You are on page 1of 11


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.

© 2013 Nature America, Inc. All rights reserved.


he MarR family of transcription factors regulates diverse genes involved in multiple antibiotic resistance, synthesis of virulence determinants and many other important biological processes1–3. 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 in vitro. 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)), β-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

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 California–Berkeley, Berkeley, California, USA. 7Shanghai Universities E-Institute for Chemical Biology, Shanghai, China. 8Current address: Cardiovascular Research Institute, University of California–San Francisco, San Francisco, California, USA. 9These authors contributed equally to this work. *e-mail: or


1b and Supplementary Fig.000 3. This. 3a).com/naturechemicalbiology .500 300 200 100 0 0 10 Control Cm Tet Nor Amp © 2013 Nature America. AU. MgrA and PerR14–17. of three independent experiments. We could therefore exclude the possibility that antibiotic-damaged Fe-S clusters release free iron.5 mM SAL with and without TETA (1 mM). cobalt(II).1380 b T DT O 2 MarRre MarRox Cu(II) + 2BCS Absorption at 483 nm (AU) 0. coli WT (K12) strain harboring the pL(marO)-GFP reporter was treated with Tet (20 μg/ml). 1 unit is equal to 1 pmol of MUG cleaved per min per OD600nm. coli. As H2O2 is a main product of the host immune response that is disproportionated from superoxide14. which forms a Cu(I)–(BCS)23− complex with a characteristic UV-absorbance peak at 483 nm (ref. Supplementary Figs. Finally. copper(II) was the only transition metal within this group to disrupt the MarR–DNA complex (Fig. SoxR. respectively). Further.500 3. FeCl3. arbitrary units. coli NATURE chEmicAl biology | ADVaNCE ONLINE PUBLICaTION | www. from three independent samples (*P < 0. We applied an electrophoretic mobility shift assay (EMSA) to 50 nM DNA in the presence of 1 μM wild-type (WT) MarR (monomer concentration). 2. which remained as a protein–DNA complex with and without the addition of 500 μM H2O2 (Fig. (f) The E. In the presence of H2O2. 21). (d) β-Gal activity of E. bathocuproine disulfonate (BCS).1038/NchEmbio. Data represent the mean ± s. (c) EMSA analysis of the DNA-binding capability of MarR6CS mutant (1 μM monomer protein and 50 nM DNA) in the absence (lane 2) and presence (lane 3) of 2. which indicated that two out of the six cysteine residues on each MarR monomer can be oxidized by copper(II). 0 Control Cu(II) Fe(III) Zn(II) Ni(II) H2O2 0 SAL (2. Titration of the protein-BCS solution (5 μM MarR monomer protein and 200 μM BCS) with aqueous copper(II) ions indicated a stoichiom­ etry of 1:2 between MarR monomer and copper(II) (Fig. AU. 1a and Supplementary Results. coli WT (M2073) strain containing the marR::lacZ reporter was determined after treatment with 100 μM CuCl2.5 μM FeCl3. coli via other pathways such as SoxS activation19. absorbance unit.  1a. we tested it first.nature. Notably. A β-galactosidase (β-Gal) assay was first conducted on an E. This result agreed with our 5. neither iron(II) nor iron(III) ions were found to cause MarR-DNA dissociation. which serves as a natural inducer for MarR. EMSA was then used to test a panel of divalent metal ions. 1 and 2). Furthermore. β-Gal activity is expressed in 4-methylumbelliferyl-D-galactopyranoside (MUG) units. This observation is in line with previous reports that indicate that oxidative stress may only indirectly derepress marRAB operon in E. Inc. the presence of MarR was found to effectively prevent radical production from copper(II) in the presence of ascorbate (Supplementary Fig.5 μM copper(II) or the same amount of copper(I) was able to efficiently dissociate MarR from its promoter DNA. 3b).5 μM CuCl2. Zn(NO3)2 or NiCl2 or 500 μM H2O2 in M9 medium for 30 min. NiCl2. copper(II) and zinc(II). bacteria such as OxyR. in combination with our 8-anilino-1-naphthalenesulfonate (ANS) assay20 monitoring the conformational change of MarR with 2 and without copper(II) (Supplementary Fig.01).d. Taken together. Data in d and e represent the mean ± s.14 c Cu(II) (2.5 μg/ml) for 1 h before being analyzed by flow cytometry. Nor (250 ng/ml) or Amp (2. strongly suggesting that MarR may efficiently compete against cellular reductants in scavenging free copper(II) ions. these MarR and DNA concentrations were used for all of the following EMSA experiments unless otherwise noted). Absorption at 483 nm (the characteristic absorption peak of the Cu(I)–(BCS)23− complex) was plotted against the copper(II)/protein ratio. Sublethal levels of antibiotics have also been shown to stimulate a ROS-induced mutagenesis that ultimately leads to bacterial multidrug resistance18. which showed that these MarR variants remained bound to the promoter DNA even after copper(II) treatment (Fig. 1c and Supplementary Fig. whereas the addition of a reducing agent such as dithiothreitol (DTT) effectively restored MarR’s DNA-binding ability.000 500 * * Cell count 2.  3c). To examine whether MarR causes the reduction of copper(II) to copper(I).000 f 500 400 MUG unit 2.5 mM) TETA (1 mM) − − − + + − + + − − + − 101 102 Fluorescence intensity (AU) Figure 1 | Copper(II) is a natural signal for MarR derepression. We next demonstrated that copper(II) is the cognitive signal for MarR derepression inside E.5′-dithiobis-2-nitrobenzoic acid (DTNB) assay22 (Supplementary Table 1). (b) Titration of aliquots of CuCl2 into the MarR-BCS solution (5 μM MarR monomer and 200 μM BCS). (a) 50 nM DNA (42 base pairs) was incubated with 1 μM WT MarR (monomer concentration. coli WT strain bearing a marR::lacZ Copper(II) is a natural inducer for MarR inside E.ARTICLE a WT MarR Nature chemical BioloGY Doi: 10. 4). including manganese(II).600 1. Cm (20 μg/ml). (e) β-Gal activity of the marR::lacZ reporter was conducted in WT strain (M2073) and the marRAB mutant strain (M2076) upon the addition of 2. our biochemical studies revealed that copper(II) ions can oxidize up to two cysteine residues on each MarR monomer and attenuate MarR’s DNA-binding ability.500 1. CuCl2 (in the presence and absence of 100 μM DTT) or 500 μM H2O2 (lanes 3–7.07 MarR–DNA complex DNA 1 2 3 4 5 6 7 0 0 1 2 3 4 Equivalent of Cu(II) M2073 M2076 5 DNA 1 2 3 d 1.d. All rights reserved.000 1. indicated that the interaction between MarR and copper(II) is redox sensitive.5 µM) MarR6CS − + Cu (II ) Cu (II )+ Cu(I)(BCS)23− I) (II ) (II Fe Ni H 2 MarR–DNA complex 0. nickel(II).200 MUG unit 800 400 * e 4. we employed a copper(I)-specific indicator. We wondered whether related species or processes could induce MarR’s dissociation from its cognitive DNA. lane 2) before being treated with 2. all of the six cysteine residues on MarR were either mutated to serine or blocked by iodoacetamide (IAM) before being subjected to EMSA analysis.

1d). 1f).1038/NchEmbio. 7a–e). SAL at a concentration of 2.27 L V C K Y4 Y3 Y2 Y1 649. and their corresponding apparent MWs are shown on top. 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.73 497. 2a). pL(marO)-GFP.10 a1+ 81′ L V C K S 590.29 515.0 462. zinc(II) or H2O2 (Fig. Further.5 mM) was also found to trigger GFP induction in the E. Fluorescence-activated cell sorting (FACS) can quantitatively measure the expression of GFP under the control of the marR promoter in living bacterial samples.d. 609.01. whereas the addition of iron(III) or H2O2 did not generate a noticeable increase (Supplementary Fig. (d) Mass spectrum of an unfractionated tryptic peptide mixture of the copper(II)-oxidized MarR5CS(80C) protein. thus verifying that SAL indirectly derepresses MarR via copper(II) species within E. Because the bacterial cytosol is a highly reduced environment under normal conditions. Finally.5 μM) effectively triggered 3 NATURE CHEMICAL BIOLOGY | ADVaNCE ONLINE PUBLICaTION | www. nickel(II). All rights reserved. the MarRC80S mutant remained bound with its promoter DNA in the presence of copper(II) ions (Fig. coli cells. that relies on MarR repression for the tight regulation of GFP expression.54 Da 86. The addition of 100 μM copper(II) ions led to a threefold increase of β-Gal activity compared to untreated cells or cells treated with the same concentration of iron(III). absorbance unit. The presence of copper(II) ions also did not cause any noticeable difference in the fluorescent signal in the marR deletion strain (Supplementary Fig. Molecular mechanism of copper(II)-induced MarR activation To elucidate the molecular details of MarR’s response mechanism to copper(II).63 442. 1e).Nature chemical BioloGY Doi: 10. We also engineered a GFP-based. we investigated potential residues involved in the MarRcopper(II) interaction. 5a). 1 unit is equal to 1 pmol of MUG cleaved per min per OD600nm. (b) β-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 μM CuCl2. β-Gal activity is expressed in 4-methylumbelliferyl-Dgalactopyranoside (MUG) units. n = 5. 6). we mutated each individual cysteine on MarR to serine for EMSA analysis to identify the cysteine residue (or residues) responsible for this process. . In contrast. coli WT (K12) strain.44 800 m/z 650 Figure 2 | The response mechanism of MarR to copper(II). (e) MS/MS fragmentation of the 2+ charged peptide (m/z: 461. The molecular weight (MW) of these apparent tetramers and dimers was calibrated with standard markers. In addition.nature. 1c and Supplementary Fig. whereas the presence of the TETA (1 mM) attenuated this MarR-related GFP induction (Supplementary Fig. To confirm that copper(II) is the real signal for SAL-triggered MarR derepression.5 m/z y2 y1 S +461. Together. we added 1 mM of the copper(II)-specific chelator triethylenetetramine (TETA) to the SAL-treated bacterial cells23. Results represent mean ± s.5 mM was also found to stimulate over threefold greater marR expression compared to untreated samples (Fig. **P < 0. 4). coli.5 μM) were unable to affect the DNA-binding capability of the MarRC80S mutant protein (1 μM). Inc.16 (b3Y2) © 2013 Nature America. coli marRAB mutant strain bearing the marR::lacZ reporter (M2076) was used as a control. reporter gene (M2073) with and without copper(II). The same amount of copper(II) ions (2.22 2+ 213. which exhibited only a slight increase of β-Gal activity after SAL treatment.45 700 750 800 m/z 100 200 282. neither Tet nor Cm was found to stimulate MarR derepression within the period of time we tested (Fig. The addition of copper(II) ions was found to markedly induce the expression of GFP in the E. 1e). 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.2807 600 700 808. we found that both Nor (250 ng/ml) and Amp (2. As we have shown that cysteine residues were required for copper(II)-triggered MarR-DNA disruption (Fig.97 Y1 or y1+ 147. the E. 1f). Similarly. (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.5 462. which can be largely eliminated by adding 1 mM TETA (Supplementary Fig.09 300 400 500 581.17 a2+ 10 11 12 Elution volume (ml) 78 b2 b3 y3 81 13 d 2 + 78 LVCK 81 78′ LVCK e Theoretical: 920. labile copper ions are thought to exist almost exclusively at the copper(I) state with few cupric species.27 81′ (y2Y4)+ 709. 5a).5 μg/ml) were able to induce GFP expression and thus MarR derepression within 1 h (Fig. 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.52 Da Experimental: 920. Inset: The 2+ charged peak (m/z: 461–463) corresponding to the disulfide-containing peptide of interest (theoretical molecular mass: 920.27). SAL (2. 5b).38 (y3Y4)+ 78′ 461. (a) EMSA analysis showing that copper(II) ions (2. coli WT (K12) strain.36 461. marR-inducible fluorescent reporter.11 b2+ (y3Y4)2+ 404.1380 a MarRC80S ARTICLE Control + Cu(II) (100 µM) b DT T Cu (II )+ O 2 800 ** c 160 MW 75 (kDa) Tetramer 44 Dimer 29 (II I) Cu (II ) 600 Absorption (mAU) MUG unit 120 80 40 0 − Cu(II) + Cu(II) Fe H 2 400 MarR–DNA complex 200 0 DNA M2076 /WT MarR M2076 /MarRC80S 9 185.29 400 450 500 550 600 671. MarR5CS(80C) dimer protein (125 μM) was incubated with (red) and without (black) 25 μM CuCl2 for 15 min before being analyzed with a Superdex 75 (10/300) column. which effectively diminished SAL-mediated MarR derepression (Fig.52 Da).

ARTICLE Nature chemical BioloGY Doi: 10. A symmetric MarR tetramer complex was observed upon copper(II) oxidation.5 μg/ml. Size-exclusion chromatography was then used to examine the oligomeric state of the MarR5CS(80C) protein before and after copper(II) oxidation. coli WT (K12) cells before (black) and after (green) treatment with different reagents were measured by flow cytometry. we performed MS mapping analysis. we were also unable to obtain the copper(II)-oxidized WT MarR protein crystals. coli cells (Supplementary Fig. Data represent the mean ± s. 4 NATURE chEmicAl biology | ADVaNCE ONLINE PUBLICaTION | www. coli K12 ∆ndh ∆ndh + Nor E.1038/NchEmbio. Electron density of the two disulfide bonds was clearly visible. Inc.d. coli marRAB mutant strain bearing the marR::lacZ reporter was complemented with a plasmid expressing either WT MarR or MarRC80S before being subjected to β-Gal analysis. 2b).and antibiotic-triggered intracellular copper elevation. 9). from three independent experiments. (b) Close-up of the two disulfide bonds between two MarR dimers. cusF. whereas a 4% copper(II) loading led to a more than 60% dimerto-tetramer conversion (Fig. 12a). 2c and Supplementary Fig. (c) Superposition of the structures of copper-oxidized MarR5CS(80C) (blue) and the previously reported SAL-complexed WT MarR (orange). 3b and Supplementary Fig. all of the DNA recognition helices (α4) in the DNA-binding domain from each MarR monomer were buried within the interface between two MarR dimers. The fluorescence (in arbitrary units (AU)) of E.52 Da) in the copper-treated MarR5CS(80C) and WT MarR proteins (Fig. Only residues Asp67(Asp67’).nature. d 500 400 Cell count 300 200 100 0 0 101 102 10 Fluorescence intensity (AU) b 400 300 200 100 0 100 101 102 Fluorescence intensity (AU) Control Nor c 400 Cell count 300 200 100 0 100 101 102 Fluorescence intensity (AU) Control Amp Normalized expression E. coli copper-dependent genes cusR. which showed an approximately 1:1 stoichiometry between copper(II) and the monomeric form of this MarR mutant (Supplementary Fig. All rights reserved. but copper(II) treatment severely disrupts its DNA binding.1380 Copper(II)-oxidized WT and mutant MarR proteins were next analyzed by nonreducing SDS-PAGE. with a measured S-S distance at 2. c) for 2 h. 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.0 Å and a dihedral angle of C-S-S-C at 90. In particular. Together.5 Å. (d) Flow cytometric analysis of copper levels as measured by the CS-1 probe inside E. The tetramer protein contains two disulfide bonds between two MarR dimers via Cys80 residues from each monomer (Fig. 2d. Finally. which showed that a covalent linkage was generated between Cys80 residues on MarR upon copper(II) treatment in vitro and inside . with the disulfide bonds shown in yellow sticks.e and Supplementary Fig. which identified disulfide-containing peptides (crosslink between Cys80 and Cys80′ on two Leu-Val-Cys-Lys peptides. copA. (a–c) Generation of copper(I) ions inside E. Figure 3 | Crystal structure of copper(II)-oxidized MarR5CS(80C). ndh and cyoB in the WT (K12) strain upon Nor treatment (250 ng/ml. Our aforementioned results indicate that the reduced MarR5CS(80C) protein can bind DNA with an affinity similar to that of WT MarR. Therefore. calculated molecular weight: 920. b) or Amp (2. Nor (250 ng/ml. coli K12 + Nor e 3 2 1 0 Control Nor cusR cusA cueO cusF copA ndh cyoB Figure 4 | SAL. Atoms in the side chains of Cys80 from all four of the monomers are shown as yellow spheres. The values are shown as the change of Nor-treated cells (black bar) in comparison with untreated cells (gray bar).3° (Fig. 7f). We found that 100 μM 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. 8). Cys80(Cys80’) and Gly82(Gly82’) from each monomer are shown for clarity. We next performed the BCS assay on MarR5CS(80C) and copper(II) ions. 10). 2 h) as determined by qRT-PCR. coli WT (K12) cells as measured by the CS-1 probe (2 μM) upon the treatment of SAL (20 mM. the behavior of MarR5CS(80C) resembles that of WT MarR with and without copper(II). Crystal structure of copper(II)-oxidized MarR5CS(80C) the dissociation of 0.5 μM MarR5CS(80C) mutant protein from 50 nM promoter DNA (Supplementary Fig. a 400 Cell count 300 200 100 0 100 101 102 Fluorescence intensity (AU) Control SAL Cell count To gain more insight into the molecular mechanism of copper(II)mediated MarR oxidation. To further reveal the nature of the disulfide-stabilized MarR tetramer produced by copper(II). a). 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. 11). rendering them incapable of DNA binding. 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. the E. Electron density map from the crosslinked Cys80-Cys80’ residues are contoured at 1σ 2Fo-Fc level. (e) The expression of E. cusA. coli WT (K12) and ndh deletion strains after Nor treatment (250 ng/ml. 12b). © 2013 Nature America. 2 h). although the aforementioned results indicated that MarR has two copper-oxidizable cysteine residues on each monomer. (a) Copper(II)-oxidized MarR5CS(80C) tetramer with two monomers from each dimer (chain A–chain B and chain A’–chain B’) colored blue and magenta. cueO. Previous work has showed that the ‘ligand-free’ MarR protein is much more challenging to crystallize because of poor crystal packing24. Taken together. a 20% loading of copper(II) was able to convert one equivalent of MarR dimer into the corresponding tetramer. we solved the crystal structure of the copper(II)-oxidized MarR protein. 3a and Supplementary Fig. We thus focused on this MarR mutant and solved the crystal structure of the copper(II)-oxidized MarR5CS(80C) protein at 2. respectively. In this tetrameric complex.

15a. which further unveiled a connection between Nor-triggered envelope stress and copper elevation. 3c. 14). Finally.b) or copper (II) chelator (Supplementary Fig. when Tet or Cm was used to treat the CS-1–bearing E. 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). Moreover.and antibiotic-induced marRAB derepression. (c) Flow cytometric analysis of E. coli cells in the presence of SAL or antibiotics10. Inc. CpxR and CpxP (all of which belong to the Cpx system) as well as DegP. In E. our copper sensor CS-1 detected a marked increase in copper in the tBHP-treated E. ST1710 and SlyA (Supplementary Fig. coli K12 ∆ndh . (d) Flow cytometric analysis of E. the cpxp gene in the Cpx regulon has been previously shown to be upregulated upon copper liberation31. we focused on this protein for further investigation. coli deletion strains we tested.m. the toxic copper ions are highly compartmentalized in the cell envelope.nature.200 Cell count Cell count 300 600 300 0 Control 100 ng/mL Nor 500 ng/mL Nor 1 µg/mL Nor 2 µg/mL Nor d 500 400 300 200 100 0 E. Among the E. Treatment of Nor increased the expression of all of these envelope stress proteins.33. Indeed. In addition. As a cytoplasmic membrane– located cupric reductase in a bacterial respiration system. the strains lacking either ndh or cyoB were found to induce copper elevation to a lesser extent than the WT strain after Nor treatment.36. AU. 5a). coli K12 + tBHP ∆ndh + tBHP E. coli K12 + TETA E.Nature chemical BioloGY Doi: 10. 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. coli K12 ∆ndh E. the CusA regulator CusR) with and without Nor treatment. coli cells bearing the pL(lexO)-GFP reporter28 demonstrated that the addition of copper(II) attenuated Nor-induced DNA damage in the E.s. tBHP is able to oxidize cuprous ions to cupric species via the Fenton-like reaction. Antibiotics trigger intracellular copper signaling we wondered whether these envelope-residing copper proteins are the targets for antibiotic-triggered copper generation. coli K12 ∆ndh E. we observed no detectable fluorescence increase (Supplementary Fig. NDH-2 can be specifically inactivated by tert-butyl hydroperoxide (tBHP). Superimposition of this dimer structure with that of the copper(II)-oxidized MarR5CS(80C) protein showed a highly similar overall topology (Fig. The previously reported SAL-complexed WT MarR structure resembles the ligand-bound form of MarR with impaired DNAbinding capability. 18). ndh caused the most noticeable decrease in copper levels when it was deleted.37).33. We next asked how this copper signal could be generated within E. 4a–c).28.35. and. Furthermore. we employed quantitative real-time RT-PCR (qRT-PCR) to examine the expression of these genes or genes encoding their regulators (for example. coli WT (K12) and ndh deletion strains bearing the pL(copA)-GFP reporter under the treatment of 750 μM tBHP for 2 h. coli WT (K12) strain but not in the marR deletion strain (Supplementary Fig. coli K12 + tBHP + TETA 100 1 102 10 Fluorescence intensity (AU) 103 10 0 1 102 103 104 10 Fluorescence intensity (AU) 10 0 10 10 Fluorescence intensity (AU) 1 2 10 10 10 Fluorescence intensity (AU) 0 1 2 Figure 5 | Antibiotic-stimulated OHP production leads to copper liberation and MarR derepression. Such an oxidative modulation of copper ions has been shown to damage the E. we have shown that copper(II) serves as a natural inducer for MarR and mediates the SAL. all of these molecules stimulated a marked increase of the CS-1 fluorescent signal.b). including NDH-2 (refs. in particular. © 2013 Nature America. coli membrane. 32. coli bacteria. coli cells. 17). both of which showed that copper(II) ions can enhance MarR-mediated bacterial drug resistance. (b) Flow cytometric analysis of E. 4e). 15c. coli WT strain relative to the ndh deletion strain. which contains multiple copper-binding proteins. including periplasmic copper-binding proteins such as CueO (a multi-copper oxidase) and CusF (a copper chaperone). and many copper-containing proteins are located within this space. So far. similar to the conversion of iron(II) to iron(III)34. We then went on to identify the copper source responsible for antibiotic-generated copper stress. indicating the generation of labile copper(I) ions upon attacking E. we showed that 5 NATURE CHEMICAL BIOLOGY | ADVaNCE ONLINE PUBLICaTION | www. Of the genes encoding copper-dependent proteins that we tested. In contrast.1038/NchEmbio. coli + tBHP ∆ndh + tBHP ARTICLE c 500 400 Cell count 300 200 100 0 E. we solved the crystal structure of reduced MarRC80S mutant protein in the absence of DNA (Supplementary Fig.54 Å for 248 Cα). we monitored the susceptibility of E. which verified that the cysteine-toserine mutations at residues 47. Furthermore. 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. 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. confirming that tBHP is able to mediate the redox cycling of copper within E.1380 a 400 300 Cell count 200 100 0 E. OHP production leads to membrane-bound copper liberation To better understand how antibiotics might promote copper release from envelope proteins. 16). 54. coli cells (Fig. (a) tBHP (750 μM)-induced copper generation inside E. a major periplasmic protein quality control factor (Supplementary Fig. All rights reserved. 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. further indicating that each of the dimer units within the copper (II)-oxidized MarR5CS(80C) tetramer adopts a conformation incapable of DNA binding. an organic hydroperoxide (OHP) known to cause the impairment of the respiration chain32. 13a). deviation = 1. arbitrary units. this structure also adopts a mode incapable of DNA binding. by using our previously developed OHP-specific fluorescent sensor-OHSer38 and FACS analysis (Supplementary Fig. coli cells harboring the genetically encoded OHP sensor OHSer upon treatment with the various indicated concentrations of Nor for 30 min. coli K12 + tBHP ∆ndh + tBHP b 1. Notably. As bactericidal antibiotics such as Nor are able to cause bacterial envelope stress30. indicating that these cytoplasmic membrane-bound copper proteins might be responsible for Nor-triggered copper release. indicating an elevated intracellular copper level triggered by antibiotics (Fig. r. 13b)25–27. coli WT (K12) and ndh deletion strains bearing the pL(marO)-GFP reporter treated by 750 μM tBHP in the presence and absence of 1 mM TETA for 1 h. Next. coli WT (K12) and marR deletion strains to Nor in the presence and absence of copper(II) (Supplementary Fig. 108 and 111 in MarR5CS(80C) did not cause major conformational changes. coli inner membrane through NDH-2 (Fig. 4d and Supplementary Fig. FACS analysis on E. We also performed qRT-PCR analysis on genes encoding envelope stress proteins such as CpxA. 51. we next studied the molecular mechanism underlying antibiotic-triggered impairment of copper-dependent envelope proteins. Particularly. 19a.500 1.d). therefore. coli WT (K12) and ndh deletion strains was examined with CS-1 probe using flow cytometry.

seems to indicate that multiple pathways may exist that either a small signaling molecule (e. FACS analysis on the pL(marO)-GFP reporter–bearing 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 cytoplasm. copper(II) may serve as a natural signal for derepression of MarR and thus for the marRAB operon. less GFP generation was observed in the ndh deletion strain treated with Nor or tBHP (Fig. 20). we show here that other types of oxidative species. transketolase A.  22). alkyl hydroperoxide. Our data presented here demonstrate that copper(II) is an environmental cue that MarR senses directly. Taken together. has been 6 Figure 6 | A new linkage of antibiotic-triggered envelope stress and copper signaling with MarR-mediated bacterial antibiotic resistance. an enzyme in central metabolism. indicating an enhanced copper level within the E. In turn. 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. Finally. further confirming that NDH-2 is a major source for Norinduced copper generation. coli WT strain (Fig. outer membrane. 19c. which subsequently produces OHPs that impair cytoplasmic membrane–bound copper proteins such as NDH-2 and CyoB.1038/NchEmbio. coli cytosol. Two clinically important antibiotics. resulting in enhanced bacterial antibiotic resistance. can be generated by fluoroquinolones or β-lactam antibiotics via lipid oxidation that will subsequently trigger the liberation of copper as a signaling molecule in E. particularly for use under stress conditions such as antibiotic treatment. We then employed EMSA to show that OHPs such as tBHP and cumene hydroperoxide could. further shown to directly interact with MarR in vivo. further verifying that copper(II) is the underlying cognitive inducer for the observed MarR derepression.40–43. mistranslation or both within 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 R 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. resulting in the release of free copper(I) ions. 21). which could trigger MarR derepression and turn on MarR-mediated antibiotic resistance. The lack of highly sensitive and selective indicators to probe these reactive and interconvertible oxidative or metal species in living cells persists. Through the use of a genetically encoded OHP fluorescent sensor. copper(II)) or an intracellular protein can modulate MarR-regulated antibiotic resistance. the presence of the copper(II) chelator TETA attenuated tBHP-triggered MarR derepression (Fig. 5d). This diagram depicts a proposed mechanistic model for copper(II)mediated MarR derepression. Together. Treatment with either Nor or tBHP can promote the expression of GFP.ARTICLE Nature chemical BioloGY Doi: 10. coli periplasm under copper stress or oxidative stress conditions44. coli. We engineered a pL(copA)-GFP reporter with GFP expression regulated by the copA promoter. 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. 5b and Supplementary Fig. Furthermore. This. SAL-mediated MarR derepression could also cycle through a similar mechanism. 19d). which subsequently oxidizes the Cys80 residues on MarR to generate two disulfide bonds between two MarR dimers. SAL-mediated MarR derepression might go through a similar mechanism. 5d and Supplementary Fig. S RO ion t duc pro Cy Copper signal Cu1+ ND Antib io resista tic nce MarR Catalytic . such as OHP. in conjunction with OHP or other oxidative species triggered by antibiotics. microorganisms have developed sensing mechanisms to detect these molecules as unified signals and turn on defense strategies against antibiotics or disinfectants16. IM. 2c). we verified that copper ions released from these inner membrane–bound copper proteins were able to promote free copper levels in the MarR-residing bacterial cytosol. coli cells. Nor and Amp. alkoxy radical. causing the derepression of the marRAB operon. OM. Antibiotic tolerance has long been linked to cellular redox and metabolic states as well as intracellular metal ion homeostasis40. Moreover. Inc.nature. Our study reveals that copper(II) is also able to trigger disulfide bond formation between MarR dimers in NATURE chEmicAl biology | ADVaNCE ONLINE PUBLICaTION | www. Therefore. 6): the treatment of Nor or Amp is able to induce ‘envelope stress’ via protein unfolding. All rights reserved. mistranslation or both within E. We found that the previously established SAL-induced MarR derepression actually goes through the copper-based mechanism.g. these OHPs or additional unknown oxidative species may serve as the oxidants to promote this copper(II)-triggered MarR dimer-totetramer conversion. 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. Notably. which can be used to sense cytosolic copper. resulting in enhanced bacterial antibiotic resistance. in conjunction with our observation that the copper(II) chelator TETA cannot completely abolish antibiotic-triggered MarR derepression. in combination with copper(I). Copper(II)-triggered MarR cysteine oxidation derepresses the MarR-regulated antibiotic resistance response in E. We further discovered that Nor and Amp.1380 Environment OM An ti t bio ics © 2013 Nature America. 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. Amp and SAL (Fig. Whether ROS can potentiate the bactericidal effects of antibiotics remains controversial. cupric ions are known to catalyze non-native disulfide bond formation in the E. causing dissociation of MarR from its cognitive DNA. Moreover. also stimulate intracellular copper elevation. 5c and Supplementary Fig. inner membrane. coli. two clinically important bactericidal antibiotics.. •OR. coli. we propose the following mechanistic model for copper(II)-modulated MarR derepression (Fig. coli cells. whereas treatment with a copper chelator eliminates SAL-triggered Mar activation. In particular. the generation of OHPs inside bacteria can be stimulated by Nor.d) but not Tet or Cm (Supplementary Fig. This copper(I) liberation. This process ultimately derepresses MarR and thus the marRAB operon. elevates oxidized intracellular copper(II). Taken together. a recent study using a bacterial two-hybrid system identified 48 potential partner proteins that may bind MarR39. our results indicate that the Nor-triggered OHP production caused oxidative impairment of cytoplasm membrane–bound copper proteins such as NDH-2. in addition to the aforementioned copper(II)-catalyzed disulfide bond formation between MarR dimers in the presence of oxygen in the air (Fig. can stimulate envelope stress via protein unfolding. which led to the generation of oxidized copper(II) species that can be sensed by MarR inside E. impair MarR’s DNA-binding ability (Supplementary Fig. ROOH.

Tanaka. A. Proc. S. 363–367 (2006). Am.R. by multiple chemicals in vitro. G. Perera. Mol. et al. Sci. campestris. C. J. D. K. J.A. T. 109–114 (2001). M. 24. Cell 135. 34. Mistranslation of membrane proteins and two-component system activation trigger antibiotic-mediated cell death. B. Proteome-wide quantification and characterization of oxidation-sensitive cysteines in pathogenic bacteria.T. & Levy. .E. 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.D. Biophys. Kershaw. M. & Kang. Jair. Transketolase A. Dwyer. Killing by bactericidal antibiotics does not depend on reactive oxygen species. Patikarnmonthon.. E. Membrane-associated redox cycling of copper mediates hydroperoxide toxicity in. USA 110. Brown. J. 22. Rodríguez-Montelongo. Enterobacter aerogenes and Klebsiella pneumoniae (Supplementary Fig. 2067–2075 (1997). L.D. The expression profile of Escherichia coli K-12 in response to minimal. Zhao. & Collins. environmentally derived stress or both. Immunol. Redox-operated genetic switches: the SoxR and OxyR transcription factors. M. Acad. Sun. A. The effects of salicylate on bacteria... Accession codes. Sci.R. accepted 13 September 2013. R.nature.. & Rosner.. Curr. M. 23. Biophys.I. Sci. 1029–1043 (2000). The characteristic Cys80 residue in MarR is highly conserved in diverse Enterobacteriaceae species. J. T.R.A..G.J. 49–60 (1983). N. Cell 37. Wu. Trends Biotechnol.L. & Collins. L. Alteration of the repressor activity of MarR.. 133–142 (1998). Levy. 2... K. Natl. Duguid. F. 405. Acta 1144.W. K.P. 383–394 (2007). Mol. & Yokoyama. Proc. 75–80 (2010)..D. 91. Biochem.3 Å resolution. & Head. Mol. 13. 1710–DNA complex crystal structure reveals the DNA binding mechanism of the MarR family of regulators. Dolan.L. Constantinidou.J. Arch. J. B.B. G. Nat. Binding of purified multiple antibiotic-resistance repressor protein (MarR) to mar operator sequences. Antimicrob. J. Science 339.L.J. L.A. Struct. 125–131 (1975). Biophys. I. Microbiology 151. J. X. Acad. Chen. et al. X. Cell death from antibiotics without the involvement of reactive oxygen species. et al. 4. 2. C. 311–320 (2010).L. 37. Received 1 March 2013.. D’Autréaux. & Helmann. 91 (2007). M. Farías. Blakeley. P. 7. 19. Frawley. 3081–3090 (2004). respectively. Chem. 27.J. J. B. Agents Chemother. E. & Collins. M.. ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis.. C.J. optimal and excess copper concentrations.B. Farías. C. & Collins. 128. Pomposiello. Sublethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis.R.A. Inc.J. Biol. Riddles. C-terminal domain of the membrane copper transporter Ctr1 from Saccharomyces cerevisiae binds four Cu(I) ions as a cuprous-thiolate polynuclear cluster: sub-femtomolar Cu(I) affinity of three proteins involved in copper trafficking. Isacoff.. Cohen. Bina. The Cu(II)-reductase NADH dehydrogenase-2 of Escherichia coli improves the bacterial growth in extreme copper concentrations and increases the resistance to the damage caused by copper and hydroperoxide. 25. 591–595 (2006).W.B. Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon. S. 28. Soc. Cell Biol.C.J. & Levy. Microbiol.. Liu. & Rosner. Chem. 178. The crystal structure of MarR. 20. P.N. Sites of electron transfer to membrane-bound copper and hydroperoxide-induced damage in the respiratory chain of Escherichia coli. FEMS Microbiol. et al. Lawrence. Uversky. J.L. Dwyer. & Brennan. A selective turn-on fluorescent sensor for imaging copper in living cells. R. 77–84 (1993). 4176–4178 (1995). M.N.A. 8. Y. C. Evidence for Cu(I)-thiolate ligation and prediction of a putative copper-binding site in the Escherichia coli NADH dehydrogenase-2.M. C. J. Alekshun.C.N. 132. R. Biochem. The significance of copper chelators in clinical and experimental application. Z. & Zerner. 451. & Massa. & Demple. Science 339..B. et al. 131–141 (2005). Alekshun.. 14. V. Shigella flexneri. Lee. Biochem. M. J. Am. 1. Nat. The mar regulon: multiple resistance to antibiotics and other toxic chemicals. Kohanski. published online 3 November 2013 ARTICLE Methods © 2013 Nature America. & Rosner. Kohanski. 66.T... V. 313. S.. et al. Miller. B. Kohanski.. T. J. 679–690 (2008). 37. Biochem. 35. I. & He. Am. Hong. J. Structure of an OhrR–ohrA operator complex reveals the DNA binding mechanism of the MarR family.J.. Martin. Biochem. J.Nature chemical BioloGY Doi: 10. Proc. Arch. Wolf. Nat. 17. R. 15. Acc. S. 1213–1216 (2013). M. & Levy.Y.. 177. L. in free and DNA-bound states. & Cunningham-Bussel. Mealy. A. & Imlay. J. 181. Mol. 3. Chem. R. 7 NATURE CHEMICAL BIOLOGY | ADVaNCE ONLINE PUBLICaTION | www. 813–824 (2007). 23). J. Xiao. Loughlin. Biol. B. at 2. 286. Bacteriol. J. 482–489 (2009)..J. Martin. Ding. 17065–17067 (2010). the negative regulator of the Escherichia coli marRAB locus. Natl. Rev. R.N. Fuangthong. G. 1210–1213 (2013). 22178–22185 (2011). Price. References 18. N. Dwyer. The PerR transcription factor senses H2O2 by metal-catalysed histidine oxidation. Zeng. Bacteriol. Molecular mechanisms of ligand-mediated attenuation of DNA binding by MarR family transcriptional regulators. 87–94 (2002). Chem.R.N. J. 32. 126.G. 243–254 (2010). & Collins. Wierzbowski. Natl.B. Nathan. A common mechanism of cellular death induced by bactericidal antibiotics.G. Cell Biol. Patel. Acta 1388. Salmonella enterica.S. 41. Alekshun. C. including various virulent E. E. E. Biol. Nature 440. Res. USA 109. 32.J. Helmann. 6. Martin. & Chang. 12. an enzyme in central metabolism. 5456–5460 (1995). derepresses the marRAB multiple antibiotic resistance operon of Escherichia coli by interaction with MarR. M. a master virulence regulator of Salmonella.. Volentini. Hayete.G. P. 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. 41. DePristo. & Wedd. R..J. Role of reactive oxygen species in antibiotic action and resistance. Cell 20. Reassessment of Ellman’s reagent.N. 358–370 (2013). Beyond oxidative stress: an immunologist’s guide to reactive oxygen species. Inocencio.. 10–11 (2006). F. & Lewis. Chem. Kumarevel. R.1038/NchEmbio.. 710–714 (2001).. S. C.D. E. Y.. Regulation of the multiple antibiotic resistance (mar) regulon by marORA sequences in Escherichia coli. 8.. L. Fenton’s reagent revisited. M. Domain. 9095–9100 (2012).R.A. J. S. T. Rodríguez-Montelongo.. All rights reserved. X.M. Self-association of 8-anilino-1naphthalene-sulfonate molecules: spectroscopic characterization and application to the investigation of protein folding. Bacteriol. Mol. 21. et al. Massa. S.A.A. 26. 2. G. Nucleic Acids Res. 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. J. Lett.L. Opin.J. V. & Hobman. Farías.J. 36. USA 92.N.W. 39. Nyantakyi. J. & Grove. Xie. Rev. D. A highly selective fluorescent probe for visualization of organic hydroperoxides in living cells. 42. A. Dwyer. Quorum-sensing agr mediates bacterial oxidation response via an intramolecular disulfide redox switch in the response regulator AgrA. Cell Biol. 11. J.. Howlett. 12054–12059 (2013). Methods and any associated references are available in the online version of the paper. J. Seaton. J. Rapisarda. Soc. 8. Autoactivation of the marRAB multiple antibiotic resistance operon by the MarA transcriptional activator in Escherichia coli. J. S. M. Salicylate induction of antibiotic-resistance in Escherichia coli: activation of the mar operon and a mar-independent pathway. J. Rodriguez-Montelongo. J. Kohanski. 19–26 (1995).M. Gyrase inhibitors induce an oxidative damage cellular death pathway in Escherichia coli. 797–810 (2007)..N.E. Arch. E. 10.J. Iron and citrate export by a major facilitator superfamily pump regulates metabolism and stress resistance in Salmonella Typhimurium. 301–310 (2011). Levy. I.. Biophys. 30.. 410–413 (1999). 40. 9. Crystal structures of SlyA protein. Nat. Nutr. 7. Hayete. R. Cell 130. F. 4669–4672 (1999).. Escherichia coli. Microbiol. E. 1–7 (2006).. M. 4723–4735 (2009). B. Keren.T. 1187–1198 (2005). Int. H. 175. 349–361 (2013). M. Mulcahy. Y. Winter. Walling. S. Mol. Bacteriol.M. 29.G. Copper ions potentiate organic hydroperoxide and hydrogen peroxide toxicity through different mechanisms in Xanthomonas campestris pv. 19. Biochim. Alekshun. coli strains. Pralle. Soc. Acad. & Rosner. Biochim...F. 7856–7862 (1993).. J. 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. Foulds.A. Lee. S..S. 3. An oxidation-sensing mechanism is used by the global regulator MgrA in Staphylococcus aureus.A. 5.. B. Chem.. J. & Massa. Kohanski. Trends Microbiol. Biophys. 323. 31. P.. de la Cruz-Rodriguez.. Cottarel.N.S. 13. a regulator of multiple antibiotic resistance.1380 a catalytic fashion. E. 22. Cell Host Microbe 13. & Levy. 2216–2223 (1996). J. Methods Enzymol. L. D. & Toledano.L. Umehara. & Gustafson.B. 8. & Rapisarda. Deng. D. George. & Löber. 16. Syst.A.B. Biol. 12. 33.

Correspondence and requests for materials should be addressed to P. Boston University) for providing the pL(lexO)GFP reporter. and X.R. All rights reserved. Chem. J.Z. We also thank the staff members of the Shanghai Synchrotron Radiation Facility and the Beijing Synchrotron Radiation Facility. Reprints and permissions information is available online at http://www.H. M. J.Z. Cell 135. Copper stress causes an in vivo requirement for the Escherichia coli disulfide isomerase DsbC. html. . Biol. determined the crystal structures of copper(II)-oxidized MarR5CS(80C) and reduced MarRC80S..R. coli) and M2076 (marRAB mutant strains) bearing the marR::lacZ reporter.).H.1038/NchEmbio. Competing financial interests Additional information The authors declare no competing financial interests. & Bardwell.. © 2013 Nature America. Winter.). University of California–Berkeley) for providing the Cu(I)-specific fluorescent probe CS-1 and S. and S. P. conceived the study. J. the National Natural Science Foundation of China Z. This work was supported by research grants from the National Basic Research Foundation of China (2010CB912302 and 2012CB917301 to P.C. U. Collet. Özcelik.. 33785–33791 (2005). D. Ilbert. J.C.nature. A.C.C.F.F. 280. The E.Z.Z.J.A.L.nature.Z. Collins (Department of Biomedical Engineering. Author contributions Acknowledgments We thank J. performed the biochemical studies and participated in the structure studies. 8 NATURE chEmicAl biology | ADVaNCE ONLINE PUBLICaTION | www.H. Rosner (National Institute of Diabetes and Digestive and Kidney Diseases.) and the E-Institutes of Shanghai Municipal Education Commission (project number E09013 to C. Supplementary information is available in the online version of the paper. interpreted data and wrote the manuscript with input from all of the authors. Japan). C.C. or C. performed the experiments for OHP detection using OHSer.C. designed experiments. H.).S.R. Reichard for editing. Bleach activates a redox-regulated chaperone by oxidative protein unfolding. P.H. Chang (College of Chemistry... Hiniker.C. 691–701 (2008). US National Institutes of Health) for providing the strains of M2073 (WT E. and P. 43. & Jakob. and C. the US National Science Foundation (CHE-1213598 to C.H. R..1380 (21225206.ARTICLE Nature chemical BioloGY Doi: 10. Graf. J.-F. 2011CB809103 to C..J. coli WT (K12) strain (BW25113) and all of the single-gene deletion mutants were obtained from the National BioResource Project (National Institute of Genetics. helped with the biochemical and/or structure experiments.C. B. 91013005 and 21001010 to P. Inc.C. synthesized the Cu(I)-specific fluorescent probe CS-1.R.R.H. J. D.

were taken on ChemDoc XRS+ (Bio-Rad). 0. We used the N-terminal His-tag cleaved protein sample for crystallization. The best MarR5CS(80C) crystal grew in 2. Nor was purchased from Fluca. SaltRx 2. All rights reserved. Crystallization. PEG/Ion 545-nm emission filter (FL1) at low flow rate. The covalently linked dimer band was separately excised from the SDS PAGE gel.000 followed by three MS/MS scans in the quadruple collision cell using the collision-induced dissociation (CID). BCS. Agilent USA).5 mM borate. which bears a pET15b vector containing the marR gene. The hydroxyl radical production upon the reaction of CuCl2 (5 μM) with sodium ascorbate (500 μM) and 3-CCA (300 μM) was also measured as a positive control. 10% glycerol and 10 mM Tris-HCl. Japan).000 r. All of the metals used in this study were purchased from J&K Scientific. Flow data were processed and analyzed with FCS express Version 2. Cells were grown in LB medium containing 50 μg/ml Amp at 37 °C to an OD600nm of 0.5′-dithiobis-2-nitrobenzoic acid (DTNB) assay.3 M NaCl and 10 mM DTT. 80%) and IAM were obtained from Alfa Aesar. The crystal was quickly dipped into a series of solutions with increased glycerol concentration up to 25%.5× TB buffer (tenfold dilution from 5× TB buffer: 50 mM Tris-HCl.m. pH 7. we used the commercially available Hampton Research screens: Index. The fluorescence probe ANS (8-anilino1-naphthalenesulfonate) was employed to indicate the conformational change of protein upon treatment with potential metal ions. 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).8). The crystal was finally flash frozen in liquid nitrogen prior to data collection. Any remaining free thiols were blocked by treatment with excess IAM for 45 min. Aliquots of CuCl2 (from 7.1 M CBTP buffer (pH 7. 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). the cell lysate was spun down at 15. Japan). The concentrations for CuCl2 and other metals were kept at 2. The best MarRC80S crystal grew in 24% PEG 3350. All pictures of protein gels.GCAGGGGATAATATTGCCC AGGCAAGTATAAGTCAAATGAAT-3′) was used for this assay. 25 μM WT MarR was treated with 500 μM CuCl2 in the presence and absence of EDTA (10 mM) for 10 min at room temperature in DTNB assay buffer (100 mM potassium phosphate. Inc. 450 nm) was measured upon the addition of WT MarR (5 μM) to the samples containing 5 μM CuCl2 and 300 μM 3-CCA in the absence and presence of sodium ascorbate (500 μM) for 25 min. Purified MarR5CS(80C) and WT MarR (all at 40 μM) were incubated with 20 μM (0. Cell pellets were collected and resuspended in NTA buffer (20 mM Tris-HCl.5 mM CaCl2. pH 7.5 μM) were titrated into the protein-BCS solution containing 5 μM MarR monomer protein and excess BCS (200 μM) in a final volume of 100 μl. 0. HITACHI. Ltd.03 M citric acid and 0.5 μM. All of the other chemicals were analytical grade or better.1 M sodium acetate trihydrate. We used step-wise soaking to avoid crystal cracking. pH 7. The analytical column was pursed from Agilent (75 μm ID. WT MarR and its mutants with a thrombin-cleavable N-terminal His tag were prepared from E.5×) CuCl2 at room temperature for 5 min.000 cells were collected for each sample. 1 μM MarR protein monomer (0.1% formic acid. Cryoprotectant was made fresh by mimicking growth conditions. Denmark). Protein expression was induced at 30 °C for 5 h before harvest. FeCl3 or NiCl2 were added to the solution containing the MarR–ANS complex (0. X-ray data collection and structure determination. Different concentrations doi:10. and mobile phase B consisted of 100% acetonitrile and 0. of CuCl2. UV-visible spectroscopy was performed using a UV-visible spectrometer (U-3010. Cleared lysate was filtrated and loaded onto a 5-ml Histrap column (GE Healthcare). Samples were loaded onto the 8% nondenaturing polyacrylamide gel before incubation at room temperature for 20 min and were run in 0. 3-coumarin carboxylic acid (3-CCA). For the crystallization conditions.5 μM to 30 μM) were titrated into the protein-BCS solution containing 15 μM MarR monomer protein and excess BCS (200 μM). Reagents and equipment. 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. pL(marO)-GFP and pL(copA)-GFP reporter genes were constructed on the basis of the design of the PLlacO-1 promoter28. 41. respectively. TETA (60%). The free thiol contents of native and copper(II)-oxidized WT MarR were determined by using the 5.5. including those of Coomassie Brilliant Blue (CBB)-stained SDS-PAGE gels and western blotting membranes. The DNA bands were visualized with UV light at 254 nm by ChemiDoc XRS+ (Bio-Rad). aliquots of CuCl2 (from 5 μM to 22. China) and the 3W1A beamline at Beijing Synchrotron Radiation Facility. The wavelength used for data NATURE CHEMICAL BIOLOGY . ANS (8-anilino-1-naphthalenesulfonate) was bought from TCI. 150-mm length.. Sitting-drop vapor diffusion at room temperature (10–20 °C) was applied by mixing 2 μl protein sample (at 7. Protein was collected and concentrated for further experimental use. The single full-scan mass spectrum was acquired in the Orbitrap from m/z 350–20. 2. The stoichiometry of copper(II)/MarR5CS(80C) was determined with the same method. and SAL was bought from Sinopharm Chemical Reagent Co. destained and digested with trypsin at 37 °C for 12 h. Electrophoretic mobility shift assays (EMSA). which was pre-equilibrated with 25 ml NTA buffer. 24). PEG/Ion 2 Screen. Addition of DTT (1 mM) or EDTA (3. 300 mM NaCl and 10% glycerol) containing 1 mM PMSF (phenylmethanesulfonyl fluoride) and 5 mM β-mercaptoethanol. pH 7.2 mg/ml or 11. HITACHI. Data sets of MarR5CS(80C) and MarRC80S were collected on the BL17U1 beamline at the Shanghai Synchrotron Radiation Facility (Shanghai. λem.5 mM ANS and 5 μM MarR in 20 mM HEPES buffer. As an indication of the hydroxyl radical production. 0.5 μM for mutant MarR5CS(80C)) in binding buffer (50 mM KCl.5. for 30 min. All of the other antibiotics were obtained from INALCO.1% formic acid. Crystal Screen.6. Mobile phase A consisted of 0. Japan). SaltRx 1.ONLINE METHODS Strains and plasmids. pH 7. For marR expression regulation or cytosolic copper stress detection.5 mM) prevented the fluorescence change of the MarR–ANS complex solution upon treatment with copper(II). λexc. and free thiol concentrations were calculated on the basis of previously published methods22. After addition of 1 mM DTNB. 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.p. coli BL21(DE3).4. 5 mM MgCl2. denaturing SDS PAGE gel. At least 70.8 mg/ml) with 2 μl well solution. 395 nm. The strains and plasmids used in this study were listed in Supplementary Tables 2 and 3. Protein expression and purification.6 before IPTG was added to a final concentration of 1 mM. 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. After sonicating on ice for 20 min. Protein samples were run on a nonreducing. DTNB assay.000-fold diluted SYBR Gold nucleic acid staining solution for 7 min. pH 4. The N-terminal His tag of MarR proteins can be removed by thrombin digestion. A 42-mer duplex DNA containing marRAB promoter sequence (5′-ATTCATTTGACTTATACTTG CCTGGGCAATATTATCCCCTGC-3′ and 5′.0). 300 mM NaCl). All of the measurements were carried out in 20 mM PBS buffer (pH 7.07 M Bis-Tris propane mixed).8) at 110 V for 1 h at 4 °C. Excess copper(II) was removed by buffer exchange. tBHP (70%). cumene hydroperoxide (CHP. the formation of the product 7-hydroxy-coumarin-3-carboxylic acid (7-OH-CCA. which was performed by incubating tagged protein with thrombin at 16 °C overnight in digestion buffer (20 mM Tris-HCl. in which the precursor mass selection window was set at 2 Da. Primers used for plasmid construction and qRT-PCR were listed in Supplementary Table 4. The sequences of these two promoters are listed in Supplementary Table 5.2. HITACHI. pH 8. Spectroscopic study. 150 mM NaCl) (Supplementary Fig.4) using a fluorescence spectro­ meter (F-4600. All of the experiments were performed in 20 mM HEPES buffer (pH 7. 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.2 M sodium chloride.0) on an UV-visible spectrometer (U-3010. All of the EMSA reaction mixtures in this study were set up in a final volume of 20 μl containing 50 nM annealed double-stranded DNA. respectively. Gels were stained in a 10. 100 mM NaCl and 1 mM EDTA). pH 7. 0. Disulfide bond detection by MS.1380 © 2013 Nature America. The absorption at 412 nm was recorded.1038/nchembio. The hydroxyl radical scavenging compound 3-CCA (3-coumarin carboxylic acid) was used to determine the copper-catalyzed hydroxyl radical production45. To determine the stoichiometry of WT MarR to copper(II).6.

Ltd. pH 8. NCS restraints52 and TLS refinement53 were applied. T. 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. Overnight cultures of E.1380 © 2013 Nature America.05 was considered to be significant. Crystallogr.. The stereochemical quality was analyzed using MolProbity54. The reactions were incubated at room temperature for 1 h before 20 μl of the samples were added to 180 μl of ABT buffer in a black 96-well plate. and the remaining aliquots (1 ml) were harvested by centrifugation. The total RNA (1 μg) was reverse transcribed to first strand cDNA by RevertAid First Strands cDNA Synthesis kit (Fermentas.­ ollection for MarR5CS(80C) and MarRC80S was 0. Osaka. Biol. The E. Amp or oxidants (tBHP and H2O2) for 30 min. Cells were then incubated with 1 μg/ml Nor in the presence and absence of CuCl2 (500 μM) or 300 μM copper(II) chelator TETA for 30 min at 37 °C. 420 (SSC) and 450 (FL1).6–0. O. Japan). Only dilutions that yielded 10–100 colonies were counted. H.6. After these treatments. λem. 413 (SSC) and 436 (FL1). USA) following the manufacturer’s guidelines. A. ZnCl2 or NiCl2 for 30 min. OHP detection.6 before being treated by NATURE chEmicAl biology SAL (5 mM). Acta Crystallogr. bacterial samples were incubated with 2 μM CS-1 for 15 min at 37 °C followed by washing with PBS buffer twice. followed by flow cytometric analysis with the following PMT voltage settings: E01 (FSC). After washing with 10 mM PBS buffer twice. Samples were analyzed by flow cytometry. The following PMT voltage settings were used: E01 (FSC). Tenfold serial dilutions of bacteria were plated for colony counts. Inc. and bacterial cells in LB medium were challenged by 2. 2. Nat. coli WT strain (M2073) or marRAB mutant strain (M2076) containing the marR::lacZ reporter fusion was used for this assay. overnight cultures of E.6 before being challenged by various stimulants. 67.G. The following PMT voltage settings were used: E01 (FSC). 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.TOYOBO Co.5 mM SAL in the presence and absence of 1 mM TETA for 30 min.5 μg/ml Amp or 750  μM tBHP in the presence and absence of 1 mM TETA for 1 h. Cells harboring pL(copA)-GFP reporter genes in exponential phase (OD600nm of 0.87% were in the allowed region. 427 (SSC) and 725(FL1).d. FeCl3 and 1 mM H2O2 for 1 h before being analyzed by flow cytometry.. bacterial cells were challenged by 2. All of the experiments were conducted in at least three independent replicates. 99. Cell viability assay. The PMT voltage settings used are: E01 (FSC). Residues 92–95 in chain A and residues 92–94 of MarR5CS(80C) were disordered.5 μg/ml Amp. DNA damage analysis. followed by incubation with 500 μM CuCl2 for an additional 1 h before flow cytometric analysis. Madison. GE Healthcare). SAL-induced marR derepression in the presence of TETA was also tested.1038/nchembio. When more than two groups were compared. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM.000) or arabinose (1:200) for 2 h before being challenged by 100 μM CuCl2 for 30 min. and relative expression levels were measured using the 2−ΔΔCt method55. 50 μl of the samples from each well were transferred to a new 96-well plate followed by addition of 10 μl MUG (0. Gel filtration was then performed with a Superdex 75 10/300 GL column (GE Healthcare). The rrsA (16S rRNA) was used as an internal control. 2 mM EDTA) containing 0. Johnson. followed by centrifugation. The fluorescence was collected every 0.7 and then incubated with 250 ng/ml Nor at 37 °C for 2 h before being harvested. 8. and 0. 4. 413 (SSC) and 500 (FL1). Meloni. BioTek). P < 0. coli cells harboring OHSer were grown overnight and then diluted 1:100 in LB medium (50 μg/ml kanamycin).5 mM SAL. we used our previously developed OHPspecific fluorescent sensor OHSer.6.8 μl primer (10 μM) were added. 250 ng/ml Nor. For each 20-μl reaction. cells in M9 medium were treated with 100 μM CuCl2. 413 (SSC) and 710 (FL1). respectively. 98. In MarRC80S. The statistical analysis was performed using an unpaired Student’s t-test when the two groups were compared.. 366–372 (2008). 10 μl 2× SYBR master mix (QPK-201. and bacterial survival rates were determined by the percentage survival of the copper. b-Gal assay. L. The following PMT voltage settings were used: E01 (FSC).76% were in the allowed region. Statistical analysis. To survey MarR derepression by SAL. Relative quantification analysis was performed as following conditions: one cycle at 94 °C for 4 min. A summary of the data statistics is shown in Supplementary Table 6.13% residues were in the most favored region. washed with 1× PBS buffer and stored at −80 °C. The upper layers were delivered to a 96-well plate and two tenfold dilutions (90 μl ABT buffer (AB buffer plus 0. Maryland. 20 μg/ml Cm and 20 μg/ml Tet) or 750 μM tBHP at 37 °C for 2 h. FeCl3. they were not modeled. Quantitative real-time RT-PCR. The molecular weight of the eluting species was calculated by standard protein marker (Gel Filtration LMW Calibration Kit. OHSer expression was induced by the addition of 1 mM IPTG (final concentration) at an OD600nm of approximately 0. To examine the MarR derepression in the presence of metal ions.R. 46. Overnight bacterial cultures were inoculated (1:100) in 5 ml fresh LB and were grown to an OD600nm of 0.1% Triton X-100) plus 10-μl sample) were set up in the plate.6 at 37 °C. cells were resuspended in 100 μl of lysis buffer (50 mM Tris-HCl. 49) and COOT50. grown at 37 °C to an OD600nm of 0. cells were pretreated with Nor (250 ng/ml) for 1 h. β-Gal activity was measured using MUG as a substrate. Powell. A specific copper(I) fluorescence sensor CS-1 was used to detect the generation of copper(I) ions inside E. Freshly purified protein of MarR5CS(80C). To assess the roles of copper signaling in MarR-mediated antibiotic resistance. therefore. 366 nm.or TETA-treated group compared to the untreated group. Total RNAs were extracted using SV Total RNA Isolation System (Promega. Kontogiannis.0. 1-ml aliquots were obtained for measurement of OD600nm. All rights reserved.9792 Å and 1 Å. At this point. Neither of the two structures have residues in the disallowed region. Refinement was carried out on the programs REFMAC5 (ref. 250 ng/ml Nor. coli cells upon exposure to various stimulants. Protein production was allowed to proceed for 2 h at 30 °C before treatment with Nor. 45. Quantitative real-time RT-PCR was performed on CFX96 Real-Time PCR (Bio-Rad). At the time of the assay. and 1. 1 unit = 1 pmol of MUG cleaved per min per OD600nm) was determined by fluorescence (λexc.5 mg/ml) into each well to start the reaction. E. Metal swap between Zn7-metallothionein-3 and amyloid-βCu protects against amyloid-β toxicity. To analyze cytosolic copper generation. To examine the MarR derepression in the presence of metal ions. antibiotics and OHPs with or without the copper(II) chelator TETA. one-way analysis of variance (ANOVA) followed by Bonferroni means comparison was used. CuCl2 was slowly added into the protein solution to avoid local precipitation. doi:10. tBHP or various antibiotics. The bacterial samples were analyzed using flow cytometry. The Ramachandran plot given by PROCHECK51 shows that in MarR5CS(80C). then one cycle at 72 °C for 20 sec.8. followed by 40 cycles at 94 °C for 20 s and 58 °C for 20 s. DmarRAB mutant strain (M2076) harboring plasmids expressing WT MarR or MarRC80S with or without the His tag were induced by IPTG (1:1. Size-exclusion chromatography. Chem. & Leslie.4 μl cDNA (100-fold dilution) and 0.6) were challenged with 250 ng/ml Nor or 750 μM tBHP for 2 h. antibiotics (2.W.2 °C. To detect OHP generation inside bacteria under treatment with SAL. coli WT (K12) and marR deletion strains were inoculated (1:100) in LB medium and grown at 37 °C to an OD600nm of 0. USA) according to the manufacturer’s protocol.G.. pL(marO)-GFP reporter assay. Overnight cell cultures of E. 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. G. 271–281 (2011). PyMOL was used to generate all of the figures. Battye. β-Gal activity (expressed in MUG units. 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. Overnight cultures were inoculated (1:100) in M9 minimal medium or LB medium and grown to an OD600nm of 0. Copper signal detection.24% residues were in the most favored region. D Biol. coli WT (K12) and marR deletion strains were inoculated (1:100) in LB medium. 445 nm) using a microplate reader (synergy4. et al. we constructed the pL(copA)-GFP reporter. For complementary experiments.G. . Changes of fluorescence were monitored by flow cytometry with the following PMT voltage settings: E01 (FSC).5 mg/ml lysozyme and were incubated at 37 °C for 15 min. M9 medium was used. All of the data are presented as mean ± s. and bacterial cells were treated with 100 μM CuCl2. Melting curve analysis started at 65 °C and went up to 94 °C. 25 mM NaCl. 420 (SSC) and 450 (FL1).

60. M.J. 240–255 (1997).N. 12–21 (2010). 283–291 (1993). V. Winn. Appl. Crystallogr. A. J.1380 NATURE CHEMICAL BIOLOGY . Methods 25. Coot: model-building tools for molecular graphics. Crystallogr. Vagin. MacArthur. M.J. Crystallogr. & Thornton. et al.D. & Schmittgen. K. K. Refinement of macromolecular structures by the maximum-likelihood method. & Merritt. Murshudov. 52.W. 52. D Biol.1038/nchembio. 51. Livak. et al. All rights reserved. doi:10. D Biol. Crystallogr. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. J. et al. 53. 66. © 2013 Nature America. Acta Crystallogr. Overview of the CCP4 suite and current developments. E. D Biol. Chen. 402–408 (2001). Crystallogr. PROCHECK: a program to check the stereochemical quality of protein structures. Phaser crystallographic software. 54. D.D. Crystallogr..B. P. 49. 55. 53. McCoy.M. A.A. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Crystallogr.A. 439–450 (2006). Acta Crystallogr. D Biol.. Crystallogr. & Cowtan. 62.S. Painter. Acta Crystallogr. T. & Dodson. 50. Kleywegt. D Biol. G. 26. R. Appl. Laskowski. Emsley. G.J. Acta Crystallogr.J. J. D Biol.A. Moss. Acta Crystallogr. 235–242 (2011).. 48. Inc. MolProbity: all-atom structure validation for macromolecular crystallography. 67. 40. 2126–2132 (2004). 658–674 (2007). Acta Crystallogr. Use of non-crystallographic symmetry in protein structure refinement. E.47. 842–857 (1996). J.