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ARTICLE

PUbliShED oNliNE: 3 NoVEmbER 2013 | Doi: 10.1038/NchEmbio.1380

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 bacterial detoxification in response to diverse antibiotics, toxic chemicals or both. The natural inducer for Escherichia coli MarR, the prototypical transcription repressor within this family, remains unknown. Here we show that copper signaling potentiates MarR derepression in E. coli. Copper(II) oxidizes a cysteine residue (Cys80) on MarR to generate disulfide bonds between two MarR dimers, thereby inducing tetramer formation and the dissociation of MarR from its cognate promoter DNA. We further discovered that salicylate, a putative MarR inducer, and the clinically important bactericidal antibiotics norfloxacin and ampicillin all stimulate intracellular copper elevation, most likely through oxidative impairment of copper-dependent envelope proteins, including NADH dehydrogenase-2. This membrane-associated copper oxidation and liberation process derepresses MarR, 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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he MarR family of transcription factors regulates diverse genes involved in multiple antibiotic resistance, synthesis of virulence determinants and many other important biological processes13. Various bacterial species employ MarR homologs to sense and exert resistance against many cellular toxins from the environment or host immune system, including multiple antibiotics, detergents, oxidative reagents and disinfectants4,5. However, despite extensive study of activation mechanisms, the true (natural) inducers for most members of the MarR family remain unknown. E. coli MarR, the prototypical member of the MarR family of proteins, resides in the chromosomally encoded Mar locus and negatively regulates the marRAB operon, an essential component that controls the Mar phenotype and various cellular responses (e.g., outer membrane permeability, superoxide stress response, DNA repair and metabolic regulation)2. Previous reports indicate that diverse phenolic compounds directly bind MarR invitro. Among these compounds, salicylate (SAL) has been further shown to trigger the dissociation of MarR from its promoter DNA and cause the derepression of the marRAB operon within E. coli cells6,7. On the basis of these results, direct binding of phenolic substrates to MarR has been proposed to cause MarR derepression inside bacteria4. However, a high concentration of SAL is required to activate MarR (5 mM), which is less likely to be physiologically relevant3,6. Meanwhile, SAL has also been found to induce the Mar phenotype in a MarR-independent fashion4,8. Moreover, MarR is known to regulate bacterial resistance to diverse, structurally unrelated antibiotics including fluoroquinolones (e.g., norfloxacin (Nor)), -lactams (e.g., ampicillin (Amp)), tetracycline (Tet) and chloramphenicol (Cm)2. As it is unlikely that a single MarR protein is capable of directly binding such a structurally dissimilar pool of compounds with specificity, it has been speculated that a common cellular product may be generated when it is exposed to any of these
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compounds. This cellular product may function as the real signal for MarR derepression2,4,6. However, the identity of such a unified signaling molecule remains elusive. Herein we report that copper(II) ions directly oxidize a cysteine residue on MarR to generate disulfide bonds between two MarR dimers, resulting in the dissociation of MarR from its cognate promoter DNA. We further demonstrate that this copper(II)-triggered MarR derepression acts as the underlying mechanism for SALmediated derepression of the marRAB operon inside E. coli. SAL as well as bactericidal antibiotics including Nor and Amp were found to increase the level of intracellular copper, most likely through oxidative impairment of the bacterial envelope and envelope-residing copper proteins that include NADH dehydrogenase-2 (NDH-2). This membrane-associated copper oxidation and release process was further shown to derepress MarR, thus leading to increased bacterial resistance to the fluoroquinolones used to kill bacterial cells. Our study reveals that copper(II) acts as a natural signal: the master multiple antibiotic resistance regulator MarR in E. coli senses copper(II) directly, which turns on bacterial defense against anti biotics, environment-derived stress or both.

RESULTS Copper(II) triggers the dissociation of MarR from DNA

To begin, we investigated a potential activation signal (or signals) for MarR. Reactive oxidative species (ROS) are produced from host immune response upon bacterial infection9. Recent research implicates ROS as common products generated by different types of bactericidal antibiotics10,11. Although whether the proposed oxidative stress or the altered iron homeostasis that can cause direct bacterial killing remains controversial12,13, these species do serve as signaling molecules to modulate diverse bacterial responses, as evidenced by the presence of various oxidation-sensing regulators in

Synthetic 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. 5 International 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: pengchen@pku.edu.cn or chuanhe@uchicago.edu
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ARTICLE
a
WT MarR

Nature chemical BioloGY Doi: 10.1038/NchEmbio.1380


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MarRre MarRox Cu(II) + 2BCS Absorption at 483 nm (AU) 0.14

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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 500 M H2O2 (lanes 37, respectively). (b) Titration of aliquots of CuCl2 into 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)23 complex) 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 MarR6CS mutant (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. coli WT (M2073) strain containing the marR::lacZ reporter was determined after treatment with 100 M CuCl2, FeCl3, Zn(NO3)2 or NiCl2 or 500 M H2O2 in 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::lacZ reporter was conducted in WT strain (M2073) and the marRAB mutant strain (M2076) upon the addition of 2.5 mM SAL with and without TETA (1 mM). Data in d and e represent the mean s.d. from three independent samples (*P < 0.01). (f) The E. coli WT (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.

bacteria such as OxyR, SoxR, MgrA and PerR1417. Sublethal levels of antibiotics have also been shown to stimulate a ROS-induced mutagenesis that ultimately leads to bacterial multidrug resistance18. We wondered whether related species or processes could induce MarRs dissociation from its cognitive DNA. As H2O2 is a main product of the host immune response that is disproportionated from superoxide14, we tested it first. We applied an electrophoretic mobility shift assay (EMSA) to 50 nM DNA in the presence of 1 M wild-type (WT) MarR (monomer concentration), which remained as a proteinDNA complex with and without the addition of 500 M H2O2 (Fig. 1a; these MarR and DNA concentrations were used for all of the following EMSA experiments unless otherwise noted). This observation is in line with previous reports that indicate that oxidative stress may only indirectly derepress marRAB operon in E. coli via other pathways such as SoxS activation19. Furthermore, neither iron(II) nor iron(III) ions were found to cause MarR-DNA dissociation. We could therefore exclude the possibility that antibiotic-damaged Fe-S clusters release free iron, which serves as a natural inducer for MarR. EMSA was then used to test a panel of divalent metal ions, including manganese(II), cobalt(II), nickel(II), copper(II) and zinc(II). Notably, copper(II) was the only transition metal within this group to disrupt the MarRDNA complex (Fig. 1a and Supplementary Results, Supplementary Figs. 1 and 2). In the presence of H2O2, 2.5 M copper(II) or the same amount of copper(I) was able to efficiently dissociate MarR from its promoter DNA, whereas the addition of a reducing agent such as dithiothreitol (DTT) effectively restored MarRs DNA-binding ability. This, in combination with our 8-anilino-1-naphthalenesulfonate (ANS) assay20 monitoring the conformational change of MarR with
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and without copper(II) (Supplementary Fig. 3a), indicated that the interaction between MarR and copper(II) is redox sensitive. To examine whether MarR causes the reduction of copper(II) to copper(I), we employed a copper(I)-specific indicator, bathocuproine disulfonate (BCS), which forms a Cu(I)(BCS)23 complex with a characteristic UV-absorbance peak at 483 nm (ref. 21). Titration of the protein-BCS solution (5 M MarR monomer protein and 200 M BCS) with aqueous copper(II) ions indicated a stoichiom etry of 1:2 between MarR monomer and copper(II) (Fig. 1b and Supplementary Fig. 3b). This result agreed with our 5,5-dithiobis-2-nitrobenzoic acid (DTNB) assay22 (Supplementary Table 1), which indicated that two out of the six cysteine residues on each MarR monomer can be oxidized by copper(II). Further, the presence of MarR was found to effectively prevent radical production from copper(II) in the presence of ascorbate (Supplementary Fig. 3c), strongly suggesting that MarR may efficiently compete against cellular reductants in scavenging free copper(II) ions. Finally, all of the six cysteine residues on MarR were either mutated to serine or blocked by iodoacetamide (IAM) before being subjected to EMSA analysis, which showed that these MarR variants remained bound to the promoter DNA even after copper(II) treatment (Fig. 1c and Supplementary Fig. 4). Taken together, our biochemical studies revealed that copper(II) ions can oxidize up to two cysteine residues on each MarR monomer and attenuate MarRs DNA-binding ability. We next demonstrated that copper(II) is the cognitive signal for MarR derepression inside E. coli. A -galactosidase (-Gal) assay was first conducted on an E. coli WT strain bearing a marR::lacZ

Copper(II) is a natural inducer for MarR inside E. coli

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a
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y2 y1 S +461.27 81 (y2Y4)+ 709.38 (y3Y4)+

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L V C K Y4 Y3 Y2 Y1

649.63 442.97

Y1 or y1+ 147.11

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(y3Y4)2+ 404.73 497.22

2+ 213.16 (b3Y2)

2013 Nature America, Inc. All rights reserved.

609.29 400 450 500 550 600

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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 MarRC80S mutant protein (1 M). (b) -Gal activity of the marR::lacZ reporter in the marRAB mutant strain (M2076) complemented with the pET15b WT MarR plasmid (expressing WT MarR) or pET15b MarRC80S plasmid (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-Dgalactopyranoside (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 25C. MarR5CS(80C) dimer protein (125 M) was incubated with (red) and without (black) 25 M CuCl2 for 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).

reporter gene (M2073) with and without copper(II). The addition of 100 M copper(II) ions led to a threefold increase of -Gal activity compared to untreated cells or cells treated with the same concentration of iron(III), nickel(II), zinc(II) or H2O2 (Fig. 1d). Similarly, SAL at a concentration of 2.5 mM was also found to stimulate over threefold greater marR expression compared to untreated samples (Fig. 1e). Because the bacterial cytosol is a highly reduced environment under normal conditions, labile copper ions are thought to exist almost exclusively at the copper(I) state with few cupric species. To confirm that copper(II) is the real signal for SAL-triggered MarR derepression, we added 1 mM of the copper(II)-specific chelator triethylenetetramine (TETA) to the SAL-treated bacterial cells23, which effectively diminished SAL-mediated MarR derepression (Fig. 1e), thus verifying that SAL indirectly derepresses MarR via copper(II) species within E. coli. Finally, the E. coli marRAB mutant strain bearing the marR::lacZ reporter (M2076) was used as a control, which exhibited only a slight increase of -Gal activity after SAL treatment. We also engineered a GFP-based, marR-inducible fluorescent reporter, pL(marO)-GFP, that relies on MarR repression for the tight regulation of GFP expression. Fluorescence-activated cell sorting (FACS) can quantitatively measure the expression of GFP under the control of the marR promoter in living bacterial samples. The addition of copper(II) ions was found to markedly induce the expression of GFP in the E. coli WT (K12) strain, whereas the addition of iron(III) or H2O2 did not generate a noticeable increase (Supplementary Fig. 5a). The presence of copper(II) ions also did not cause any noticeable difference in the fluorescent signal in the marR deletion strain (Supplementary Fig. 5a).

Further, SAL (2.5 mM) was also found to trigger GFP induction in the E. coli WT (K12) strain, which can be largely eliminated by adding 1 mM TETA (Supplementary Fig. 5b). In addition, we found that both Nor (250 ng/ml) and Amp (2.5 g/ml) were able to induce GFP expression and thus MarR derepression within 1 h (Fig. 1f), whereas the presence of the TETA (1 mM) attenuated this MarR-related GFP induction (Supplementary Fig. 6). In contrast, neither Tet nor Cm was found to stimulate MarR derepression within the period of time we tested (Fig. 1f). Together, we verify that copper(II) not only is an underlying signal for MarR derepression triggered by SAL but also serves as a signal for clinically important antibiotics such as Nor and Amp within E. coli cells.

Molecular mechanism of copper(II)-induced MarR activation

To elucidate the molecular details of MarRs response mechanism to copper(II), we investigated potential residues involved in the MarRcopper(II) interaction. As we have shown that cysteine residues were required for copper(II)-triggered MarR-DNA disruption (Fig. 1c and Supplementary Fig. 4), we mutated each individual cysteine on MarR to serine for EMSA analysis to identify the cysteine residue (or residues) responsible for this process. Whereas copper(II) caused the dissociation of five of the six MarR mutants we created from DNA in a similar fashion as WT MarR (Supplementary Fig.7ae), the MarRC80S mutant remained bound with its promoter DNA in the presence of copper(II) ions (Fig. 2a). We then mutated the other five cysteine residues on MarR to serine and kept only a single cysteine at residue 80 to produce the MarR5CS(80C) mutant. The same amount of copper(II) ions (2.5 M) effectively triggered
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Copper(II)-oxidized WT and mutant MarR proteins were next analyzed by nonreducing SDS-PAGE, which showed that a covalent linkage was generated between Cys80 residues on MarR upon copper(II) treatment in vitro and inside E. coli cells (Supplementary Fig. 9). Size-exclusion chromatography was then used to examine the oligomeric state of the MarR5CS(80C) protein before and after copper(II) oxidation. The naturally occurring MarR dimer protein was found to convert into a disulfide-stabilized tetramer in the presence of catalytic amount of copper(II) under ambient conditions. In particular, a 20% loading of copper(II) was able to convert one equivalent of MarR dimer into the corresponding tetramer, whereas a 4% copper(II) loading led to a more than 60% dimerto-tetramer conversion (Fig. 2c and Supplementary Fig. 10). To further reveal the nature of the disulfide-stabilized MarR tetramer produced by copper(II), we performed MS mapping analysis, which identified disulfide-containing peptides (crosslink between Cys80 and Cys80 on two Leu-Val-Cys-Lys peptides, calculated molecular weight: 920.52 Da) in the copper-treated MarR5CS(80C) and WT MarR proteins (Fig. 2d,e and Supplementary Fig. 11). Together, these observations indicate that copper(II) may trigger the formation of a covalent dimer-of-dimer of MarR via Cys80 residues in a catalytic fashion and that the formation of the first disulfide bond would promote the second disulfide bond formation at the other dimer-dimer interface upon oxidation.

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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 two disulfide bonds between two MarR dimers. Electron density map from the crosslinked Cys80-Cys80 residues are contoured at 1 2Fo-Fc level, 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).

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

the dissociation of 0.5 M MarR5CS(80C) mutant protein from 50 nM promoter DNA (Supplementary Fig. 7f). We next performed the BCS assay on MarR5CS(80C) and copper(II) ions, which showed an approximately 1:1 stoichiometry between copper(II) and the monomeric form of this MarR mutant (Supplementary Fig. 8). Finally, the E. coli marRAB mutant strain bearing the marR::lacZ reporter was complemented with a plasmid expressing either WT MarR or MarRC80S before being subjected to -Gal analysis. We found that 100 M copper(II) caused a threefold derepression of marR in the marRAB/WT MarR strain but exhibited a negligible effect on marR level in the marRAB/MarRC80S strain (Fig. 2b). Taken together, although the aforementioned results indicated that MarR has two copper-oxidizable cysteine residues on each monomer, all of our further in vitro and bacteria-based results revealed that Cys80 is the key residue that is required and sufficient for copper(II)-induced MarR-DNA dissociation.
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To gain more insight into the molecular mechanism of copper(II)mediated MarR oxidation, we solved the crystal structure of the copper(II)-oxidized MarR protein. Previous work has showed that the ligand-free MarR protein is much more challenging to crystallize because of poor crystal packing24; we were also unable to obtain the copper(II)-oxidized WT MarR protein crystals. Our aforementioned results indicate that the reduced MarR5CS(80C) protein can bind DNA with an affinity similar to that of WT MarR, but copper(II) treatment severely disrupts its DNA binding. Therefore, the behavior of MarR5CS(80C) resembles that of WT MarR with and without copper(II). We thus focused on this MarR mutant and solved the crystal structure of the copper(II)-oxidized MarR5CS(80C) protein at 2.5 . A symmetric MarR tetramer complex was observed upon copper(II) oxidation. The tetramer protein contains two disulfide bonds between two MarR dimers via Cys80 residues from each monomer (Fig. 3a and Supplementary Fig. 12a). Electron density of the two disulfide bonds was clearly visible, with a measured S-S distance at 2.0 and a dihedral angle of C-S-S-C at 90.3 (Fig. 3b and Supplementary Fig. 12b). In this tetrameric complex, all of the DNA recognition helices (4) in the DNA-binding domain from each MarR monomer were buried within the interface between two MarR dimers, rendering them incapable of DNA binding.
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Figure 4 | SAL- and antibiotic-triggered intracellular copper elevation. (ac) Generation of copper(I) ions inside E. coli WT (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. coli WT (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. coli WT (K12) and ndh deletion strains after Nor treatment (250 ng/ml; 2 h). (e) The expression of E. coli copper-dependent genes cusR, cusA, cueO, cusF, copA, ndh and cyoB in 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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Figure 5 | Antibiotic-stimulated OHP production leads to copper liberation and MarR derepression. (a) tBHP (750 M)-induced copper generation inside E. coli WT (K12) and ndh deletion strains was examined with CS-1 probe using flow cytometry. AU, arbitrary units. (b) Flow cytometric analysis of E. coli cells 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. coli WT (K12) and ndh deletion strains bearing the pL(copA)-GFP reporter under the treatment of 750 M tBHP for 2 h. (d) Flow cytometric analysis of E. coli WT (K12) and ndh deletion 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 previously reported SAL-complexed WT MarR structure resembles the ligand-bound form of MarR with impaired DNAbinding capability. Superimposition of this dimer structure with that of the copper(II)-oxidized MarR5CS(80C) protein showed a highly similar overall topology (Fig. 3c; r.m.s. deviation = 1.54 for 248 C), further indicating that each of the dimer units within the copper (II)-oxidized MarR5CS(80C) tetramer adopts a conformation incapable of DNA binding. Finally, we solved the crystal structure of reduced MarRC80S mutant protein in the absence of DNA (Supplementary Fig. 13a), which verified that the cysteine-toserine mutations at residues 47, 51, 54, 108 and 111 in MarR5CS(80C) did not cause major conformational changes. Moreover, this structure also adopts a mode incapable of DNA binding, which is a conserved feature among the apo forms of MarR family proteins that has been revealed by previously solved apo and DNA-bound structures of OhrR, ST1710 and SlyA (Supplementary Fig. 13b)2527. So far, we have shown that copper(II) serves as a natural inducer for MarR and mediates the SAL- and antibiotic-induced marRAB derepression. We next asked how this copper signal could be generated within E. coli cells in the presence of SAL or antibiotics10,28. We first used a cell-permeable fluorogenic copper(I) sensor (CS-1)29 to test whether the amount of intracellular copper could be changed upon treatment of SAL or bactericidal antibiotics such as Nor and Amp. Notably, all of these molecules stimulated a marked increase of the CS-1 fluorescent signal, indicating the generation of labile copper(I) ions upon attacking E. coli cells (Fig. 4ac). In contrast, when Tet or Cm was used to treat the CS-1bearing E. coli cells, we observed no detectable fluorescence increase (Supplementary Fig. 14). In addition, we monitored the susceptibility of E. coli WT (K12) and marR deletion strains to Nor in the presence and absence of copper(II) (Supplementary Fig. 15a,b) or copper (II) chelator (Supplementary Fig. 15c,d), both of which showed that copper(II) ions can enhance MarR-mediated bacterial drug resistance. Furthermore, FACS analysis on E. coli cells bearing the pL(lexO)-GFP reporter28 demonstrated that the addition of copper(II) attenuated Nor-induced DNA damage in the E. coli WT (K12) strain but not in the marR deletion strain (Supplementary Fig. 16). We then went on to identify the copper source responsible for antibiotic-generated copper stress. In E. coli bacteria, the toxic copper ions are highly compartmentalized in the cell envelope, and many copper-containing proteins are located within this space, including periplasmic copper-binding proteins such as CueO (a multi-copper oxidase) and CusF (a copper chaperone), inner membrane copper transporters such as CopA and CusA and membrane-bound copperdependent proteins such as NDH-2 (a cupric reductase) and CyoB (cytochrome bo(3) ubiquinol oxidase subunit I). As bactericidal antibiotics such as Nor are able to cause bacterial envelope stress30,

Antibiotics trigger intracellular copper signaling

we wondered whether these envelope-residing copper proteins are the targets for antibiotic-triggered copper generation. We employed the CS-1 probe to examine Nor-induced intracellular labile copper production by individually deleting genes encoding the aforementioned representative copper-dependent envelope proteins (Fig. 4d and Supplementary Fig. 17). Among the E. coli deletion strains we tested, the strains lacking either ndh or cyoB were found to induce copper elevation to a lesser extent than the WT strain after Nor treatment, indicating that these cytoplasmic membrane-bound copper proteins might be responsible for Nor-triggered copper release. Next, we employed quantitative real-time RT-PCR (qRT-PCR) to examine the expression of these genes or genes encoding their regulators (for example, the CusA regulator CusR) with and without Nor treatment. All of the copper-related genes we tested were upregulated in the presence of a sublethal amount of Nor (250 ng/ml) in 2 h, indicating an elevated intracellular copper level triggered by antibiotics (Fig. 4e). We also performed qRT-PCR analysis on genes encoding envelope stress proteins such as CpxA, CpxR and CpxP (all of which belong to the Cpx system) as well as DegP, a major periplasmic protein quality control factor (Supplementary Fig. 18). Treatment of Nor increased the expression of all of these envelope stress proteins, and, in particular, the cpxp gene in the Cpx regulon has been previously shown to be upregulated upon copper liberation31, which further unveiled a connection between Nor-triggered envelope stress and copper elevation.

OHP production leads to membrane-bound copper liberation

To better understand how antibiotics might promote copper release from envelope proteins, we next studied the molecular mechanism underlying antibiotic-triggered impairment of copper-dependent envelope proteins. Of the genes encoding copper-dependent proteins that we tested, ndh caused the most noticeable decrease in copper levels when it was deleted; therefore, we focused on this protein for further investigation. As a cytoplasmic membrane located cupric reductase in a bacterial respiration system, NDH-2 can be specifically inactivated by tert-butyl hydroperoxide (tBHP), an organic hydroperoxide (OHP) known to cause the impairment of the respiration chain32,33. Particularly, tBHP is able to oxidize cuprous ions to cupric species via the Fenton-like reaction, similar to the conversion of iron(II) to iron(III)34,35. Such an oxidative modulation of copper ions has been shown to damage the E. coli membrane, which contains multiple copper-binding proteins, including NDH-2 (refs. 32,33,36,37). Indeed, our copper sensor CS-1 detected a marked increase in copper in the tBHP-treated E. coli WT strain relative to the ndh deletion strain, confirming that tBHP is able to mediate the redox cycling of copper within E. coli inner membrane through NDH-2 (Fig. 5a). Furthermore, by using our previously developed OHP-specific fluorescent sensor-OHSer38 and FACS analysis (Supplementary Fig. 19a,b), we showed that
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Environment OM
An ti t bio ics

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the generation of OHPs inside bacteria can be stimulated by Nor, Amp and SAL (Fig. 5b and Supplementary Fig. 19c,d) but not Tet or Cm (Supplementary Fig. 19d). We then employed EMSA to show that OHPs such as tBHP and cumene hydroperoxide could, in combination with copper(I), impair MarRs DNA-binding ability (Supplementary Fig. 20). Therefore, in addition to the aforementioned copper(II)-catalyzed disulfide bond formation between MarR dimers in the presence of oxygen in the air (Fig. 2c), these OHPs or additional unknown oxidative species may serve as the oxidants to promote this copper(II)-triggered MarR dimer-totetramer conversion. Further, we verified that copper ions released from these inner membranebound copper proteins were able to promote free copper levels in the MarR-residing bacterial cytosol. We engineered a pL(copA)-GFP reporter with GFP expression regulated by the copA promoter, which can be used to sense cytosolic copper. Treatment with either Nor or tBHP can promote the expression of GFP, indicating an enhanced copper level within the E. coli cytoplasm. Furthermore, less GFP generation was observed in the ndh deletion strain treated with Nor or tBHP (Fig. 5c and Supplementary Fig. 21), further confirming that NDH-2 is a major source for Norinduced copper generation. Finally, FACS analysis on the pL(marO)-GFP reporterbearing bacteria showed that both Nor treatment and OHP stress increased the fluorescent signal to a much lesser extent in the ndh deletion stain than in the E. coli WT strain (Fig. 5d and Supplementary Fig. 22). Moreover, the presence of the copper(II) chelator TETA attenuated tBHP-triggered MarR derepression (Fig. 5d), further verifying that copper(II) is the underlying cognitive inducer for the observed MarR derepression. Taken together, our results indicate that the Nor-triggered OHP production caused oxidative impairment of cytoplasm membranebound copper proteins such as NDH-2, which led to the generation of oxidized copper(II) species that can be sensed by MarR inside E. coli.

Envelope stress

OHPs Membrane damage


H2 Co pA oB

Cu

eO

Periplasm IM

dy stu is Th
H2O2 or ROOH OH or OR

M
Cu2+

ar

Cy s8 0

Cy s8 0

MarR

Discussion

This study uncovers a new connection between antibiotic-triggered envelope stress and copper signaling and MarR-mediated antibiotic tolerance in E. coli. Our data presented here demonstrate that copper(II) is an environmental cue that MarR senses directly. Copper(II)-triggered MarR cysteine oxidation derepresses the MarR-regulated antibiotic resistance response in E. coli. We found that the previously established SAL-induced MarR derepression actually goes through the copper-based mechanism, whereas treatment with a copper chelator eliminates SAL-triggered Mar activation. We further discovered that Nor and Amp, two clinically important bactericidal antibiotics, also stimulate intracellular copper elevation, which could trigger MarR derepression and turn on MarR-mediated antibiotic resistance. Taken together, we propose the following mechanistic model for copper(II)-modulated MarR derepression (Fig. 6): the treatment of Nor or Amp is able to induce envelope stress via protein unfolding, mistranslation or both within E. coli cells, which subsequently produces OHPs that impair cytoplasmic membranebound copper proteins such as NDH-2 and CyoB, resulting in the release of free copper(I) ions. This copper(I) liberation, in conjunction with OHP or other oxidative species triggered by antibiotics, elevates oxidized intracellular copper(II), which subsequently oxidizes the Cys80 residues on MarR to generate two disulfide bonds between two MarR dimers, causing dissociation of MarR from its cognitive DNA. This process ultimately derepresses MarR and thus the marRAB operon, resulting in enhanced bacterial antibiotic resistance. SAL-mediated MarR derepression could also cycle through a similar mechanism. Notably, a recent study using a bacterial two-hybrid system identified 48 potential partner proteins that may bind MarR39. In particular, transketolase A, an enzyme in central metabolism, has been
6

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. coli cells, which may subsequently cause lipid oxidation and produce OHPs that lead to the impairment of cytoplasmic membranebound 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. coli cytosol. 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 marRAB operon, 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.

further shown to directly interact with MarR in vivo, causing the derepression of the marRAB operon. This, in conjunction with our observation that the copper(II) chelator TETA cannot completely abolish antibiotic-triggered MarR derepression, seems to indicate that multiple pathways may exist that either a small signaling molecule (e.g., copper(II)) or an intracellular protein can modulate MarR-regulated antibiotic resistance. Whether ROS can potentiate the bactericidal effects of antibiotics remains controversial. Through the use of a genetically encoded OHP fluorescent sensor, we show here that other types of oxidative species, such as OHP, can be generated by fluoroquinolones or -lactam antibiotics via lipid oxidation that will subsequently trigger the liberation of copper as a signaling molecule in E. coli. Antibiotic tolerance has long been linked to cellular redox and metabolic states as well as intracellular metal ion homeostasis40. A low level of certain oxidative species is sufficient to have direct signaling roles or to trigger the liberation of a secondary signaling molecule within the cell. The lack of highly sensitive and selective indicators to probe these reactive and interconvertible oxidative or metal species in living cells persists, particularly for use under stress conditions such as antibiotic treatment. In turn, microorganisms have developed sensing mechanisms to detect these molecules as unified signals and turn on defense strategies against antibiotics or disinfectants16,4043. Moreover, cupric ions are known to catalyze non-native disulfide bond formation in the E. coli periplasm under copper stress or oxidative stress conditions44. Our study reveals that copper(II) is also able to trigger disulfide bond formation between MarR dimers in

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S RO ion t duc pro

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a catalytic fashion. E. coli may thus adapt this unique mechanism to the cytoplasm-residing transcription regulator MarR to directly sense the copper(II) signal and cope with antibiotics, environmentally derived stress or both. The characteristic Cys80 residue in MarR is highly conserved in diverse Enterobacteriaceae species, including various virulent E. coli strains, Shigella flexneri, Salmonella enterica, Enterobacter aerogenes and Klebsiella pneumoniae (Supplementary Fig. 23). This suggests that copper(II)-mediated cysteine oxidation and disulfide bond formation may be a general mechanism for derepression of these MarR transcription factors in Enterobacteriaceae. The exact role of MarR in defending host immune-derived copper poisoning as well as the crosstalk between MarR-mediated antibiotic resistance and copper defense merits further investigation.
Received 1 March 2013; accepted 13 September 2013; published online 3 November 2013

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Methods
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Methods and any associated references are available in the online version of the paper. Accession codes. Protein Data Bank: atomic coordinates and structure factors for the crystal structures of the copper(II)-oxidized MarR5CS(80C) and reduced MarRC80S proteins have been deposited under accession codes 4JBA and 3VOD, respectively.
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Rodriguez-Montelongo, L., de la Cruz-Rodriguez, L.C., Faras, R.N. & Massa, E.M. Membrane-associated redox cycling of copper mediates hydroperoxide toxicity in. Escherichia coli. Biochim. Biophys. Acta 1144, 7784 (1993). 33. Rodrguez-Montelongo, L., Faras, R.N. & Massa, E.M. Sites of electron transfer to membrane-bound copper and hydroperoxide-induced damage in the respiratory chain of Escherichia coli. Arch. Biochem. Biophys. 323, 1926 (1995). 34. Walling, C. Fentons reagent revisited. Acc. Chem. Res. 8, 125131 (1975). 35. Patikarnmonthon, N. et al. Copper ions potentiate organic hydroperoxide and hydrogen peroxide toxicity through different mechanisms in Xanthomonas campestris pv. campestris. FEMS Microbiol. Lett. 313, 7580 (2010). 36. Rapisarda, V.A. et al. Evidence for Cu(I)-thiolate ligation and prediction of a putative copper-binding site in the Escherichia coli NADH dehydrogenase-2. Arch. Biochem. Biophys. 405, 8794 (2002). 37. 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(21225206, 91013005 and 21001010 to P.R.C.), the US National Science Foundation (CHE-1213598 to C.H.) and the E-Institutes of Shanghai Municipal Education Commission (project number E09013 to C.H. and P.R.C.).

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Author contributions

Acknowledgments

We thank J. Rosner (National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health) for providing the strains of M2073 (WT E. coli) and M2076 (marRAB mutant strains) bearing the marR::lacZ reporter, J.J. Collins (Department of Biomedical Engineering, Boston University) for providing the pL(lexO)GFP reporter, C.J. Chang (College of Chemistry, University of CaliforniaBerkeley) for providing the Cu(I)-specific fluorescent probe CS-1 and S.F. Reichard for editing. The E. coli WT (K12) strain (BW25113) and all of the single-gene deletion mutants were obtained from the National BioResource Project (National Institute of Genetics, Japan). We also thank the staff members of the Shanghai Synchrotron Radiation Facility and the Beijing Synchrotron Radiation Facility. This work was supported by research grants from the National Basic Research Foundation of China (2010CB912302 and 2012CB917301 to P.R.C.; 2011CB809103 to C.H.), the National Natural Science Foundation of China

Z.H. performed the biochemical studies and participated in the structure studies. H.L. determined the crystal structures of copper(II)-oxidized MarR5CS(80C) and reduced MarRC80S. R.Z., J.Z. and X.C. helped with the biochemical and/or structure experiments. D.Z., B.S.Z. and S.Z. performed the experiments for OHP detection using OHSer. J.C. synthesized the Cu(I)-specific fluorescent probe CS-1. P.R.C. and C.H. conceived the study, designed experiments, interpreted data and wrote the manuscript with input from all of the authors.

Competing financial interests Additional information

The authors declare no competing financial interests. Supplementary information is available in the online version of the paper. Reprints and permissions information is available online at http://www.nature.com/reprints/index. html. Correspondence and requests for materials should be addressed to P.R.C. or C.H.

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8

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ONLINE METHODS

Strains and plasmids. The strains and plasmids used in this study were listed in Supplementary Tables 2 and 3, respectively. Primers used for plasmid construction and qRT-PCR were listed in Supplementary Table 4. For marR expression regulation or cytosolic copper stress detection, pL(marO)-GFP and pL(copA)-GFP reporter genes were constructed on the basis of the design of the PLlacO-1 promoter28. The gfpmut3b gene (expressing green fluorescent protein) was placed under the transcriptional control of synthesized marR promoter or copA promoter (restriction site: XhoI and KpnI). The sequences of these two promoters are listed in Supplementary Table 5. Reagents and equipment. All of the metals used in this study were purchased from J&K Scientific. Nor was purchased from Fluca, and SAL was bought from Sinopharm Chemical Reagent Co., Ltd. All of the other antibiotics were obtained from INALCO. ANS (8-anilino-1-naphthalenesulfonate) was bought from TCI. BCS, TETA (60%), 3-coumarin carboxylic acid (3-CCA), tBHP (70%), cumene hydroperoxide (CHP, 80%) and IAM were obtained from Alfa Aesar. All of the other chemicals were analytical grade or better. All pictures of protein gels, including those of Coomassie Brilliant Blue (CBB)-stained SDS-PAGE gels and western blotting membranes, were taken on ChemDoc XRS+ (Bio-Rad). UV-visible spectroscopy was performed using a UV-visible spectrometer (U-3010, HITACHI, Japan). All fluorescent spectroscopic mea surements were recorded at 25 C on a Hitachi F4500 fluorescence spectrophotometer equipped with a LPS-220B 75-W xenon lamp and power supply. All of the data in flow cytometry analysis were collected using a Becton Dickinson FACSCalibur flow cytometer (BD bioscience) with a 488-nm argon laser and a 515- to 545-nm emission filter (FL1) at low flow rate. At least 70,000 cells were collected for each sample. Flow data were processed and analyzed with FCS express Version 2. Protein expression and purification. WT MarR and its mutants with a thrombin-cleavable N-terminal His tag were prepared from E. coli BL21(DE3), which bears a pET15b vector containing the marR gene. Cells were grown in LB medium containing 50 g/ml Amp at 37 C to an OD600nm of 0.6 before IPTG was added to a final concentration of 1 mM. Protein expression was induced at 30 C for 5 h before harvest. Cell pellets were collected and resuspended in NTA buffer (20 mM Tris-HCl, pH 7.5, 300 mM NaCl and 10% glycerol) containing 1 mM PMSF (phenylmethanesulfonyl fluoride) and 5 mM -mercaptoethanol. After sonicating on ice for 20 min, the cell lysate was spun down at 15,000 r.p.m. for 30 min. Cleared lysate was filtrated and loaded onto a 5-ml Histrap column (GE Healthcare), which was pre-equilibrated with 25 ml NTA buffer. MarR protein was then eluted with a linear imidazole gradient followed by a further purification step using HiLoad 16/60 Superdex 200 column (GE Healthcare) equilibrated with buffer A (20 mM Tris-HCl, pH 7.5, 300 mM NaCl). Protein was collected and concentrated for further experimental use. The N-terminal His tag of MarR proteins can be removed by thrombin digestion, which was performed by incubating tagged protein with thrombin at 16 C overnight in digestion buffer (20 mM Tris-HCl, pH 8.4, 2.5 mM CaCl2, 150 mM NaCl) (Supplementary Fig. 24). Electrophoretic mobility shift assays (EMSA). A 42-mer duplex DNA containing marRAB promoter sequence (5-ATTCATTTGACTTATACTTG CCTGGGCAATATTATCCCCTGC-3 and 5- GCAGGGGATAATATTGCCC AGGCAAGTATAAGTCAAATGAAT-3) was used for this assay. All of the EMSA reaction mixtures in this study were set up in a final volume of 20 l containing 50 nM annealed double-stranded DNA, 1 M MarR protein monomer (0.5 M for mutant MarR5CS(80C)) in binding buffer (50 mM KCl, 5 mM MgCl2, 10% glycerol and 10 mM Tris-HCl, pH 7.8). The concentrations for CuCl2 and other metals were kept at 2.5 M. Samples were loaded onto the 8% nondenaturing polyacrylamide gel before incubation at room temperature for 20 min and were run in 0.5 TB buffer (tenfold dilution from 5 TB buffer: 50 mM Tris-HCl, 41.5 mM borate, pH 7.8) at 110 V for 1 h at 4 C. Gels were stained in a 10,000-fold diluted SYBR Gold nucleic acid staining solution for 7min. The DNA bands were visualized with UV light at 254 nm by ChemiDoc XRS+ (Bio-Rad). Spectroscopic study. The fluorescence probe ANS (8-anilino1-naphthalenesulfonate) was employed to indicate the conformational change of protein upon treatment with potential metal ions. Different concentrations
doi:10.1038/nchembio.1380

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of CuCl2, FeCl3 or NiCl2 were added to the solution containing the MarRANS complex (0.5 mM ANS and 5 M MarR in 20 mM HEPES buffer, pH 7.0). Addition of DTT (1 mM) or EDTA (3.5 mM) prevented the fluorescence change of the MarRANS complex solution upon treatment with copper(II). To determine the stoichiometry of WT MarR to copper(II), aliquots of CuCl2 (from 5 M to 22.5 M) were titrated into the protein-BCS solution containing 5 M MarR monomer protein and excess BCS (200 M) in a final volume of 100 l. The absorption at 483 nm (characteristic absorption of Cu(I)(BCS)23 complex) were plotted versus the molar ratio of copper(II) to protein (monomer concentration). All of the experiments were performed in 20 mM HEPES buffer (pH 7.0) on an UV-visible spectrometer (U-3010, HITACHI, Japan). The stoichiometry of copper(II)/MarR5CS(80C) was determined with the same method. Aliquots of CuCl2 (from 7.5 M to 30 M) were titrated into the protein-BCS solution containing 15 M MarR monomer protein and excess BCS (200 M). The hydroxyl radical scavenging compound 3-CCA (3-coumarin carboxylic acid) was used to determine the copper-catalyzed hydroxyl radical production45. As an indication of the hydroxyl radical production, the formation of the product 7-hydroxy-coumarin-3-carboxylic acid (7-OH-CCA, exc, 395 nm; em, 450 nm) was measured upon the addition of WT MarR (5 M) to the samples containing 5 M CuCl2 and 300 M 3-CCA in the absence and presence of sodium ascorbate (500 M) for 25 min. The hydroxyl radical production upon the reaction of CuCl2 (5 M) with sodium ascorbate (500 M) and 3-CCA (300 M) was also measured as a positive control. All of the measurements were carried out in 20 mM PBS buffer (pH 7.4) using a fluorescence spectro meter (F-4600, HITACHI, Japan). DTNB assay. The free thiol contents of native and copper(II)-oxidized WT MarR were determined by using the 5,5-dithiobis-2-nitrobenzoic acid (DTNB) assay. 25 M WT MarR was treated with 500 M CuCl2 in the presence and absence of EDTA (10 mM) for 10 min at room temperature in DTNB assay buffer (100 mM potassium phosphate, pH 7.2, 100 mM NaCl and 1 mM EDTA). Excess copper(II) was removed by buffer exchange. After addition of 1 mM DTNB, The absorption at 412 nm was recorded, and free thiol concentrations were calculated on the basis of previously published methods22. Disulfide bond detection by MS. Purified MarR5CS(80C) and WT MarR (all at 40 M) were incubated with 20 M (0.5) CuCl2 at room temperature for 5 min. Any remaining free thiols were blocked by treatment with excess IAM for 45 min. Protein samples were run on a nonreducing, denaturing SDS PAGE gel. The covalently linked dimer band was separately excised from the SDS PAGE gel, destained and digested with trypsin at 37 C for 12 h. The resulting tryptic peptide was extracted and subjected to MS analysis using a Thermo LTQ-Orbitrap velos mass spectrometer equipped with the EASYnLCII integrated nano-HPLC system (Proxeon, Denmark). The analytical column was pursed from Agilent (75 m ID, 150-mm length; Agilent USA). Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 100% acetonitrile and 0.1% formic acid. The single full-scan mass spectrum was acquired in the Orbitrap from m/z 35020,000 followed by three MS/MS scans in the quadruple collision cell using the collision-induced dissociation (CID), in which the precursor mass selection window was set at 2 Da. Crystallization. For the crystallization conditions, we used the commercially available Hampton Research screens: Index, Crystal Screen, SaltRx 1, SaltRx 2, PEG/Ion Screen, PEG/Ion 2 Screen. Sitting-drop vapor diffusion at room temperature (1020 C) was applied by mixing 2 l protein sample (at 7.2 mg/ml or 11.8 mg/ml) with 2 l well solution. We used the N-terminal His-tag cleaved protein sample for crystallization. The best MarR5CS(80C) crystal grew in 2.2 M sodium chloride, 0.1 M sodium acetate trihydrate, pH 4.6. The best MarRC80S crystal grew in 24% PEG 3350, 0.1 M CBTP buffer (pH 7.6; 0.03 M citric acid and 0.07 M Bis-Tris propane mixed), 0.3 M NaCl and 10 mM DTT. X-ray data collection and structure determination. Cryoprotectant was made fresh by mimicking growth conditions. We used step-wise soaking to avoid crystal cracking. The crystal was quickly dipped into a series of solutions with increased glycerol concentration up to 25%. The crystal was finally flash frozen in liquid nitrogen prior to data collection. Data sets of MarR5CS(80C) and MarRC80S were collected on the BL17U1 beamline at the Shanghai Synchrotron Radiation Facility (Shanghai, China) and the 3W1A beamline at Beijing Synchrotron Radiation Facility, respectively. The wavelength used for data
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ollection for MarR5CS(80C) and MarRC80S was 0.9792 and 1 , respectively. c Both data sets were initially indexed by iMOSFLM46 and were further processed with the CCP4 package47. The structures were solved through molecular replacement using the dimer form of WT MarR (Protein Data Bank code 1JGS) as the search model with the program PHASER48. Refinement was carried out on the programs REFMAC5 (ref. 49) and COOT50. The Ramachandran plot given by PROCHECK51 shows that in MarR5CS(80C), 99.24% residues were in the most favored region, and 0.76% were in the allowed region. In MarRC80S, 98.13% residues were in the most favored region, and 1.87% were in the allowed region. Neither of the two structures have residues in the disallowed region. Residues 9295 in chain A and residues 9294 of MarR5CS(80C) were disordered; therefore, they were not modeled. NCS restraints52 and TLS refinement53 were applied. The stereochemical quality was analyzed using MolProbity54. PyMOL was used to generate all of the figures. A summary of the data statistics is shown in Supplementary Table 6. Size-exclusion chromatography. Freshly purified protein of MarR5CS(80C), MarRC80S and MarR6CS from the Histrap column (GE Healthcare) was instantly incubated with 20% CuCl2 (5% CuCl2 was also tested for MarR5CS(80C)) at room temperature (25 C) for 15 min. CuCl2 was slowly added into the protein solution to avoid local precipitation. Gel filtration was then performed with a Superdex 75 10/300 GL column (GE Healthcare). The molecular weight of the eluting species was calculated by standard protein marker (Gel Filtration LMW Calibration Kit, GE Healthcare). b-Gal assay. The E. coli WT strain (M2073) or marRAB mutant strain (M2076) containing the marR::lacZ reporter fusion was used for this assay. -Gal activity was measured using MUG as a substrate. Overnight cultures were inoculated (1:100) in M9 minimal medium or LB medium and grown to an OD600nm of 0.6 at 37 C. To examine the MarR derepression in the presence of metal ions, cells in M9 medium were treated with 100 M CuCl2, FeCl3, ZnCl2 or NiCl2 for 30 min. For complementary experiments, DmarRAB mutant strain (M2076) harboring plasmids expressing WT MarR or MarRC80S with or without the His tag were induced by IPTG (1:1,000) or arabinose (1:200) for 2 h before being challenged by 100 M CuCl2 for 30 min. SAL-induced marR derepression in the presence of TETA was also tested, and bacterial cells in LB medium were challenged by 2.5 mM SAL in the presence and absence of 1 mM TETA for 30 min. After these treatments, 1-ml aliquots were obtained for measurement of OD600nm, and the remaining aliquots (1 ml) were harvested by centrifugation, washed with 1 PBS buffer and stored at 80 C. At the time of the assay, cells were resuspended in 100 l of lysis buffer (50 mM Tris-HCl, pH 8.0, 25 mM NaCl, 2 mM EDTA) containing 0.5 mg/ml lysozyme and were incubated at 37C for 15 min, followed by centrifugation. The upper layers were delivered to a 96-well plate and two tenfold dilutions (90 l ABT buffer (AB buffer plus 0.1% Triton X-100) plus 10-l sample) were set up in the plate. 50 l of the samples from each well were transferred to a new 96-well plate followed by addition of 10 l MUG (0.5 mg/ml) into each well to start the reaction. The reactions were incubated at room temperature for 1 h before 20 l of the samples were added to 180 l of ABT buffer in a black 96-well plate. -Gal activity (expressed in MUG units; 1 unit = 1 pmol of MUG cleaved per min per OD600nm) was determined by fluorescence (exc, 366 nm; em, 445 nm) using a microplate reader (synergy4, BioTek). pL(marO)-GFP reporter assay. An overnight bacterial culture harboring the pL(marO)-GFP reporter gene was inoculated (1:100) in growth medium and grown to an OD600nm of 0.6 before being challenged by various stimulants. To examine the MarR derepression in the presence of metal ions, M9 medium was used, and bacterial cells were treated with 100 M CuCl2, FeCl3 and 1 mM H2O2 for 1 h before being analyzed by flow cytometry. The PMT voltage settings used are: E01 (FSC), 420 (SSC) and 450 (FL1). To survey MarR derepression by SAL, antibiotics and OHPs with or without the copper(II) chelator TETA, bacterial cells were challenged by 2.5 mM SAL, 250 ng/ml Nor, 2.5 g/ml Amp or 750 M tBHP in the presence and absence of 1 mM TETA for 1 h, followed by flow cytometric analysis with the following PMT voltage settings: E01 (FSC), 413 (SSC) and 436 (FL1). Copper signal detection. A specific copper(I) fluorescence sensor CS-1 was used to detect the generation of copper(I) ions inside E. coli cells upon exposure to various stimulants. Overnight bacterial cultures were inoculated (1:100) in 5 ml fresh LB and were grown to an OD600nm of 0.6 before being treated by
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SAL (5 mM), antibiotics (2.5 g/ml Amp, 250 ng/ml Nor, 20 g/ml Cm and 20 g/ml Tet) or 750 M tBHP at 37 C for 2 h. After washing with 10 mM PBS buffer twice, bacterial samples were incubated with 2 M CS-1 for 15 min at 37 C followed by washing with PBS buffer twice. Changes of fluorescence were monitored by flow cytometry with the following PMT voltage settings: E01 (FSC), 413 (SSC) and 710 (FL1). To analyze cytosolic copper generation, we constructed the pL(copA)-GFP reporter. Cells harboring pL(copA)-GFP reporter genes in exponential phase (OD600nm of 0.6) were challenged with 250 ng/ml Nor or 750 M tBHP for 2 h. The bacterial samples were analyzed using flow cytometry. The following PMT voltage settings were used: E01 (FSC), 420 (SSC) and 450 (FL1). DNA damage analysis. Overnight cell cultures of E. coli WT (K12) and marR deletion strains harboring the pL(lexO)-GFP reporter were inoculated (1:100) in 5 ml LB medium and further grown to an OD600nm of approximately 0.6. At this point, cells were pretreated with Nor (250 ng/ml) for 1 h, followed by incubation with 500 M CuCl2 for an additional 1 h before flow cytometric analysis. The following PMT voltage settings were used: E01 (FSC), 413 (SSC) and 500 (FL1). Quantitative real-time RT-PCR. Overnight cultures of E. coli WT (K12) and marR deletion strains were inoculated (1:100) in LB medium, grown at 37 C to an OD600nm of 0.7 and then incubated with 250 ng/ml Nor at 37 C for 2 h before being harvested. Total RNAs were extracted using SV Total RNA Isolation System (Promega, Madison, USA) according to the manufacturers protocol. The total RNA (1 g) was reverse transcribed to first strand cDNA by RevertAid First Strands cDNA Synthesis kit (Fermentas, Maryland, USA) following the manufacturers guidelines. Quantitative real-time RT-PCR was performed on CFX96 Real-Time PCR (Bio-Rad). For each 20-l reaction, 10 l 2 SYBR master mix (QPK-201,TOYOBO Co., Ltd. Osaka, Japan), 8.4 l cDNA (100-fold dilution) and 0.8 l primer (10 M) were added. Relative quantification analysis was performed as following conditions: one cycle at 94 C for 4 min, followed by 40 cycles at 94 C for 20 s and 58 C for 20 s, then one cycle at 72 C for 20 sec. Melting curve analysis started at 65 C and went up to 94 C. The fluorescence was collected every 0.2 C. The rrsA (16S rRNA) was used as an internal control. All of the experiments were conducted in at least three independent replicates, and relative expression levels were measured using the 2Ct method55. Cell viability assay. To assess the roles of copper signaling in MarR-mediated antibiotic resistance, overnight cultures of E. coli WT (K12) and marR deletion strains were inoculated (1:100) in LB medium and grown at 37 C to an OD600nm of 0.6. Cells were then incubated with 1 g/ml Nor in the presence and absence of CuCl2 (500 M) or 300 M copper(II) chelator TETA for 30 min at 37 C. Tenfold serial dilutions of bacteria were plated for colony counts. Only dilutions that yielded 10100 colonies were counted, and bacterial survival rates were determined by the percentage survival of the copper- or TETA-treated group compared to the untreated group. OHP detection. To detect OHP generation inside bacteria under treatment with SAL, tBHP or various antibiotics, we used our previously developed OHPspecific fluorescent sensor OHSer. E. coli cells harboring OHSer were grown overnight and then diluted 1:100 in LB medium (50 g/ml kanamycin). OHSer expression was induced by the addition of 1 mM IPTG (final concentration) at an OD600nm of approximately 0.60.8. Protein production was allowed to proceed for 2 h at 30 C before treatment with Nor, Amp or oxidants (tBHP and H2O2) for 30 min. Samples were analyzed by flow cytometry. The following PMT voltage settings were used: E01 (FSC), 427 (SSC) and 725(FL1). Statistical analysis. All of the data are presented as mean s.d. The statistical analysis was performed using an unpaired Students t-test when the two groups were compared. When more than two groups were compared, one-way analysis of variance (ANOVA) followed by Bonferroni means comparison was used. P < 0.05 was considered to be significant.
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, T.G.G., Kontogiannis, L., Johnson, O., Powell, H.R. & Leslie, A.G.W. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D Biol. Crystallogr. 67, 271281 (2011).
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47. Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235242 (2011). 48. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658674 (2007). 49. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240255 (1997). 50. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 21262132 (2004). 51. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283291 (1993).

52. Kleywegt, G.J. Use of non-crystallographic symmetry in protein structure refinement. Acta Crystallogr. D Biol. Crystallogr. 52, 842857 (1996). 53. Painter, J. & Merritt, E.A. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D Biol. Crystallogr. 62, 439450 (2006). 54. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 1221 (2010). 55. Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2CT method. Methods 25, 402408 (2001).

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