 A). Since MarR protects closelyspaced sequences on both strands from DNase I digestion, itis unlikely that the DNA wraps around the surface of the MarRmolecule. However, the extent of each site could be explainedif each were protected by a dimer of MarR in which themonomeric unit had an axial ratio of -1.2 (Fig. 1). Consistentwith this hypothesis we find that MarR appears to exist indilute solution as oligomers. The TTGCC pentanucleotidepresent as inverted repeats within DR-1 and DR-i' mightprovide a recognition site for each MarR monomer.mar regulation differs from that of the multidrug efflux

system of Bacillus subtilis in which a single protein, BmrR,binds to a wide variety of inducers (17). Chloramphenicol and

*Seoane, A. S. & Levy, S. B., Abstracts of the General Meeting of theAmerican Society for Microbiology, Las Vegas, NV, 1994, p. 204(abstr. H-26).

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Biochemistry: Martin and Rosner

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5460 Biochemistry: Martin and Rosner

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activation of mammalian transcription factor NF-KB (21), andactivate DNA binding by heat shock transcription factor (22)resists explanation. Detailed analysis of the mar system mayprovide insights.

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FIG. 7. Retardation of electrophoretic movement of marO byMarR in the absence (A) or presence (B) of 5 mM sodium salicylate.Approximately S ng of 32P-labeled marO and 50 ng of salmon spermDNA were incubated with MarR (0, 0.008, 0.024, 0.08, 0.24, 0.8, 2.4,and 8 ,g; lanes 1-8, respectively) and analyzed by electrophoresis andradioautography.

tetracycline, antibiotics that are both inducers and targets ofthe mar operon, do not bind MarR significantly, suggestingthat a metabolite generated by the inhibition of proteinsynthesis is the "true" mar inducer. The high (millimolar)concentrations of salicylate, needed to bind to MarR andweaken the MarR-marO interaction, may indicate that salic-ylate fortuitously resembles this "true" inducer. Curiously,acetylsalicylate did not bind MarR in vitro but may become aninducer in vivo upon hydrolysis to salicylate.

Salicylate plays important regulatory roles in several otherbiological systems. That it regulates genes required for catab-olism of salicylate in Pseudomonas (18) and for resistance tolipophilic protonophores in E. coli (19) is readily rationalizedsince it is a target of these systems. But that it should engendersystemic acquired resistance to infection in plants (20), inhibit

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Proc. Natl. Acad. Sci. USA 92 (1995)