hao et al nat chem biol 2013

8/13/2019 Hao Et Al Nat Chem Biol 2013

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NATURE CHEMICAL BIOLOGY| ADVANCE ONLINE PUBLICATION | www.nature.com/naturechemicalbiology 1

ARTICLEPUBLISHED ONLINE: 3 NOVEMBER 2013 |DOI: 10.1038/NCHEMBIO.1380

The MarR amily o transcription actors regulates diversegenes involved in multiple antibiotic resistance, synthesis ovirulence determinants and many other important biologi-

cal processes13. Various bacterial species employ MarR homologsto sense and exert resistance against many cellular toxins rom theenvironment or host immune system, including multiple antibiotics,detergents, oxidative reagents and disinectants4,5. However, despiteextensive study o activation mechanisms, the true (natural) induc-ers or most members o the MarR amily remain unknown.

E. coliMarR, the prototypical member o the MarR amily oproteins, resides in the chromosomally encoded Mar locus and

negatively regulates the marRABoperon, an essential componentthat controls the Mar phenotype and various cellular responses(e.g., outer membrane permeability, superoxide stress response,DNA repair and metabolic regulation)2. Previous reports indi-cate that diverse phenolic compounds directly bind MarR in vitro.Among these compounds, salicylate (SAL) has been urther shownto trigger the dissociation o MarR rom its promoter DNA andcause the derepression o the marRABoperon within E. colicells6,7.On the basis o these results, direct binding o phenolic substratesto MarR has been proposed to cause MarR derepression insidebacteria4. However, a high concentration o SAL is required toactivate MarR (5 mM), which is less likely to be physiologicallyrelevant3,6. Meanwhile, SAL has also been ound to induce the Marphenotype in a MarR-independent ashion4,8. Moreover, MarR

is known to regulate bacterial resistance to diverse, structurallyunrelated antibiotics including luoroquinolones (e.g., norloxacin(Nor)), -lactams (e.g., ampicillin (Amp)), tetracycline (et) andchloramphenicol (Cm)2. As it is unlikely that a single MarR proteinis capable o directly binding such a structurally dissimilar pool ocompounds with speciicity, it has been speculated that a commoncellular product may be generated when it is exposed to any o these

compounds. his cellular product may unction as the real signalor MarR derepression2,4,6. However, the identity o such a uniiedsignaling molecule remains elusive.

Herein we report that copper(II) ions directly oxidize a cysteineresidue on MarR to generate disulide bonds between two MarRdimers, resulting in the dissociation o MarR rom its cognate pro-moter DNA. We urther demonstrate that this copper(II)-triggeredMarR derepression acts as the underlying mechanism or SAL-mediated derepression o the marRABoperon inside E. coli. SAL aswell as bactericidal antibiotics including Nor and Amp were oundto increase the level o intracellular copper, most likely through oxi-

dative impairment o the bacterial envelope and envelope-residingcopper proteins that include NADH dehydrogenase-2 (NDH-2).his membrane-associated copper oxidation and release processwas urther shown to derepress MarR, thus leading to increasedbacterial resistance to the luoroquinolones used to kill bacterialcells. Our study reveals that copper(II) acts as a natural signal: themaster multiple antibiotic resistance regulator MarR in E. colisensescopper(II) directly, which turns on bacterial deense against anti-biotics, environment-derived stress or both.

RESULTSCopper(II) triggers the dissociation of MarR from DNAo begin, we investigated a potential activation signal (or signals)or MarR. Reactive oxidative species (ROS) are produced rom

host immune response upon bacterial inection9

. Recent researchimplicates ROS as common products generated by dierent typeso bactericidal antibiotics10,11. Although whether the proposed oxi-dative stress or the altered iron homeostasis that can cause directbacterial killing remains controversial12,13, these species do serve assignaling molecules to modulate diverse bacterial responses, as evi-denced by the presence o various oxidation-sensing regulators in

1Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry

and Molecular Engineering, Peking University, Beijing, China. 2Peking-Tsinghua Center for Life Sciences, Beijing, China. 3College of Biological Sciences, China

Agricultural University, Beijing, China. 4Department of Chemistry and Institute for Biophysical Dynamics, University of Chicago, Chicago, Illinois, USA.5International Curriculum Center, High School affiliated to Renmin University, Beijing, China. 6Department of Chemistry, University of CaliforniaBerkeley,

Berkeley, California, USA. 7Shanghai Universities E-Institute for Chemical Biology, Shanghai, China. 8Current address: Cardiovascular Research Institute,

University of CaliforniaSan Francisco, San Francisco, California, USA. 9These authors contributed equally to this work. *e-mail: [email protected]

[email protected]

The multiple antibiotic resistance regulator MarR

is a copper sensor in Escherichia coli

Ziyang Hao1,9, Hubing Lou1,8,9, Rongfeng Zhu2, Jiuhe Zhu3, Dianmu Zhang1, Boxuan Simen Zhao1,4,

Shizhe Zeng5, Xing Chen1, Jefferson Chan6, Chuan He1,4,7* & Peng R Chen1,2,7*

The widely conserved multiple antibiotic resistance regulator (MarR) family of transcription factors modulates bacterialdetoxification in response to diverse antibiotics, toxic chemicals or both. The natural inducer for Escherichia coliMarR, theprototypical transcription repressor within this family, remains unknown. Here we show that copper signaling potentiatesMarR derepression in E. coli. Copper(II) oxidizes a cysteine residue (Cys80) on MarR to generate disulfide bonds between twoMarR dimers, thereby inducing tetramer formation and the dissociation of MarR from its cognate promoter DNA. We fur-ther discovered that salicylate, a putative MarR inducer, and the clinically important bactericidal antibiotics norfloxacin andampicillin all stimulate intracellular copper elevation, most likely through oxidative impairment of copper-dependent envelopeproteins, including NADH dehydrogenase-2. This membrane-associated copper oxidation and liberation process derepressesMarR, causing increased bacterial antibiotic resistance. Our study reveals that this bacterial transcription regulator senses

copper(II) as a natural signal to cope with stress caused by antibiotics or the environment.
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ARTICLE NATURE CHEMICAL BIOLOGYDOI: 10.1038/NCHEMBIO.1380

bacteria such as OxyR, SoxR, MgrA and PerR1417

. Sublethal levelso antibiotics have also been shown to stimulate a ROS-inducedmutagenesis that ultimately leads to bacterial multidrug resis-tance18. We wondered whether related species or processes couldinduce MarRs dissociation rom its cognitive DNA. As H2O2 is amain product o the host immune response that is disproportion-ated rom superoxide14, we tested it irst. We applied an electropho-retic mobility shit assay (EMSA) to 50 nM DNA in the presenceo 1 M wild-type (W) MarR (monomer concentration), whichremained as a proteinDNA complex with and without the addi-tion o 500 M H2O2 (Fig. 1a; these MarR and DNA concentra-tions were used or all o the ollowing EMSA experiments unlessotherwise noted). his observation is in line with previous reportsthat indicate that oxidative stress may only indirectly derepress

marRABoperon in E. colivia other pathways such as SoxS activa-tion19. Furthermore, neither iron(II) nor iron(III) ions were ound tocause MarR-DNA dissociation. We could thereore exclude the pos-sibility that antibiotic-damaged Fe-S clusters release ree iron, whichserves as a natural inducer or MarR. EMSA was then used to testa panel o divalent metal ions, including manganese(II), cobalt(II),nickel(II), copper(II) and zinc(II). Notably, copper(II) was the onlytransition metal within this group to disrupt the MarRDNA com-plex (Fig. 1aand Supplementary Results, Supplementary Figs. 1and 2). In the presence o H2O2, 2.5 M copper(II) or the sameamount o copper(I) was able to eiciently dissociate MarR romits promoter DNA, whereas the addition o a reducing agent such asdithiothreitol (D) eectively restored MarRs DNA-binding ability.his, in combination with our 8-anilino-1-naphthalenesulonate(ANS) assay20monitoring the conormational change o MarR with

and without copper(II) (Supplementary Fig. 3a), indicated that theinteraction between MarR and copper(II) is redox sensitive.o examine whether MarR causes the reduction o copper(II)

to copper(I), we employed a copper(I)-speciic indicator, batho-cuproine disulonate (BCS), which orms a Cu(I)(BCS)23 com-plex with a characteristic UV-absorbance peak at 483 nm (re. 21).itration o the protein-BCS solution (5 M MarR monomer proteinand 200M BCS) with aqueous copper(II) ions indicated a stoichiom-etry o 1:2 between MarR monomer and copper(II) (Fig. 1b andSupplementary Fig. 3b). his result agreed with our 5,5-dithio-bis-2-nitrobenzoic acid (DNB) assay22 (Supplementary Table 1),which indicated that two out o the six cysteine residues on eachMarR monomer can be oxidized by copper(II). Further, the presenceo MarR was ound to eectively prevent radical production rom

copper(II) in the presence o ascorbate (Supplementary Fig. 3c),strongly suggesting that MarR may eiciently compete against cel-lular reductants in scavenging ree copper(II) ions. Finally, all othe six cysteine residues on MarR were either mutated to serine orblocked by iodoacetamide (IAM) beore being subjected to EMSAanalysis, which showed that these MarR variants remained boundto the promoter DNA even ater copper(II) treatment (Fig. 1candSupplementary Fig. 4). aken together, our biochemical studiesrevealed that copper(II) ions can oxidize up to two cysteine residueson each MarR monomer and attenuate MarRs DNA-binding ability.

Copper(II) is a natural inducer for MarR inside E. coliWe next demonstrated that copper(II) is the cognitive signal orMarR derepression inside E. coli. A -galactosidase (-Gal) assaywas irst conducted on an E. coliW strain bearing a marR::lacZ

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Figure 1 | Copper(II) is a natural signal for MarR derepression. (a) 50 nM DNA (42 base pairs) was incubated with 1 M WT MarR (monomer concentration,

lane 2) before being treated with 2.5 M FeCl3, NiCl2, CuCl2(in the presence and absence of 100 M DTT) or 500M H2O2(lanes 37, respectively).

(b) Titration of aliquots of CuCl2into the MarR-BCS solution (5 M MarR monomer and 200 M BCS). Absorption at 483 nm (the characteristic absorption

peak of the Cu(I)(BCS)23complex) was plotted against the copper(II)/protein ratio. Data represent the mean s.d. of three independent experiments.

AU, absorbance unit. (c) EMSA analysis of the DNA-binding capability of MarR6CSmutant (1 M monomer protein and 50 nM DNA) in the absence (lane 2)

and presence (lane 3) of 2.5 M CuCl2. (d) -Gal activity of E. coliWT (M2073) strain containing the marR::lacZreporter was determined after treatment with

100 M CuCl2, FeCl3, Zn(NO3)2or NiCl2or 500 M H2O2in M9 medium for 30 min. -Gal activity is expressed in 4-methylumbelliferyl-D-galactopyranoside

(MUG) units; 1 unit is equal to 1 pmol of MUG cleaved per min per OD600nm. (e) -Gal activity of the marR::lacZreporter was conducted in WT strain (M2073)

and the marRABmutant strain (M2076) upon the addition of 2.5 mM SAL with and without TETA (1 mM). Data in dand erepresent the mean s.d. from three

independent samples (*P< 0.01). (f) The E. coliWT (K12) strain harboring the pL(marO)-GFP reporter was treated with Tet (20 g/ml), Cm (20 g/ml),

Nor (250 ng/ml) or Amp (2.5 g/ml) for 1 h before being analyzed by flow cytometry. AU, arbitrary units.


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ARTICLENATURE CHEMICAL BIOLOGYDOI: 10.1038/NCHEMBIO.1380

reporter gene (M2073) with and without copper(II). he addition o100 M copper(II) ions led to a threeold increase o -Gal activitycompared to untreated cells or cells treated with the same concen-tration o iron(III), nickel(II), zinc(II) or H2O2 (Fig. 1d). Similarly,SAL at a concentration o 2.5 mM was also ound to stimulate overthreeold greater marRexpression compared to untreated samples(Fig. 1e). Because the bacterial cytosol is a highly reduced environ-ment under normal conditions, labile copper ions are thought toexist almost exclusively at the copper(I) state with ew cupric spe-cies. o conirm that copper(II) is the real signal or SAL-triggeredMarR derepression, we added 1 mM o the copper(II)-speciic chela-tor triethylenetetramine (EA) to the SAL-treated bacterial cells23,which eectively diminished SAL-mediated MarR derepression(Fig. 1e), thus veriying that SAL indirectly derepresses MarR via

copper(II) species within E. coli. Finally, the E. coli marRABmutantstrain bearing the marR::lacZreporter (M2076) was used as a con-trol, which exhibited only a slight increase o -Gal activity aterSAL treatment.

We also engineered a GFP-based, marR-inducible luorescentreporter, pL(marO)-GFP, that relies on MarR repression or thetight regulation o GFP expression. Fluorescence-activated cellsorting (FACS) can quantitatively measure the expression o GFPunder the control o the marRpromoter in living bacterial sam-ples. he addition o copper(II) ions was ound to markedly inducethe expression o GFP in the E. coliW (K12) strain, whereasthe addition o iron(III) or H2O2 did not generate a noticeableincrease (Supplementary Fig. 5a). he presence o copper(II)ions also did not cause any noticeable dierence in the luores-cent signal in the marRdeletion strain (Supplementary Fig. 5a).

Further, SAL (2.5 mM) was also ound to trigger GFP inductionin the E. coliW (K12) strain, which can be largely eliminatedby adding 1 mM EA (Supplementary Fig. 5b). In addition, weound that both Nor (250 ng/ml) and Amp (2.5 g/ml) were ableto induce GFP expression and thus MarR derepression within1 h (Fig. 1f), whereas the presence o the EA (1 mM) attenu-ated this MarR-related GFP induction (Supplementary Fig. 6).In contrast, neither et nor Cm was ound to stimulate MarRderepression within the period o time we tested (Fig. 1f).ogether, we veriy that copper(II) not only is an underlying sig-nal or MarR derepression triggered by SAL but also serves as asignal or clinically important antibiotics such as Nor and Ampwithin E. colicells.

Molecular mechanism of copper(II

)-induced MarR activationo elucidate the molecular details o MarRs response mechanism tocopper(II), we investigated potential residues involved in the MarR-copper(II) interaction. As we have shown that cysteine residues wererequired or copper(II)-triggered MarR-DNA disruption (Fig. 1cand Supplementary Fig. 4), we mutated each individual cysteineon MarR to serine or EMSA analysis to identiy the cysteine resi-due (or residues) responsible or this process. Whereas copper(II)caused the dissociation o ive o the six MarR mutants we cre-ated rom DNA in a similar ashion as W MarR (SupplementaryFig. 7ae), the MarRC80Smutant remained bound with its promoterDNA in the presence o copper(II) ions (Fig. 2a). We then mutatedthe other ive cysteine residues on MarR to serine and kept onlya single cysteine at residue 80 to produce the MarR5CS(80C)mutant.he same amount o copper(II) ions (2.5 M) eectively triggered

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Figure 2 | The response mechanism of MarR to copper(II).(a) EMSA analysis showing that copper(II) ions (2.5 M) were unable to affect the

DNA-binding capability of the MarRC80Smutant protein (1 M). (b) -Gal activity of the marR::lacZreporter in the marRABmutant strain (M2076)

complemented with the pET15b WT MarR plasmid (expressing WT MarR) or pET15b MarRC80Splasmid (expressing MarRC80S) was measured in

M9 medium with and without 100 M CuCl2. Results represent mean s.d. n= 5; **P< 0.01. -Gal activity is expressed in 4-methylumbelliferyl-D-

galactopyranoside (MUG) units; 1 unit is equal to 1 pmol of MUG cleaved per min per OD600nm. (c) Size-exclusion chromatography illustrating the

formation of disulfide-stabilized tetramer from MarR5CS(80C)protein in the presence of a catalytic amount of copper(II) under aerobic conditions and at

25 C. MarR5CS(80C)dimer protein (125 M) was incubated with (red) and without (black) 25 M CuCl2for 15 min before being analyzed with a Superdex

75 (10/300) column. The molecular weight (MW) of these apparent tetramers and dimers was calibrated with standard markers, and their corresponding

apparent MWs are shown on top. AU, absorbance unit. (d) Mass spectrum of an unfractionated tryptic peptide mixture of the copper(II)-oxidized

MarR5CS(80C)protein. Inset: The 2+charged peak (m/z: 461463) corresponding to the disulfide-containing peptide of interest (theoretical molecular mass:

920.52 Da). (e) MS/MS fragmentation of the 2+charged peptide (m/z: 461.27).


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the dissociation o 0.5 M MarR5CS(80C)mutant protein rom 50 nMpromoter DNA (Supplementary Fig. 7f). We next perormed theBCS assay on MarR5CS(80C) and copper(II) ions, which showed anapproximately 1:1 stoichiometry between copper(II) and the mono-meric orm o this MarR mutant (Supplementary Fig. 8). Finally,

the E. coli marRABmutant strain bearing the marR::lacZreporterwas complemented with a plasmid expressing either W MarR orMarRC80Sbeore being subjected to -Gal analysis. We ound that100 M copper(II) caused a threeold derepression o marRin themarRAB/W MarR strain but exhibited a negligible eect on marRlevel in the marRAB/MarRC80S strain (Fig. 2b). aken together,although the aorementioned results indicated that MarR has twocopper-oxidizable cysteine residues on each monomer, all o oururther in vitroand bacteria-based results revealed that Cys80 is thekey residue that is required and suicient or copper(II)-inducedMarR-DNA dissociation.

Copper(II)-oxidized W and mutant MarR proteins were nextanalyzed by nonreducing SDS-PAGE, which showed that a cova-lent linkage was generated between Cys80 residues on MarR uponcopper(II) treatment in vitroand inside E. colicells (SupplementaryFig. 9). Size-exclusion chromatography was then used to examinethe oligomeric state o the MarR5CS(80C) protein beore and atercopper(II) oxidation. he naturally occurring MarR dimer proteinwas ound to convert into a disulide-stabilized tetramer in thepresence o catalytic amount o copper(II) under ambient condi-

tions. In particular, a 20% loading o copper(II) was able to convertone equivalent o MarR dimer into the corresponding tetramer,whereas a 4% copper(II) loading led to a more than 60% dimer-to-tetramer conversion (Fig. 2c and Supplementary Fig. 10). ourther reveal the nature o the disulide-stabilized MarR tetramerproduced by copper(II), we perormed MS mapping analysis, whichidentiied disulide-containing peptides (crosslink between Cys80and Cys80on two Leu-Val-Cys-Lys peptides, calculated molecularweight: 920.52 Da) in the copper-treated MarR5CS(80C)and W MarRproteins (Fig. 2d,e and Supplementary Fig. 11). ogether, theseobservations indicate that copper(II) may trigger the ormation o acovalent dimer-o-dimer o MarR via Cys80 residues in a catalyticashion and that the ormation o the irst disulide bond would pro-mote the second disulide bond ormation at the other dimer-dimer

interace upon oxidation.

Crystal structure of copper(II)-oxidized MarR5CS(80C)

o gain more insight into the molecular mechanism o copper( II)-mediated MarR oxidation, we solved the crystal structure o thecopper(II)-oxidized MarR protein. Previous work has showed thatthe ligand-ree MarR protein is much more challenging to crystal-lize because o poor crystal packing24; we were also unable to obtainthe copper(II)-oxidized W MarR protein crystals. Our aoremen-tioned results indicate that the reduced MarR5CS(80C)protein can bindDNA with an ainity similar to that o W MarR, but copper( II)treatment severely disrupts its DNA binding. hereore, the behav-ior o MarR5CS(80C) resembles that o W MarR with and withoutcopper(II). We thus ocused on this MarR mutant and solved the

crystal structure o the copper(II)-oxidized MarR5CS(80C)

protein at2.5 . A symmetric MarR tetramer complex was observed uponcopper(II) oxidation. he tetramer protein contains two disulidebonds between two MarR dimers via Cys80 residues rom eachmonomer (Fig. 3aand Supplementary Fig. 12a). Electron densityo the two disulide bonds was clearly visible, with a measured S-Sdistance at 2.0 and a dihedral angle o C-S-S-C at 90.3 (Fig. 3band Supplementary Fig. 12b). In this tetrameric complex, all o theDNA recognition helices (4) in the DNA-binding domain romeach MarR monomer were buried within the interace betweentwo MarR dimers, rendering them incapable o DNA binding.

Figure 3 | Crystal structure of copper(II)-oxidized MarR5CS(80C).

(a) Copper(II)-oxidized MarR5CS(80C)tetramer with two monomers from

each dimer (chain Achain B and chain Achain B) colored blue and

magenta, respectively. Atoms in the side chains of Cys80 from all four

of the monomers are shown as yellow spheres. (b) Close-up of the twodisulfide bonds between two MarR dimers. Electron density map from the

crosslinked Cys80-Cys80 residues are contoured at 12Fo-Fclevel, with

the disulfide bonds shown in yellow sticks. Only residues Asp67(Asp67),

Cys80(Cys80) and Gly82(Gly82) from each monomer are shown for

clarity. (c) Superposition of the structures of copper-oxidized MarR5CS(80C)

(blue) and the previously reported SAL-complexed WT MarR (orange).

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Figure 4 | SAL- and antibiotic-triggered intracellular copper elevation. (ac) Generation of copper(I) ions inside E. coliWT (K12) cells as measured by

the CS-1 probe (2 M) upon the treatment of SAL (20 mM; a), Nor (250 ng/ml; b) or Amp (2.5 g/ml; c) for 2 h. The fluorescence (in arbitrary units (AU))

of E. coliWT (K12) cells before (black) and after (green) treatment with different reagents were measured by flow cytometry. (d) Flow cytometric analysis

of copper levels as measured by the CS-1 probe inside E. coliWT (K12) and ndhdeletion strains after Nor treatment (250 ng/ml; 2 h). (e) The expression of

E. colicopper-dependent genes cusR, cusA, cueO, cusF, copA, ndhand cyoBin the WT (K12) strain upon Nor treatment (250 ng/ml; 2 h) as determined

by qRT-PCR. The values are shown as the change of Nor-treated cells (black bar) in comparison with untreated cells (gray bar). Data represent the

mean s.d. from three independent experiments.


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he previously reported SAL-complexed W MarR structureresembles the ligand-bound orm o MarR with impaired DNA-binding capability. Superimposition o this dimer structure withthat o the copper(II)-oxidized MarR5CS(80C)protein showed a highlysimilar overall topology (Fig. 3c; r.m.s. deviation = 1.54 or248 C), urther indicating that each o the dimer units within the

copper (II)-oxidized MarR5CS(80C) tetramer adopts a conormationincapable o DNA binding. Finally, we solved the crystal struc-ture o reduced MarRC80S mutant protein in the absence o DNA(Supplementary Fig. 13a), which veriied that the cysteine-to-serine mutations at residues 47, 51, 54, 108 and 111 in MarR5CS(80C)did not cause major conormational changes. Moreover, this struc-ture also adopts a mode incapable o DNA binding, which is a con-served eature among the apo orms o MarR amily proteins thathas been revealed by previously solved apo and DNA-bound struc-tures o OhrR, S1710 and SlyA (Supplementary Fig. 13b)2527.

Antibiotics trigger intracellular copper signalingSo ar, we have shown that copper(II) serves as a natural inducer orMarR and mediates the SAL- and antibiotic-inducedmarRABdere-

pression. We next asked how this copper signal could be generatedwithin E. colicells in the presence o SAL or antibiotics10,28. We irstused a cell-permeable luorogenic copper(I) sensor (CS-1)29to testwhether the amount o intracellular copper could be changed upontreatment o SAL or bactericidal antibiotics such as Nor and Amp.Notably, all o these molecules stimulated a marked increase o theCS-1 luorescent signal, indicating the generation o labile copper(I)ions upon attacking E. colicells (Fig. 4ac). In contrast, when et orCm was used to treat the CS-1bearing E. colicells, we observed nodetectable luorescence increase (Supplementary Fig. 14). In addi-tion, we monitored the susceptibility o E. coliW (K12) and marRdeletion strains to Nor in the presence and absence o copper(II)(Supplementary Fig. 15a,b) or copper (II) chelator (SupplementaryFig. 15c,d), both o which showed that copper(II) ions can enhance

MarR-mediated bacterial drug resistance. Furthermore, FACS anal-ysis on E. coli cells bearing the pL(lexO)-GFP reporter28demon-strated that the addition o copper(II) attenuated Nor-induced DNAdamage in the E. coliW (K12) strain but not in the marRdeletionstrain (Supplementary Fig. 16).

We then went on to identiy the copper source responsible orantibiotic-generated copper stress. In E. colibacteria, the toxic cop-per ions are highly compartmentalized in the cell envelope, and manycopper-containing proteins are located within this space, includingperiplasmic copper-binding proteins such as CueO (a multi-copperoxidase) and CusF (a copper chaperone), inner membrane coppertransporters such as CopA and CusA and membrane-bound copper-dependent proteins such as NDH-2 (a cupric reductase) and CyoB(cytochrome bo(3) ubiquinol oxidase subunit I). As bactericidalantibiotics such as Nor are able to cause bacterial envelope stress30,

we wondered whether these envelope-residing copper proteins arethe targets or antibiotic-triggered copper generation. We employedthe CS-1 probe to examine Nor-induced intracellular labile copperproduction by individually deleting genes encoding the aoremen-tioned representative copper-dependent envelope proteins (Fig. 4dand Supplementary Fig. 17). Among the E. colideletion strains we

tested, the strains lacking either ndhor cyoBwere ound to inducecopper elevation to a lesser extent than the W strain ater Nortreatment, indicating that these cytoplasmic membrane-bound cop-per proteins might be responsible or Nor-triggered copper release.Next, we employed quantitative real-time R-PCR (qR-PCR) toexamine the expression o these genes or genes encoding their regu-lators (or example, the CusA regulator CusR) with and without Nortreatment. All o the copper-related genes we tested were upregu-lated in the presence o a sublethal amount o Nor (250 ng/ml) in2 h, indicating an elevated intracellular copper level triggered byantibiotics (Fig. 4e). We also perormed qR-PCR analysis on genesencoding envelope stress proteins such as CpxA, CpxR and CpxP(all o which belong to the Cpx system) as well as DegP, a majorperiplasmic protein quality control actor (Supplementary Fig. 18).

reatment o Nor increased the expression o all o these envelopestress proteins, and, in particular, the cpxpgene in the Cpx regulonhas been previously shown to be upregulated upon copper libera-tion31, which urther unveiled a connection between Nor-triggeredenvelope stress and copper elevation.

OHP production leads to membrane-bound copper liberationo better understand how antibiotics might promote copper releaserom envelope proteins, we next studied the molecular mechanismunderlying antibiotic-triggered impairment o copper-dependentenvelope proteins. O the genes encoding copper-dependent pro-teins that we tested, ndh caused the most noticeable decrease incopper levels when it was deleted; thereore, we ocused on thisprotein or urther investigation. As a cytoplasmic membrane

located cupric reductase in a bacterial respiration system, NDH-2can be speciically inactivated by tert-butyl hydroperoxide (tBHP),an organic hydroperoxide (OHP) known to cause the impairmento the respiration chain32,33. Particularly, tBHP is able to oxidizecuprous ions to cupric species via the Fenton-like reaction, simi-lar to the conversion o iron(II) to iron(III)34,35. Such an oxidativemodulation o copper ions has been shown to damage the E. colimembrane, which contains multiple copper-binding proteins,including NDH-2 (res. 32,33,36,37). Indeed, our copper sensorCS-1 detected a marked increase in copper in the tBHP-treatedE. coliW strain relative to the ndhdeletion strain, conirming thattBHP is able to mediate the redox cycling o copper within E. coliinner membrane through NDH-2 (Fig. 5a). Furthermore, by usingour previously developed OHP-speciic luorescent sensor-OHSer38and FACS analysis (Supplementary Fig. 19a,b), we showed that

Cellcount

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+ TETA

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Figure 5 | Antibiotic-stimulated OHP production leads to copper liberation and MarR derepression. (a) tBHP (750 M)-induced copper generation inside

E. coliWT (K12) and ndhdeletion strains was examined with CS-1 probe using flow cytometry. AU, arbitrary units. (b) Flow cytometric analysis of E. colicells

harboring the genetically encoded OHP sensor OHSer upon treatment with the various indicated concentrations of Nor for 30 min. (c) Flow cytometric analysis

of E. coliWT (K12) and ndhdeletion strains bearing the pL(copA)-GFP reporter under the treatment of 750 M tBHP for 2 h. (d) Flow cytometric analysis of

E. coliWT (K12) and ndhdeletion strains bearing the pL(marO)-GFP reporter treated by 750 M tBHP in the presence and absence of 1 mM TETA for 1 h.


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the generation o OHPs inside bacteria can be stimulated by Nor,Amp and SAL (Fig. 5band Supplementary Fig. 19c,d) but not etor Cm (Supplementary Fig. 19d). We then employed EMSA toshow that OHPs such as tBHP and cumene hydroperoxide could,in combination with copper(I), impair MarRs DNA-binding abil-ity (Supplementary Fig. 20). hereore, in addition to the aore-mentioned copper(II)-catalyzed disulide bond ormation betweenMarR dimers in the presence o oxygen in the air (Fig. 2c), theseOHPs or additional unknown oxidative species may serve as the

oxidants to promote this copper(II)-triggered MarR dimer-to-tetramer conversion.

Further, we veriied that copper ions released rom these innermembranebound copper proteins were able to promote ree cop-per levels in the MarR-residing bacterial cytosol. We engineered apL(copA)-GFP reporter with GFP expression regulated by the copApromoter, which can be used to sense cytosolic copper. reatmentwith either Nor or tBHP can promote the expression o GFP, indi-cating an enhanced copper level within the E. coli cytoplasm.Furthermore, less GFP generation was observed in the ndh dele-tion strain treated with Nor or tBHP (Fig. 5cand SupplementaryFig. 21), urther conirming that NDH-2 is a major source or Nor-induced copper generation.

Finally, FACS analysis on the pL(marO)-GFP reporterbearing

bacteria showed that both Nor treatment and OHP stress increasedthe luorescent signal to a much lesser extent in the ndhdeletionstain than in the E. coli W strain (Fig. 5d and SupplementaryFig. 22). Moreover, the presence o the copper(II) chelator EAattenuated tBHP-triggered MarR derepression (Fig. 5d), urtherveriying that copper(II) is the underlying cognitive inducer or theobserved MarR derepression. aken together, our results indicatethat the Nor-triggered OHP production caused oxidative impair-ment o cytoplasm membranebound copper proteins such asNDH-2, which led to the generation o oxidized copper(II) speciesthat can be sensed by MarR inside E. coli.

DISCUSSIONhis study uncovers a new connection between antibiotic-triggered

envelope stress and copper signaling and MarR-mediated anti-biotic tolerance in E. coli. Our data presented here demonstratethat copper(II) is an environmental cue that MarR senses directly.Copper(II)-triggered MarR cysteine oxidation derepresses theMarR-regulated antibiotic resistance response in E. coli. We oundthat the previously established SAL-induced MarR derepressionactually goes through the copper-based mechanism, whereas treat-ment with a copper chelator eliminates SAL-triggered Mar acti-vation. We urther discovered that Nor and Amp, two clinicallyimportant bactericidal antibiotics, also stimulate intracellular cop-per elevation, which could trigger MarR derepression and turn onMarR-mediated antibiotic resistance. aken together, we proposethe ollowing mechanistic model or copper(II)-modulated MarRderepression (Fig. 6): the treatment o Nor or Amp is able to induce

envelope stress via protein unolding, mistranslation or bothwithin E. colicells, which subsequently produces OHPs that impaircytoplasmic membranebound copper proteins such as NDH-2 andCyoB, resulting in the release o ree copper(I) ions. his copper(I)liberation, in conjunction with OHP or other oxidative species trig-gered by antibiotics, elevates oxidized intracellular copper(II), whichsubsequently oxidizes the Cys80 residues on MarR to generate twodisulide bonds between two MarR dimers, causing dissociation oMarR rom its cognitive DNA. his process ultimately derepressesMarR and thus the marRABoperon, resulting in enhanced bacterialantibiotic resistance. SAL-mediated MarR derepression could alsocycle through a similar mechanism.

Notably, a recent study using a bacterial two-hybrid system iden-tiied 48 potential partner proteins that may bind MarR39. In par-ticular, transketolase A, an enzyme in central metabolism, has been

urther shown to directly interact with MarR in vivo, causing thederepression o the marRABoperon. his, in conjunction with ourobservation that the copper(II) chelator EA cannot completelyabolish antibiotic-triggered MarR derepression, seems to indicatethat multiple pathways may exist that either a small signalingmolecule (e.g., copper(II)) or an intracellular protein can modulateMarR-regulated antibiotic resistance.

Whether ROS can potentiate the bactericidal eects o antibiot-ics remains controversial. hrough the use o a genetically encodedOHP luorescent sensor, we show here that other types o oxida-tive species, such as OHP, can be generated by luoroquinolones or-lactam antibiotics via lipid oxidation that will subsequently trigger

the liberation o copper as a signaling molecule in E. coli. Antibiotictolerance has long been linked to cellular redox and metabolic statesas well as intracellular metal ion homeostasis40. A low level o cer-tain oxidative species is suicient to have direct signaling roles or totrigger the liberation o a secondary signaling molecule within thecell. he lack o highly sensitive and selective indicators to probethese reactive and interconvertible oxidative or metal species in liv-ing cells persists, particularly or use under stress conditions such asantibiotic treatment. In turn, microorganisms have developed sens-ing mechanisms to detect these molecules as uniied signals andturn on deense strategies against antibiotics or disinectants16,4043.Moreover, cupric ions are known to catalyze non-native disulidebond ormation in the E. coliperiplasm under copper stress or oxi-dative stress conditions44. Our study reveals that copper(II) is alsoable to trigger disulide bond ormation between MarR dimers in

Antibiotic

s

CueO

CyoB

NDH

-2

CopA

Environment

Periplasm

Envelope

stressOHPs

Thisstu

dy

ROS

productio

n

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damage

Copper

signal Antibioticresistance

Catalytic

MarR

Cys80

Cys80

Cu1+

Cu2+

H2O2or ROOH

OH

or OR

OM

IM

MarR

MarR

Figure 6 | A new linkage of antibiotic-triggered envelope stress and

copper signaling with MarR-mediated bacterial antibiotic resistance.

This diagram depicts a proposed mechanistic model for copper(II)-

mediated MarR derepression. Two clinically important antibiotics, Nor and

Amp, can stimulate envelope stress via protein unfolding, mistranslation

or both within E. colicells, which may subsequently cause lipid oxidation

and produce OHPs that lead to the impairment of cytoplasmic membrane-

bound copper proteins such as NDH-2 and CyoB. This process may

cause the release and oxidation of membrane-bound copper(I) ions to

generate a higher level of copper( II) species within the E. colicytosol. Such

a copper signal could catalytically oxidize the unique Cys80 residue on

MarR to generate two disulfide bonds to produce MarR tetramers with an

attenuated DNA-binding ability. Together, copper(II) may serve as a natural

signal for derepression of MarR and thus for the marRABoperon, resulting

in enhanced bacterial antibiotic resistance. SAL-mediated MarR derepression

might go through a similar mechanism. ROOH, alkyl hydroperoxide;

OR, alkoxy radical; OM, outer membrane; IM, inner membrane.


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a catalytic ashion. E. colimay thus adapt this unique mechanismto the cytoplasm-residing transcription regulator MarR to directlysense the copper(II) signal and cope with antibiotics, environmen-tally derived stress or both.

he characteristic Cys80 residue in MarR is highly conserved indiverse Enterobacteriaceae species, including various virulent E. colistrains, Shigella flexneri, Salmonella enterica, Enterobacter aerogenesand Klebsiella pneumoniae(Supplementary Fig. 23). his suggeststhat copper(II)-mediated cysteine oxidation and disulide bond or-

mation may be a general mechanism or derepression o these MarRtranscription actors in Enterobacteriaceae . he exact role o MarRin deending host immune-derived copper poisoning as well as thecrosstalk between MarR-mediated antibiotic resistance and copperdeense merits urther investigation.

Received 1 March 2013; accepted 13 September 2013;published online 3 November 2013

METHODSMethods and any associated reerences are available in the onlineversion o the paper.

Accession codes.Protein Data Bank: atomic coordinates and struc-

ture actors or the crystal structures o the copper( II)-oxidizedMarR5CS(80C) and reduced MarRC80S proteins have been depositedunder accession codes 4JBAand 3VOD, respectively.

References1. Martin, .G., Nyantayi, P.S. & osner, J.L. egulation o the multiple

antibiotic resistance (mar) regulon by marOAsequences in Escherichia coli.J. Bacteriol.177, 41764178 (1995).

2. Aleshun, M.N. & Levy, S.B. egulation o chromosomally mediated multipleantibiotic resistance: the marregulon.Antimicrob. Agents Chemother.41,20672075 (1997).

3. Perera, I.C. & Grove, A. Molecular mechanisms o ligand-mediatedattenuation o DNA binding by Mar amily transcriptional regulators.J. Mol. Cell Biol.2, 243254 (2010).

4. Cohen, S.P., Levy, S.B., Foulds, J. & osner, J.L. Salicylate induction oantibiotic-resistance in Escherichia coli: activation o the maroperon and a

mar-independent pathway.J. Bacteriol.175, 78567862 (1993).5. Aleshun, M.N. & Levy, S.B. Te marregulon: multiple resistance to

antibiotics and other toxic chemicals. Trends Microbiol.7, 410413 (1999).6. Martin, .G. & osner, J.L. Binding o purified multiple antibiotic-resistance

repressor protein (Mar) to maroperator sequences. Proc. Natl. Acad. Sci.USA92, 54565460 (1995).

7. Aleshun, M.N. & Levy, S.B. Alteration o the repressor activity o Mar, thenegative regulator o the Escherichia coli marABlocus, by multiplechemicals in vitro.J. Bacteriol.181, 46694672 (1999).

8. Price, C..D., Lee, I.. & Gustason, J.E. Te effects o salicylate on bacteria.Int. J. Biochem. Cell Biol.32, 10291043 (2000).

9. Nathan, C. & Cunningham-Bussel, A. Beyond oxidative stress: animmunologists guide to reactive oxygen species. Nat. ev. Immunol.13,349361 (2013).

10. ohansi, M.A., Dwyer, D.J., Hayete, B., Lawrence, C.A. & Collins, J.J.A common mechanism o cellular death induced by bactericidal antibiotics.Cell130, 797810 (2007).

11. Dwyer, D.J., ohansi, M.A. & Collins, J.J. ole o reactive oxygen species inantibiotic action and resistance. Curr. Opin. Microbiol.12, 482489 (2009).

12. Liu, Y. & Imlay, J.A. Cell death rom antibiotics without the involvement oreactive oxygen species. Science339, 12101213 (2013).

13. eren, I., Wu, Y., Inocencio, J., Mulcahy, L.. & Lewis, . illing bybactericidal antibiotics does not depend on reactive oxygen species. Science339, 12131216 (2013).

14. DAutraux, B. & oledano, M.B. OS as signalling molecules: mechanismsthat generate specificity in OS homeostasis. Nat. ev. Mol. Cell Biol.8,813824 (2007).

15. Pomposiello, P.J. & Demple, B. edox-operated genetic switches: the Soxand Oxy transcription actors. Trends Biotechnol.19, 109114 (2001).

16. Chen, P.. et al.An oxidation-sensing mechanism is used by the globalregulator MgrA in Staphylococcus aureus. Nat. Chem. Biol.2, 591595(2006).

17. Lee, J.-W. & Helmann, J.D. Te Per transcription actor senses H2O2bymetal-catalysed histidine oxidation. Nature440, 363367 (2006).

18. ohansi, M.A., DePristo, M.A. & Collins, J.J. Sublethal antibiotic treatmentleads to multidrug resistance via radical-induced mutagenesis. Mol. Cell37,311320 (2010).

19. Martin, .G., Jair, .W., Wol, .E. & osner, J.L. Autoactivation o themarABmultiple antibiotic resistance operon by the MarA transcriptionalactivator in Escherichia coli.J. Bacteriol.178, 22162223 (1996).

20. Uversy, V.N., Winter, S. & Lber, G. Sel-association o 8-anilino-1-naphthalene-sulonate molecules: spectroscopic characterization andapplication to the investigation o protein olding. Biochim. Biophys. Acta1388, 133142 (1998).

21. Xiao, Z., Loughlin, F., George, G.N., Howlett, G.J. & Wedd, A.G. C-terminaldomain o the membrane copper transporter Ctr1 rom Saccharomycescerevisiaebinds our Cu(I) ions as a cuprous-thiolate polynuclear cluster:sub-emtomolar Cu(I) affinity o three proteins involved in copper trafficing.J. Am. Chem. Soc.126, 30813090 (2004).

22. iddles, P.W., Blaeley, .L. & Zerner, B. eassessment o Ellmans reagent.Methods Enzymol.91, 4960 (1983).

23. Ding, X., Xie, H. & ang, Y.J. Te significance o copper chelators in clinicaland experimental application.J. Nutr. Biochem.22, 301310 (2011).

24. Aleshun, M.N., Levy, S.B., Mealy, .., Seaton, B.A. & Head, J.F. Te crystalstructure o Mar, a regulator o multiple antibiotic resistance, at 2.3 resolution. Nat. Struct. Biol.8, 710714 (2001).

25. Hong, M., Fuangthong, M., Helmann, J.D. & Brennan, .G. Structure o anOhrohrA operator complex reveals the DNA binding mechanism o theMar amily.Mol. Cell20, 131141 (2005).

26. umarevel, ., anaa, ., Umehara, . & Yooyama, S.S.. 1710DNAcomplex crystal structure reveals the DNA binding mechanism o the Maramily o regulators. Nucleic Acids es.37, 47234735 (2009).

27. Dolan, .., Duguid, E.M. & He, C. Crystal structures o SlyA protein, amaster virulence regulator o Salmonella, in ree and DNA-bound states.J. Biol. Chem.286, 2217822185 (2011).

28. Dwyer, D.J., ohansi, M.A., Hayete, B. & Collins, J.J. Gyrase inhibitorsinduce an oxidative damage cellular death pathway in Escherichia coli.Mol. Syst. Biol.3, 91 (2007).

29. Zeng, L., Miller, E.W., Pralle, A., Isacoff, E.Y. & Chang, C.J. A selectiveturn-on fluorescent sensor or imaging copper in living cells.J. Am. Chem.Soc.128, 1011 (2006).

30. ohansi, M.A., Dwyer, D.J., Wierzbowsi, J., Cottarel, G. & Collins, J.J.Mistranslation o membrane proteins and two-component system activationtrigger antibiotic-mediated cell death. Cell135, 679690 (2008).

31. ershaw, C.J., Brown, N.L., Constantinidou, C., Patel, M.D. & Hobman, J.L.Te expression profile o Escherichia coli-12 in response to minimal,optimal and excess copper concentrations. Microbiology151, 11871198(2005).

32. odriguez-Montelongo, L., de la Cruz-odriguez, L.C., Faras, .N. & Massa,

E.M. Membrane-associated redox cycling o copper mediates hydroperoxidetoxicity in. Escherichia coli. Biochim. Biophys. Acta1144, 7784 (1993).

33. odrguez-Montelongo, L., Faras, .N. & Massa, E.M. Sites o electrontranser to membrane-bound copper and hydroperoxide-induced damage inthe respiratory chain o Escherichia coli.Arch. Biochem. Biophys.323, 1926(1995).

34. Walling, C. Fentons reagent revisited.Acc. Chem. es.8, 125131 (1975).35. Patiarnmonthon, N. et al.Copper ions potentiate organic hydroperoxide and

hydrogen peroxide toxicity through different mechanisms inXanthomonascampestrispv. campestris. FEMS Microbiol. Lett.313, 7580 (2010).

36. apisarda, V.A. et al.Evidence or Cu(I)-thiolate ligation and prediction o aputative copper-binding site in the Escherichia coliNADH dehydrogenase-2.Arch. Biochem. Biophys.405, 8794 (2002).

37. odrguez-Montelongo, L., Volentini, S.I., Faras, .N., Massa, E.M. &apisarda, V.A. Te Cu(II)-reductase NADH dehydrogenase-2 o Escherichiacoliimproves the bacterial growth in extreme copper concentrations andincreases the resistance to the damage caused by copper and hydroperoxide.

Arch. Biochem. Biophys.451, 17 (2006).38. Zhao, B.S. et al.A highly selective fluorescent probe or visualization o

organic hydroperoxides in living cells. J. Am. Chem. Soc.132, 1706517067(2010).

39. Domain, F., Bina, X.. & Levy, S.B. ransetolase A, an enzyme in centralmetabolism, derepresses the marABmultiple antibiotic resistance operono Escherichia coliby interaction with Mar. Mol. Microbiol.66, 383394(2007).

40. Frawley, E.. et al.Iron and citrate export by a major acilitator superamilypump regulates metabolism and stress resistance in Salmonellayphimurium.Proc. Natl. Acad. Sci. USA110, 1205412059 (2013).

41. Sun, F. et al.Quorum-sensing agrmediates bacterial oxidation response viaan intramolecular disulfide redox switch in the response regulator AgrA.Proc. Natl. Acad. Sci. USA109, 90959100 (2012).

42. Deng, X. et al.Proteome-wide quantification and characterization ooxidation-sensitive cysteines in pathogenic bacteria. Cell Host Microbe13,358370 (2013).
http://www.nature.com/doifinder/10.1038/nchembio.1380http://www.nature.com/doifinder/10.1038/nchembio.1380http://www.nature.com/doifinder/10.1038/nchembio.1380http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4JBAhttp://www.rcsb.org/pdb/explore/explore.do?structureId=3VODhttp://www.rcsb.org/pdb/explore/explore.do?structureId=3VODhttp://www.rcsb.org/pdb/search/structidSearch.do?structureId=4JBAhttp://www.nature.com/doifinder/10.1038/nchembio.1380http://www.nature.com/doifinder/10.1038/nchembio.1380http://www.nature.com/doifinder/10.1038/nchembio.1380


8/11



43. Winter, J., Ilbert, M., Gra, P.C.F., zceli, D. & Jaob, U. Bleach activates aredox-regulated chaperone by oxidative protein unolding. Cell135, 691701(2008).

44. Hinier, A., Collet, J.-F. & Bardwell, J.C.A. Copper stress causes an in vivorequirement or the Escherichia colidisulfide isomerase DsbC. J. Biol. Chem.280, 3378533791 (2005).

AcknowledgmentsWe thank J. Rosner (National Institute o Diabetes and Digestive and Kidney Diseases,US National Institutes o Health) or providing the strains o M2073 (W E. coli)and M2076 (marRABmutant strains) bearing the marR::lacZreporter, J.J. Collins(Department o Biomedical Engineering, Boston University) or providing the pL(lexO)-GFP reporter, C.J. Chang (College o Chemist ry, University o CaliorniaBerkeley) orproviding the Cu(I)-speciic luorescent probe CS-1 and S.F. Reichard or editing. heE. coliW (K12) strain (BW25113) and all o the single-gene deletion mutants wereobtained rom the National BioResource Project (National Institute o Genetics, Japan).We also thank the sta members o the Shanghai Synchrotron Radiation Facility and theBeijing Synchrotron Radiation Facility. his work was supported by research grants romthe National Basic Research Foundation o China (2010CB912302 and 2012CB917301to P.R.C.; 2011CB809103 to C.H.), the National Natural Science Foundation o China

(21225206, 91013005 and 21001010 to P.R.C.), the US National Science Foundation(CHE-1213598 to C.H.) and the E-Institutes o Shanghai Municipal EducationCommission (project number E09013 to C.H. and P.R.C.).

Author contributionsZ.H. perormed the biochemical studies and participated in the structure studies.H.L. determined the crystal structures o copper(II)-oxidized MarR5CS(80C)and reducedMarRC80S. R.Z., J.Z. and X.C. helped with the biochemical and/or structure experiments.D.Z., B.S.Z. and S.Z. perormed the experiments or OHP detection using OHSer.J.C. synthesized the Cu(I)-speciic luorescent probe CS-1. P.R.C. and C.H. conceived thestudy, designed experiments, interpreted data and wrote the manuscript with input romall o the authors.

Competing financial interestshe authors declare no competing inancial interests.

Additional informationSupplementary inormation is available in the online version o the paper.Reprints andpermissions inormation is available online at http://www.nature.com/reprints/index.html. Correspondence and requests or materials should be addressed to P.R.C. or C.H.
http://www.nature.com/doifinder/10.1038/nchembio.1380http://www.nature.com/doifinder/10.1038/nchembio.1380http://www.nature.com/doifinder/10.1038/nchembio.1380http://www.nature.com/reprints/index.htmlhttp://www.nature.com/reprints/index.htmlhttp://www.nature.com/reprints/index.htmlhttp://www.nature.com/reprints/index.htmlhttp://www.nature.com/doifinder/10.1038/nchembio.1380http://www.nature.com/doifinder/10.1038/nchembio.1380


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NATURE CHEMICAL BIOLOGYdoi:10.1038/nchembio.1380

ONLINE METHODSStrains and plasmids.he strains and plasmids used in this study were listedin Supplementary Tables 2 and 3, respectively. Primers used or plasmidconstruction and qR-PCR were listed in Supplementary Table 4. For marRexpression regulation or cytosolic copper stress detection, pL(marO)-GFP andpL(copA)-GFP reporter genes were constructed on the basis o the design othe PLlacO-1 promoter28. he gfpmut3bgene (expressing green luorescentprotein) was placed under the transcriptional control o synthesized marRpromoter or copApromoter (restriction site: XhoI and KpnI). he sequences o

these two promoters are listed in Supplementary Table 5.Reagents and equipment.All o the metals used in this study were purchasedrom J&K Scientiic. Nor was purchased rom Fluca, and SAL was boughtrom Sinopharm Chemical Reagent Co., Ltd. All o the other antibiotics wereobtained rom INALCO. ANS (8-anilino-1-naphthalenesulonate) was boughtrom CI. BCS, EA (60%), 3-coumarin carboxylic acid (3-CCA), tBHP(70%), cumene hydroperoxide (CHP, 80%) and IAM were obtained rom AlaAesar. All o the other chemicals were analytical grade or better. All pictureso protein gels, including those o Coomassie Brilliant Blue (CBB)-stainedSDS-PAGE gels and western blotting membranes, were taken on ChemDocXRS+ (Bio-Rad). UV-visible spectroscopy was perormed using a UV-visiblespectrometer (U-3010, HIACHI, Japan). All luorescent spectroscopic mea-surements were recorded at 25 C on a Hitachi F4500 luorescence spectropho-tometer equipped with a LPS-220B 75-W xenon lamp and power supply. All o

the data in low cytometry analysis were collected using a Becton DickinsonFACSCalibur low cytometer (BD bioscience) with a 488-nm argon laser and a515- to 545-nm emission ilter (FL1) at low low rate. At least 70,000 cells werecollected or each sample. Flow data were processed and analyzed with FCSexpress Version 2.

Protein expression and purification. W MarR and its mutants witha thrombin-cleavable N-terminal His tag were prepared rom E. coliBL21(DE3), which bears a pE15b vector containing the marR gene.Cells were grown in LB medium containing 50 g/ml Amp at 37 C to anOD600nmo 0.6 beore IPG was added to a inal concentration o 1 mM.Protein expression was induced at 30 C or 5 h beore harvest. Cell pelletswere collected and resuspended in NA buer (20 mM ris-HCl, pH 7.5,300 mM NaCl and 10% glycerol) containing 1 mM PMSF (phenylmeth-anesulonyl luoride) and 5 mM -mercaptoethanol. Ater sonicating on ice

or 20 min, the cell lysate was spun down at 15,000 r.p.m. or 30 min. Clearedlysate was iltrated and loaded onto a 5-ml Histrap column (GE Healthcare),which was pre-equilibrated with 25 ml NA buer. MarR protein was theneluted with a linear imidazole gradient ollowed by a urther puriicationstep using HiLoad 16/60 Superdex 200 column (GE Healthcare) equili-brated with buer A (20 mM ris-HCl, pH 7.5, 300 mM NaCl). Protein wascollected and concentrated or urther experimental use. he N-terminalHis tag o MarR proteins can be removed by thrombin digestion, which wasperormed by incubating tagged protein with thrombin at 16 C overnightin digestion buer (20 mM ris-HCl, pH 8.4, 2.5 mM CaCl2, 150 mM NaCl)(Supplementary Fig. 24).

Electrophoretic mobility shift assays (EMSA).A 42-mer duplex DNA con-taining marRAB promoter sequence (5-ACAGACAACGCCGGGCAAAACCCCGC-3and 5- GCAGGGGAAAAGCCC

AGGCAAGAAAGCAAAGAA-3 ) was used or this assay. All o theEMSA reaction mixtures in this study were set up in a inal volume o 20 lcontaining 50 nM annealed double-stranded DNA, 1 M MarR protein mono-mer (0.5 M or mutant MarR5CS(80C)) in binding buer (50 mM KCl, 5 mMMgCl2, 10% glycerol and 10 mM ris-HCl, pH 7.8). he concentrations orCuCl2and other metals were kept at 2.5 M. Samples were loaded onto the 8%nondenaturing polyacrylamide gel beore incubation at room temperature or20 min and were run in 0.5 B buer (tenold dilution rom 5 B buer:50 mM ris-HCl, 41.5 mM borate, pH 7.8) at 110 V or 1 h at 4 C. Gels werestained in a 10,000-old diluted SYBR Gold nucleic acid staining solution or7 min. he DNA bands were visualized with UV l ight at 254 nm by ChemiDocXRS+ (Bio-Rad).

Spectroscopic study. he luorescence probe ANS (8-anilino-1-naphthalenesulonate) was employed to indicate the conormational change

o protein upon treatment with potential metal ions. Dierent concentrations

o CuCl2, FeCl3or NiCl2were added to the solution containing the MarRANScomplex (0.5 mM ANS and 5 M MarR in 20 mM HEPES buer, pH 7.0).Addition o D (1 mM) or EDA (3.5 mM) prevented the luorescencechange o the MarRANS complex solution upon treatment with copper(II).o determine the stoichiometry o W MarR to copper(II), aliquots o CuCl2(rom 5 M to 22.5 M) were titrated into the protein-BCS solution containing5 M MarR monomer protein and excess BCS (200 M) in a inal volume o100 l. he absorption at 483 nm (characteristic absorption o Cu(I)(BCS)23complex) were plotted versus the molar ratio o copper( II) to protein (mono-mer concentration). All o the experiments were perormed in 20 mM HEPESbuer (pH 7.0) on an UV-visible spectrometer (U-3010, HIACHI, Japan). hestoichiometry o copper(II)/MarR5CS(80C)was determined with the same method.Aliquots o CuCl2(rom 7.5 M to 30 M) were titrated into the protein-BCSsolution containing 15 M MarR monomer protein and excess BCS (200 M).he hydroxyl radical scavenging compound 3-CCA (3-coumarin carboxylicacid) was used to determine the copper-catalyzed hydroxyl radical produc-tion45. As an indication o the hydroxyl radical production, the ormation o theproduct 7-hydroxy-coumarin-3-carboxylic acid (7-OH-CCA,exc, 395 nm; em,450 nm) was measured upon the addition o W MarR (5 M) to the samplescontaining 5 M CuCl2and 300 M 3-CCA in the absence and presence osodium ascorbate (500 M) or 25 min. he hydroxyl radical production uponthe reaction o CuCl2 (5 M) with sodium ascorbate (500 M) and 3-CCA(300 M) was also measured as a positive control. All o the measurementswere carried out in 20 mM PBS buer (pH 7.4) using a luorescence spectro-

meter (F-4600, HIACHI, Japan).DTNB assay. he ree thiol contents o native and copper(II)-oxidized WMarR were determined by using the 5,5 -dithiobis-2-nitrobenzoic acid(DNB) assay. 25 M W MarR was treated with 500 M CuCl2in the pres-ence and absence o EDA (10 mM) or 10 min at room temperature in DNBassay buer (100 mM potassium phosphate, pH 7.2, 100 mM NaCl and 1 mMEDA). Excess copper(II) was removed by buer exchange. Ater addition o1 mM DNB, he absorption at 412 nm was recorded, and ree thiol concen-trations were calculated on the basis o previously published methods22.

Disulfide bond detection by MS.Puriied MarR5CS(80C)and W MarR (all at40 M) were incubated with 20 M (0.5) CuCl2 at room temperature or5 min. Any remaining ree thiols were blocked by treatment with excess IAMor 45 min. Protein samples were run on a nonreducing, denaturing SDS PAGEgel. he covalently linked dimer band was separately excised rom the SDSPAGE gel, destained and digested with trypsin at 37 C or 12 h. he resultingtryptic peptide was extracted and subjected to MS analysis using a hermoLQ-Orbitrap velos mass spectrometer equipped with the EASYnLCII inte-grated nano-HPLC system (Proxeon, Denmark). he analytical column waspursed rom Agilent (75 m ID, 150-mm length; Agilent USA). Mobile phase Aconsisted o 0.1% ormic acid, and mobile phase B consisted o 100% ace-tonitrile and 0.1% ormic acid.

he single ull-scan mass spectrum was acquired in the Orbitrap rom m/z35020,000 ollowed by three MS/MS scans in the quadruple collision cellusing the collision-induced dissociation (CID), in which the precursor massselection window was set at 2 Da.

Crystallization.For the crystallization conditions, we used the commerciallyavailable Hampton Research screens: Index, Crystal Screen, SaltRx 1, SaltRx 2,PEG/Ion Screen, PEG/Ion 2 Screen. Sitting-drop vapor diusion at room tem-perature (1020 C) was applied by mixing 2 l protein sample (at 7.2 mg/mlor 11.8 mg/ml) with 2 l well solution. We used the N-terminal His-tag cleavedprotein sample or crystallization. he best MarR5CS(80C)crystal grew in 2.2 Msodium chloride, 0.1 M sodium acetate trihydrate, pH 4.6. he best MarRC80Scrystal grew in 24% PEG 3350, 0.1 M CBP buer (pH 7.6; 0.03 M citric acidand 0.07 M Bis-ris propane mixed), 0.3 M NaCl and 10 mM D.

X-ray data collection and structure determination.Cryoprotectant was maderesh by mimicking growth conditions. We used step-wise soaking to avoidcrystal cracking. he crystal was quickly dipped into a series o solutions withincreased glycerol concentration up to 25%. he crystal was inally lash ro-zen in liquid nitrogen prior to data collection. Data sets o MarR5CS(80C) andMarRC80Swere collected on the BL17U1 beamline at the Shanghai SynchrotronRadiation Facility (Shanghai, China) and the 3W1A beamline at Beijing

Synchrotron Radiation Facility, respectively. he wavelength used or data


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NATURE CHEMICAL BIOLOGY doi:10.1038/nchembio.1380

collection or MarR5CS(80C) and MarRC80Swas 0.9792 and 1 , respectively.Both data sets were initially indexed by iMOSFLM46and were urther proc-essed with the CCP4 package47. he structures were solved through molecularreplacement using the dimer orm o W MarR (Protein Data Bank code1JGS)as the search model with the program PHASER48. Reinement was carried outon the programs REFMAC5 (re. 49) and COO50. he Ramachandran plotgiven by PROCHECK51shows that in MarR5CS(80C), 99.24% residues were in themost avored region, and 0.76% were in the allowed region. In MarRC80S, 98.13%residues were in the most avored region, and 1.87% were in the allowed region.Neither o the two structures have residues in the disallowed region. Residues9295 in chain A and residues 9294 o MarR5CS(80C)were disordered; thereore,they were not modeled. NCS restraints52and LS reinement53were applied.he stereochemical quality was analyzed using MolProbity54. PyMOL wasused to generate all o the igures. A summary o the data statistics is shown inSupplementary Table 6.

Size-exclusion chromatography. Freshly puriied protein o MarR5CS(80C),MarRC80Sand MarR6CSrom the Histrap column (GE Healthcare) was instantlyincubated with 20% CuCl2(5% CuCl2was also tested or MarR5CS(80C)) at roomtemperature (25 C) or 15 min. CuCl2was slowly added into the protein solu-tion to avoid local precipitation. Gel iltration was then perormed with aSuperdex 75 10/300 GL column (GE Healthcare). he molecular weight o theeluting species was calculated by standard protein marker (Gel Filtration LMWCalibration Kit, GE Healthcare).

b-Gal assay.he E. coliW strain (M2073) or marRABmutant strain (M2076)containing the marR::lacZ reporter usion was used or this assay. -Gal activitywas measured using MUG as a substrate. Overnight cultures were inoculated(1:100) in M9 minimal medium or LB medium and grown to an OD 600nmo0.6 at 37 C. o examine the MarR derepression in the presence o metal ions,cells in M9 medium were treated with 100M CuCl2, FeCl3, ZnCl2or NiCl2or30 min. For complementary experiments, DmarRABmutant strain (M2076)harboring plasmids expressing W MarR or MarRC80Swith or without the Histag were induced by IPG (1:1,000) or arabinose (1:200) or 2 h beore beingchallenged by 100 M CuCl2or 30 min. SAL-induced marRderepression inthe presence o EA was also tested, and bacterial cells in LB medium werechallenged by 2.5 mM SAL in the presence and absence o 1 mM EA or30 min. Ater these treatments, 1-ml aliquots were obtained or measuremento OD600nm, and the remaining aliquots (1 ml) were harvested by centriuga-tion, washed with 1 PBS buer and stored at 80 C. At the time o the assay,cells were resuspended in 100 l o lysis buer (50 mM ris-HCl, pH 8.0,25 mM NaCl, 2 mM EDA) containing 0.5 mg/ml lysozyme and were incu-bated at 37 C or 15 min, ollowed by centriugation. he upper layers weredelivered to a 96-well plate and two tenold dilutions (90 l AB buer(AB buer plus 0.1% riton X-100) plus 10-l sample) were set up in the plate.50 l o the samples rom each well were transerred to a new 96-well plateollowed by addition o 10 l MUG (0.5 mg/ml) into each well to start thereaction. he reactions were incubated at room temperature or 1 h beore20 l o the samples were added to 180 l o AB buer in a black 96-well plate.-Gal activity (expressed in MUG units; 1 unit = 1 pmol o MUG cleaved permin per OD600nm) was determined by luorescence (exc, 366 nm; em, 445 nm)using a microplate reader (synergy4, Bioek).

pL(marO)-GFP reporter assay.An overnight bacterial culture harboring thepL(marO)-GFP reporter gene was inoculated (1:100) in growth medium andgrown to an OD600nmo 0.6 beore being challenged by various stimulants. oexamine the MarR derepression in the presence o metal ions, M9 medium wasused, and bacterial cells were treated with 100M CuCl2, FeCl3and 1 mM H2O2or 1 h beore being analyzed by low cytometry. he PM voltage settingsused are: E01 (FSC), 420 (SSC) and 450 (FL1). o survey MarR derepressionby SAL, antibiotics and OHPs with or without the copper(II) chelator EA,bacterial cells were challenged by 2.5 mM SAL, 250 ng/ml Nor, 2.5 g/ml Ampor 750 M tBHP in the presence and absence o 1 mM EA or 1 h, ol-lowed by low cytometric analysis with the ollowing PM voltage settings: E01(FSC), 413 (SSC) and 436 (FL1).

Copper signal detection.A speciic copper(I) luorescence sensor CS-1 wasused to detect the generation o copper(I) ions inside E. colicells upon exposureto various stimulants. Overnight bacterial cultures were inoculated (1:100) in5 ml resh LB and were grown to an OD600nmo 0.6 beore being treated by

SAL (5 mM), antibiotics (2.5 g/ml Amp, 250 ng/ml Nor, 20 g/ml Cm and20 g/ml et) or 750 M tBHP at 37 C or 2 h. Ater washing with 10 mMPBS buer twice, bacterial samples were incubated with 2 M CS-1 or 15 minat 37 C ollowed by washing with PBS buer twice. Changes o luorescencewere monitored by low cytometry with the ollowing PM voltage settings:E01 (FSC), 413 (SSC) and 710 (FL1). o analyze cytosolic copper generation,we constructed the pL(copA)-GFP reporter. Cells harboring pL(copA)-GFPreporter genes in exponential phase (OD600nm o 0.6) were challenged with250 ng/ml Nor or 750 M tBHP or 2 h. he bacterial samples were analyzedusing low cytometry. he ollowing PM voltage settings were used: E01(FSC), 420 (SSC) and 450 (FL1).

DNA damage analysis.Overnight cell cultures o E. coliW (K12) and marRdeletion strains harboring the pL(lexO)-GFP reporter were inoculated (1:100)in 5 ml LB medium and urther grown to an OD600nm o approximately 0.6.At this point, cells were pretreated with Nor (250 ng/ml) or 1 h, ollowed byincubation with 500 M CuCl2or an additional 1 h beore low cytometricanalysis. he ollowing PM voltage settings were used: E01 (FSC), 413 (SSC)and 500 (FL1).

Quantitative real-time RT-PCR. Overnight cultures o E. coli W (K12)and marRdeletion strains were inoculated (1:100) in LB medium, grown at37 C to an OD600nmo 0.7 and then incubated with 250 ng/ml Nor at 37 C or2 h beore being harvested. otal RNAs were extracted using SV otal RNAIsolation System (Promega, Madison, USA) according to the manuacturers

protocol. he total RNA (1 g) was reverse transcribed to irst strand cDNA byRevertAid First Strands cDNA Synthesis kit (Fermentas, Maryland, USA) ol-lowing the manuacturers guidelines. Quantitative real-time R-PCR was per-ormed on CFX96 Real-ime PCR (Bio-Rad). For each 20-l reaction, 10 l 2SYBR master mix (QPK-201,OYOBO Co., Ltd. Osaka, Japan), 8.4 l cDNA(100-old dilution) and 0.8 l primer (10 M) were added. Relative quantii-cation analysis was perormed as ollowing conditions: one cycle at 94 C or4 min, ollowed by 40 cycles at 94 C or 20 s and 58 C or 20 s, then one cycleat 72 C or 20 sec. Melting curve analysis started at 65 C and went up to94 C. he luorescence was collected every 0.2 C. he rrsA(16S rRNA) wasused as an internal control. All o the experiments were conducted in at leastthree independent replicates, and relative expression levels were measuredusing the 2DDCtmethod55.

Cell viability assay.o assess the roles o copper signaling in MarR-mediated

antibiotic resistance, overnight cultures o E. coliW (K12) and marRdele-tion strains were inoculated (1:100) in LB medium and grown at 37 C toan OD600nmo 0.6. Cells were then incubated with 1 g/ml Nor in the pres-ence and absence o CuCl2(500 M) or 300 M copper(II) chelator EA or30 min at 37 C. enold serial dilutions o bacteria were plated or colonycounts. Only dilutions that yielded 10100 colonies were counted, and bacte-rial survival rates were determined by the percentage survival o the copper- orEA-treated group compared to the untreated group.

OHP detection.o detect OHP generation inside bacteria under treatmentwith SAL, tBHP or various antibiotics, we used our previously developed OHP-speciic luorescent sensor OHSer. E. colicells harboring OHSer were grownovernight and then diluted 1:100 in LB medium (50 g/ml kanamycin). OHSerexpression was induced by the addition o 1 mM IPG (inal concentration) atan OD600nmo approximately 0.60.8. Protein production was allowed to pro-

ceed or 2 h at 30 C beore treatment with Nor, Amp or oxidants (tBHP andH2O2) or 30 min. Samples were analyzed by low cytometry. he ollowingPM voltage settings were used: E01 (FSC), 427 (SSC) and 725(FL1).

Statistical analysis.All o the data are presented as mean s.d. he statisticalanalysis was perormed using an unpaired Students t-test when the two groupswere compared. When more than two groups were compared, one-way analy-sis o variance (ANOVA) ollowed by Bonerroni means comparison was used.P< 0.05 was considered to be signiicant.

45. Meloni, G. et al.Metal swap between Zn7-metallothionein-3 and amyloid--Cu protects against amyloid- toxicity. Nat. Chem. Biol.4, 366372 (2008).

46. Battye, .G.G., ontogiannis, L., Johnson, O., Powell, H.. & Leslie, A.G.W.iMOSFLM: a new graphical interace or diffraction-image processing withMOSFLM.Acta Crystallogr. D Biol. Crystallogr.67, 271281 (2011).
http://www.rcsb.org/pdb/explore/explore.do?structureId=1JGShttp://www.rcsb.org/pdb/explore/explore.do?structureId=1JGShttp://www.rcsb.org/pdb/explore/explore.do?structureId=1JGS


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NATURE CHEMICAL BIOLOGYdoi:10 1038/nchembio 1380

47. Winn, M.D. et al.Overview o the CCP4 suite and current developments.Acta Crystallogr. D Biol. Crystallogr.67, 235242 (2011).

48. McCoy, A.J. et al.Phaser crystallographic sofware.J. Appl. Crystallogr.40,658674 (2007).

49. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. efinement o macromolecularstructures by the maximum-lielihood method. Acta Crystallogr. D Biol.Crystallogr.53, 240255 (1997).

50. Emsley, P. & Cowtan, . Coot: model-building tools or molecular graphics.Acta Crystallogr. D Biol. Crystallogr.60, 21262132 (2004).

51. Lasowsi, .A., MacArthur, M.W., Moss, D.S. & Tornton, J.M.POCHEC: a program to chec the stereochemical quality o proteinstructures.J. Appl. Crystallogr.26, 283291 (1993).

52. leywegt, G.J. Use o non-crystallographic symmetry in proteinstructure refinement. Acta Crystallogr. D Biol. Crystallogr.52, 842857(1996).

53. Painter, J. & Merritt, E.A. Optimal description o a protein structure in termso multiple groups undergoing LS motion. Acta Crystallogr. D Biol.Crystallogr. 62, 439450 (2006).

54. Chen, V.B. et al.MolProbity: all-atom structure validation ormacromolecular crystallography.Acta Crystallogr. D Biol. Crystallogr.66,1221 (2010).

55. Liva, .J. & Schmittgen, .D. Analysis o relative gene expression data usingreal-time quantitative PC and the 2DDCmethod.Methods25, 402408(2001).

hao et al nat chem biol 2013

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