Molecular Microbiology (2015) 97(5), 808–821 ■

doi:10.1111/mmi.13068 First published online 12 June 2015

Factors affecting redox potential and differential sensitivity of SoxR to redox-active compounds Kang-Lok Lee,1 Atul K. Singh,1 Lim Heo,2 Chaok Seok2 and Jung-Hye Roe1* 1 Laboratory of Molecular Microbiology, School of Biological Sciences, and Institute of Microbiology, Seoul National University, Seoul 151-742, Korea. 2 Department of Chemistry, Seoul National University, Seoul 151-747, Korea.

Summary SoxR is a [2Fe-2S]-containing sensor-regulator, which is activated through oxidation by redox-active compounds (RACs). SoxRs show differential sensitivity to RACs, partly due to different redox potentials, such that Escherichia coli (Ec) SoxR with lower potential respond to broader range of RACs than Streptomyces coelicolor (Sc) SoxR. In S. coelicolor, the RACs that do not activate ScSoxR did not inhibit growth, suggesting that ScSoxR is tuned to respond to growth-inhibitory RACs. Based on sequence comparison and mutation studies, two critical amino acids around the [2Fe-2S] binding site were proposed as key determinants of sensitivity. ScSoxR-like mutation (R127L/P131V) in EcSoxR changed its sensitivity profile as ScSoxR, whereas EcSoxR-like mutation (L126R/V130P) in ScSoxR caused relaxed response. In accordance, the redox potentials of EcSoxRR127L/P131V and ScSoxRL126R/ V130P were estimated to be −192 ± 8 mV and −273 ± 10 mV, respectively, approaching that of ScSoxR (−185 mV) and EcSoxR (−290 mV). Molecular dynamics simulations revealed that the R127L and P131V substitutions in EcSoxR caused more electropositive environment around [2Fe-2S], making it harder to get oxidized. This reveals a mechanism to modulate redox-potential in [Fe-S]-containing sensors by point mutations and to evolve a sensor with differential sensitivity to achieve optimal cellular physiology.

Introduction Bacteria are exposed to a variety of redox-active compounds (RACs) in the environment that can harm cellular Accepted 20 May, 2015. *For correspondence. E-mail jhroe@ snu.ac.kr; Tel. (+82) 2 880 6706; Fax (+82) 2 882 6706.

© 2015 John Wiley & Sons Ltd

constituents when present at high concentrations. They include various phenazines and quinones produced from plants, fungi and bacteria in the natural habitat, or synthetic derivatives applied as environmentally contaminating xenobiotics. In natural ecosystem, RACs could serve as signaling molecules, physiological modulators or toxic chemical weapons. Phenazines could act as cellular redox modifier and regulate gene expression to change physiology of the producing bacteria and neighboring organisms (Pierson and Pierson, 2010). To human microbiota and pathogens in animal hosts, RACs are administered as pharmaceutical drugs. They can redox-cycle by abstracting electrons from flavins or metal centers of various enzymes in the cell, thus continuously producing superoxides and reactive oxygen species (ROS) (Imlay, 2013). In bacteria, RACs are sensed by redox-sensitive sensor/ regulator proteins directly, or indirectly through ROS they produce via redox-cycling or through perturbed redox poise in the cell (Okegbe et al., 2012). SoxR is a transcription factor conserved across proteobacteria and actinobacteria. It functions as a dimer containing a redox-sensitive [2Fe-2S] cluster per monomer. Its direct target gene is not many, but its physiological function spans a broader range. For example, in Escherichia coli, where it was first found and named as a superoxide-sensor SoxR (Greenberg et al., 1990; Tsaneva and Weiss, 1990), its sole known direct target is the adjacent gene soxS whose product induces transcription of about 100 genes that function in anti-oxidative and anti-superoxide response, as well as in diminishing the concentration of RACs by pumping them out or altering envelope permeability (Wu and Weiss, 1991; 1992; Nunoshiba et al., 1992; Ma et al., 1995; Pomposiello et al., 2001; Lee et al., 2009). In comparison, SoxRs from non-enteric bacteria such as pseudomonads and streptomycetes activate several target genes whose primary function appears defense against the toxicity of inducing compounds, without involving anti-superoxide activity such as superoxide dismutase (Palma et al., 2005; Dietrich et al., 2008; Shin et al., 2011; Naseer et al., 2014). The crystal structure of SoxR from E. coli (EcSoxR) revealed that each monomer consists of an N-terminal DNA-binding domain, a dimerization helix domain and a C-terminal domain with a [2Fe-2S] cluster (Watanabe et al., 2008). The [2Fe-2S] cluster is located at the surface

SoxR activation by redox-active compounds 809

of the protein, and its two Fe atoms are fully exposed to the solvent, enabling rapid electron transfer with various RACs. EcSoxR responds to a variety of RACs including O2− (Tsaneva and Weiss, 1990; Ding and Demple, 2000; Gu and Imlay, 2011; Fujikawa et al., 2012). Even though the structures of SoxRs from other bacteria are predicted to be quite similar to EcSoxR due to sequence conservation, the range of chemicals that can activate each SoxR differs, as revealed from studies of SoxRs from Pseudomonas aeruginosa (PaSoxR) and Streptomyces coelicolor (ScSoxR) (Dietrich et al., 2008; Sheplock et al., 2013; Singh et al., 2013). Compared with EcSoxR that responds to nearly all RACs examined so far, PaSoxR is slightly more selective (Sheplock et al., 2013; Singh et al., 2013), responding with slower kinetics to certain RACs such as paraquat (PQ; methyl viologen) and menadione bisulfite (Singh et al., 2013). ScSoxR is most selective among the three, being nearly insensitive to PQ and menadione bisulfite (Singh et al., 2013). Differential sensitivity could arise from several factors, such as redox potential, accessibility and reactivity of the [2Fe-2S] cluster. For each bacterial SoxR, combined contribution from each determining factors will fine-tune the range of chemicals it responds to effectively. The finding that PaSoxR, with the same redox potential as EcSoxR (Eh, −290 mV; Ding and Demple, 1996; Gaudu and Weiss, 1996; Kobayashi and Tagawa, 2004), responds much slowly to PQ (Eh, −440 mM; Steckhan and Kuwana, 1974) than EcSoxR (Singh et al., 2013) suggests the importance of kinetic factors in determining differential sensitivity. For ScSoxR, insensitivity toward PQ is determined primarily by its high redox potential (−187 mV), which demands more energy to get oxidized by PQ, in comparison with EcSoxR (Singh et al., 2013). How specific features in SoxR sequence determine selectivity is an interesting question to understand the function and evolution of SoxR system in bacteria. In this study, we examined the effect of different RACs on the growth of S. coelicolor, correlating their physiological effect with the sensitivity of ScSoxR toward them. We also investigated the contribution of specific amino acid residues around the [2Fe-2S] site to determining differential sensitivity and redox potential.

Results RACs that do not activate ScSoxR are not toxic to S. coelicolor Previous study demonstrated that SoxR plays a role to protect S. coelicolor against SoxR-activating RACs such as γ-actinorhodin, a secreted antibiotic produced by S. coelicolor, and plumbagin (PL) (Singh et al., 2013). We further examined growth inhibitory effect of various © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 97, 808–821

RACs: phenazine methosulfate (5-methylphenazinium methyl sulfate; PMS), paraquat (methyl viologen, 1,1′dimethyl-4,4′-bipyridinium; PQ), menadione (2-methyl1,4-naphthoquinone; MD) and menadione sodium bisulfite (MDs), in addition to actinorhodin (ACT) and PL (5-hydroxy-2-methyl-1,4-naphthoquinone). Among these chemicals, PQ and menadione bisulfite are ineffective in activating ScSoxR (Singh et al., 2013). Exponentially growing S. coelicolor cells (at OD600 ∼ 0.3 in liquid YEME) were treated with various concentrations of RACs and monitored for growth for 8 h by following OD600. Results in Fig. 1 demonstrated that SoxRactivating chemicals are all potent inhibitors of S. coelicolor growth at sub-micromolar (ACT) or sub-millimolar (MD, PL and PMS) concentrations. On the other hand, the growth rate of S. coelicolor was not affected at all by PQ and MDs even at 1 mM. Generation of peroxides by toxic RACs in S. coelicolor Some RACs can generate ROS inside the cell through redox cycling between oxidized and reduced states under aerobic condition, producing superoxide and other ROS. They can be oxidized by giving an electron to oxygen but requires reducers from which to abstract electrons and undergo successive redox-cycling. Whether various RACs we used in this study generate ROS in S. coelicolor was assessed by using peroxide-reactive fluorescence probe 2′-7′-dichlorodihydrofluorescein diacetate (DCFHDA). Exponentially grown cells were treated with RACs for 30 min before DCFH-DA treatment. Results in Fig. 2 demonstrated that the peroxide levels in cells treated with non-toxic PQ (200 μM) and menadione bisulfite (MDs; 500 μM) were similar to that of non-treated sample. On the other hand, the growth-inhibiting γ-ACT (400 nM), MD (200 μM), PL (50 μM) and PMS (50 μM) produced significantly higher peroxides by 2.1, 1.6, 4.1 and 4.0 folds, respectively, compared with the non-treated control. This suggests that the toxicity of SoxR-activating RACs may involve ROS-derived oxidative damage. It can also be hypothesized that the non-toxic and ScSoxR-inert RACs do not undergo redox-cycling, possibly due to the absence of their reducers in S. coelicolor. In an effort to estimate redox-cycling activity, we measured cyanideresistant oxygen consumption rate of cells treated with MD, PMS, PQ and MDs as above, based on the principle that redox-cycling agents consume oxygen in the presence of cyanide that inhibits respiratory oxygen consumption (Hassan and Fridovich, 1979). The results revealed that MD and PMS increased oxygen consumption rate significantly by 80 and 60%, respectively, whereas the effect of PQ and MDs was marginal (supplementary Table S1). This coincides with the implication from the results of DCF fluorescence measurement (Fig. 2).

810 K-L. Lee et al. ■

Methyl Viologen (PQ)

Menadione bisulfite (MDs)

1.6

Non-treated

1.4

Non-treated

PQ 250 μM

1.2

MDs 250 μM

PQ 500 μM

1

MDs 500 μM

0.8

MDs 750 μM

0.6

MDs 1000 μM

1.2 1 0.8

PQ 750 μM

0.6

PQ 1000 μM

A600

A600

1.6 1.4

0.4

0.4

0.2

0.2

0

0 15

17

19

21

23

15

17

19

Time (h) 1.6

Actinorhodin (ACT)

Non-treated

1.2

ACT 0.1 μM

1.2

MD 100 μM

1

ACT 0.2 μM

1

MD 200 μM

0.8

ACT 0.4 μM

0.8

MD 300 μM

0.6

ACT 0.8 μM

0.6

MD 400 μM

0.4

0.4

0.2

0.2

0

0 15

17

19

21

23

15

17

19

Time (h)

Non-treated

1.4

1.2

PL 25 μM

1.2

PMS 25 μM

1

PL 50 μM

1

PMS 50 μM

0.6

PL 200 μM

A600

1.4

0.8

23

Phenazine methosulfate (PMS)

1.6

Non-treated

PL 100 μM

21

Time (h)

Plumbagin (PL)

1.6

A600

23

Menadione (MD)

Non-treated

1.4

A600

A600

1.6 1.4

21

Time (h)

0.8

PMS 100 μM

0.6

PMS 200 μM

0.4

0.4

0.2

0.2 0

0 15

17

19

21

23

Time (h)

15

17

19 Time (h)

21

23

Fig. 1. Effect of various RACs on the growth of S. coelicolor. Wild-type S. coelicolor (M145) cells were grown in YEME liquid medium by inoculating 108 spores per 100 ml medium in 1 L flask, until OD600 reaches 0.2–0.3 (mid exponential phase) after 15 h incubation by shaking at 30°C. Chemicals at increasing final concentrations were then added: methyl viologen (paraquat, PQ; 250, 500, 750, 1000 μM), menadione bisulfite (MDs; 250, 500, 750, 1000 μM), actinorhodin (Act; 0.1, 0.2, 0.4, 0.8 μM), menadione (MD; 100, 200, 300, 400 μM), plumbagin (PL; 25, 50, 100, 200 μM) or phenazine methosulfate (PMS; 25, 50, 100, 200 μM). Cell growth was subsequently monitored by measuring OD600. Growth of non-treated cells was monitored in parallel. At least three independent experiments were performed for each compound at each concentration, to present average values with standard deviations.

Prediction of residues affecting differential sensitivity of SoxR homologues to RACs In an effort to find key determinants of differential sensitivity, especially between EcSoxR and ScSoxR, we examined amino acid sequences around the [2Fe-2S] cluster binding sites in SoxRs from representative bacteria of all SoxR-containing groups using CLUSTAL W algorithm (Thompson et al., 1994). HMM logo was generated by Skylign (Wheeler et al., 2014). The alignment demonstrated highly conserved sequence pattern around the four conserved cysteines with group-specific variations (Fig. 3A). The most salient difference that distinguishes

enterobacterial group (except Proteus mirabilis) from the others lies in residues that precede the 4th conserved cysteine. The corresponding residues in EcSoxR and ScSoxR are 127RSDCP131 and 126LETCV130 respectively. The location of these residues in 3D structural model of dimeric EcSoxR (PDB No., 2zhh; Watanabe et al., 2008) was demonstrated in Fig. 3B. Activation profile of ScSoxR mutants with EcSoxR-like substitutions We first examined the contribution of C-terminal region of ScSoxR in determining differential sensitivity. The deletion © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 97, 808–821

SoxR activation by redox-active compounds 811

Fluorescence/A600

18000 16000 14000 12000

*

10000 8000

*

6000 4000 2000 0

None

PQ

MDs

Act

MD

of C-terminal 18 residues of ScSoxR (ΔC) was previously demonstrated not to affect sensitivity pattern of ScSoxR toward RACs that was examined (Sheplock et al., 2013; Singh et al., 2013). We therefore generated a mutant ScSoxR where the C-terminal tail from L126 to the end (D175) was substituted with that of EcSoxR from R127 to N154 (Swap1; [1–126]Sc[127–154]Ec, Fig. 3B). The swap mutant gene was chromosomally integrated into the ΔsoxR strain through the att site using the pSET152derived integration vector. Exponentially grown recombinant S. coelicolor cells (at OD600 of ∼ 0.4 to 0.5 in YEME liquid media) were treated with PMS, PQ or MDs for 30 min, followed by S1 mapping of transcripts from SCO2478, a SoxR target gene encoding a putative NADPH-dependent reductase. Results in Fig. 4A demonstrated that in contrast to ScSoxRΔC, which exhibited the wild-type sensitivity pattern, the Swap1 mutant exhibited EcSoxR-like sensitivity, responding to all three RACs. The untreated basal level of SCO2478 was also elevated in the Swap1 mutant. We compared the sensitivity profile of Swap1 with Swap2 mutant, whose replaced portion is shorter by four residues than Swap1 and contains E. coli sequence from P131 to N154 ([1–130]Sc[131–154]Ec, Fig. 3B). The mutants were introduced to E. coli (ΔsoxR) on pTac4-based plasmid, and the activation profile was monitored by measuring soxSp-driven LacZ activity (Fig. 4B). In E. coli, the ΔC and Swap1 mutants replicated the sensitivity profiles observed in S. coelicolor background. The Swap2 mutant exhibited sensitivity profile similar to the wild type or ΔC mutant ScSoxR (Fig. 4B). This reveals the importance of the three residues preceding the fourth cysteine in determining differential sensitivity. Among the three 126LET128 residues, substitution of E127 to S and T128 to D in Swap2 mutant still exhibited the wild type sensitivity profile, whereas changing L126 to R in Swap2 changed the sensitivity behavior as Swap1 mutant (Supplementary Fig. S1). We then proceeded to examine the effect of substituting 126 LETCV130 residues in ScSoxR to the corresponding ones © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 97, 808–821

PL

PMS

Fig. 2. Peroxide generation by RACs in S. coelicolor. Exponentially grown wild-type cells (OD600 ∼ 0.2 to 0.3) were treated with paraquat (PQ, 200 μM), menadione sodium bisulfite (MDs, 500 μM), actinorhodin (Act, 0.4 μM), plumbagin (PL, 50 μM), phenazine methosulfate (PMS, 50 μM) and menadione (MD, 200 μM) for 30 min. Accumulated peroxides in the cell were detected by peroxide-reactive fluorescence probe, 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA), with excitation at 492 nm and emission at 535 nm. At least three different experiments were done to obtain average values with standard deviations. P-values for actinorhodin- and menadione-treated samples (marked with *) were less than 0.001 by Student’s t-test.

of EcSoxR (127RSDCP131). The substitution mutations were made in ScSoxRΔC, which behaves like the wild type. The target (SCO2478) RNA analysis in S. coelicolor demonstrated that the L126R mutant behaved somewhat similarly to the Swap1 mutant, with elevated basal expression and slight induction by PQ and MDs, even though with lower induction fold than Swap1 (Fig. 5A). Expression and activation of ScSoxRL126R in E. coli showed similar profile (Fig. 5B). The V130P substitution hardly affected the sensitivity profile in S. coelicolor as well as in E. coli, except slightly elevated induction by MDs in E. coli. The L126R + V130P double mutant, however, exhibited greatly elevated basal expression and constitutive activation in S. coelicolor as well as in E. coil. Activation profile of EcSoxR mutants with ScSoxR-like substitutions We then examined the sensitivity profile of EcSoxR variants, whose R127 and P131 residues were changed to ScSoxR-like ones. Activation was monitored by S1 mapping of SCO2478 transcripts in S. coelicolor (Fig. 6A), or soxSp-driven LacZ assay in E. coli (Fig. 6B). Figure 6A demonstrated that in S. coelicolor, each single R127L and P131V mutant retained almost similar sensitivity profile as the wild-type EcSoxR, even though somewhat less activated by PQ and MDs. The double mutant EcSoxRR127L/ P131V, on the other hand, behaved very much like ScSoxR, not being activated by either PQ or MDs. The untreated basal level was lowered in the double mutant. In E. coli cells, the overall activation profiles of the mutants were similarly observed as in S. coelicolor cells, with some minor differences (Fig. 6B). Compared with the wild-type EcSoxR, the R127L and P131V single mutants became less sensitive to PQ while retaining the sensitivity to MDs. The double mutant showed a very low basal level activation, and nearly no activation by PQ as observed in S. coelicolor. Activation by MDs was observed for all mutants in E. coli, suggesting some difference in intracellular

812 K-L. Lee et al. ■

Actinobacteria

A

α

Proteobacteria

β

γ

δ

B

ScSoxR EcSoxR

•

Enterobacteria

Pseudomonads

1 89 (1) -MPQIPEKIQELTVGQLADRSGAAVSALHFYESKGLITSRRTSGNQRRFARDALRRVAFVRAAQRVGIPLATIREALAELPEGRTPTEDD (1) MEKKLPRIKALLTPGEVAKRSGVAVSALHFYESKGLITSIRNSGNQRRYKRDVLRYVAIIKIAQRIGIPLATIGEAFGVLPEGHTLSAKE

H1

ScSoxR EcSoxR

• ••

Arthrobacter aurescens Salinispora tropica Gordonia sihwensis Mycobacterium smegmatis Nocardia farcinica Streptomyces coelicolor Streptomyces venezuelae Nocardia farcinica SoxR1 Agrobacterium tumefaciens Rhizobium leguminosarum Erythrobacter litoralis Mesorhizobium loti Sinorhizobium meliloti Bordetella pertussis Ralstonia eutropha Collimonas fungivorans Burkholderia glumae Xanthomonas campestris Vibrio cholerae Shewanella denitrificans Escherichia coli Shigella flexneri Shigella sonnei Salmonella enterica Enterobacter aerogenes Klebsiella pneumoniae Proteus mirabilis Pseudomonas putida Pseudomonas aeruginosa Pseudomonas fluorescens Pseudomonas stutzeri Bdellovibrio bacteriovorus Stigmatella aurantiaca

H4

H3

H2

90 126 130 140 150 ∆ C (G158-D175) (90) WARLSESWRSELDERIKQLNRLRDHLTDCIGCGCLSLETCVLSNPDDAFGERSAGSRLLVERRGSTARGGRAPSRAREPEVPCCGD 175 (91) WKQLSSQWREELDRRIHTLVALRDELDGCIGCGCLSRSDCPLRNPGDRLGEEGTGARLLEDEQN 154

Swap1; [1-126]Sc[127-154]Ec Swap2; [1-130]Sc[131-154]Ec

H5

H5

H5

P-131 C-130

C-124

H3 H4

R-127 H2

E. coli SoxR

H1 H1

© 2015 John Wiley & Sons Ltd, Molecular Microbiology, 97, 808–821

SoxR activation by redox-active compounds 813

Fig. 3. Comparison of residues around the [2Fe-2S] cluster binding site in SoxRs. A. Sequence comparison of 45 amino acids containing the conserved cysteines for [2Fe-2S] cluster binding in representative SoxRs from actinobacteria and proteobacteria. Alignment was done by CLUSTAL W algorithm (Thompson et al., 1994), and the HMM logo was generated by Skylign (Wheeler et al., 2014). The position of four conserved cysteines was marked with dots on top. B. Residues mutated in ScSoxR and EcSoxR. The primary, secondary, and tertiary structures of EcSoxR (PDB code, 2zhh) were demonstrated along with the sequence of ScSoxR. Alpha helices (H1, H2, H3, H4 and H5) and residues around the [2Fe-2S] cluster were shown. C-terminal regions that were deleted (ΔC, truncation from G158 to D175) or swapped by E. coli residues (Swap1 from R127 or Swap2 from P131 to N154) in ScSoxR were indicated. The position of substituted residues were indicated by red (L126R in ScSoxR or R127L in EcSoxR) or blue (V130P in ScSoxR or P131V in EcSoxR) arrows.

chemistry that MDs undergoes in two organisms. Overall, the activation profile of EcSoxR variants demonstrated a striking contribution of the two residues flanking C130 in EcSoxR, whose substitution to ScSoxR-like residues switched the sensitivity profile from that of E. coli- to the S. coelicolor-type. Changes in redox potential of ScSoxR and EcSoxR by switching two amino acids The more selective activation of ScSoxR is likely to be due to its elevated redox potential, which is 100 mV higher than that of EcSoxR. Therefore, we hypothesized

that the two amino acid changes might have shifted the redox potential of ScSoxRL126R/V130P (Ec-mimic) and EcSoxRR127L/P131V (Sc-mimic) to approach that of EcSoxR and ScSoxR respectively. To evaluate this hypothesis, we measured redox potential of the two proteins expressed and purified anaerobically from E. coli as described in Experimental procedures. The UV-VIS absorption spectra of air-oxidized EcSoxRR127L/P131V and ScSoxRL126R/V130P indicated characteristic oxidized [2Fe-2S] peaks that disappeared upon adding sodium dithionite (Fig. S2). The redox titration was done by following absorbance at 415 nm in the presence of safranin-O and varying amounts of sodium dithionite. The mid-point reduction

Fig. 4. Activation profile of ScSoxR C-terminal mutants in S. coelicolor and in E. coli. A. Activation of ScSoxR variants with C-terminal deletion (ΔC) or swapping (Swap1) in S. coelicolor. The mutated soxR gene was cloned in pSET152-based vector pSET162 and chromosomally integrated into S. coelicolor ΔsoxR cells. Activation of SoxR was monitored by analyzing its target gene transcripts (SCO2478) by S1 mapping, following addition of PMS (50 μM), paraquat (PQ, 200 μM) or menadione sodium bisulfite (MDs, 500 μM) to exponentially grown cells for 30 min. Expression levels relative to untreated sample were obtained from at least three independent experiments. Average values with standard deviations were presented. B. Activation profile of ScSoxRΔC, ScSoxRSwap1 and ScSoxRSwap2 mutants in E. coli. Each gene was cloned in the multicopy pTac4 plasmid (Table 1). The recombinant plasmids were introduced into E. coli ΔsoxR strain that contains the soxSp-driven β-galactosidase (LacZ) reporter gene in the chromosome (MS1343, Table 1). The transformed cells were grown in LB to early exponential phase (OD600 ∼ 0.2) and either were left untreated or were treated with 50 μM of PMS, 200 μM of PQ or 500 μM of MDs for 60 min, followed by β-galactosidase activity assay. The mean values of activity in Miller units were obtained from three independent experiments. For each mutant strain, the induction fold relative to the untreated level was indicated above each graphic bar. © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 97, 808–821

814 K-L. Lee et al. ■

A

S. coelicolor D soxR + pSET–ScSoxRD C L126R

L126R+V130P

V130P

0

PMS PQ MDs

0

1.0

2.1 1.4 2.3 ± 0.3 ± 0.2 ± 1.2

1.0

PMS PQ MDs 0

17.6 1.0 1.4 ± 14.2 ± 0.1 ± 0.3

1.0

PMS PQ MDs

0.9 ± 0.1

1.0 1.1 ± 0.1 ± 0.7

B b-galactosidase activity (MU)

2000 1800 1600 1400 1200 1000 800 600 400 200 0

1

1.2 1.2 1

Non-treated PMS PQ

14.3 1.9 1

1.2 1.4

Fig. 5. Activation profile of ScSoxR substitution mutants in S. coelicolor and in E. coli. A. Activation profile in S. coelicolor. Amino acid substitution mutations (L126R, V130P or L126R/V130P double) were introduced into ScSoxRΔC. The mutated soxR gene was cloned in pSET162 and chromosomally integrated into S. coelicolor ΔsoxR cells. Activation assay was done as described in Fig. 4A. B. Activation profile in E. coli. The same mutant soxR genes described above were cloned in pTac4 plasmid, introduced into E. coli and measured for activation as described in Fig. 4B.

MDs 5.7 1.2

1

L126R

V130P

L126R V130P

E. coli D soxR + pTac-ScSoxRD C

potentials of EcSoxRR127L/P131V and ScSoxRL126R/V130P were estimated to be −192 ± 8 mV and −273 ± 10 mV respectively (Fig. 7). Therefore, the Sc-mimicking substitutions in EcSoxR increased its redox-potential from −287 mV by ∼ 100 mV, approaching that of the full-length wild-type ScSoxR (−187 ± 10 mV; Singh et al., 2013) or ScSoxRΔC (−179 ± 11 mV; Supplementary Fig. S3). The EcSoxRmimicking substitutions in ScSoxRΔC decreased its redox potential by 94 mV from −179 to −273 mV, approaching closely to that of EcSoxR (−287 ± 4 mV; Singh et al., 2013). The redox potential of EcSoxRR127L was estimated to be −249 ± 9 mV (Supplementary Fig. S3), revealing that the R127L single mutation in EcSoxR caused an increase in potential by 38 mV.

WT 0

1.0

R127L

PMS PQ MDs

11.4 ± 8.7

1600

14.0 9.5 ± 9.0 ± 6.5

0

1.0

PMS PQ MDs

8.1 7.9 ± 1.5 ± 1.5

R127L+P131V

P131V

2.4 ± 1.1

0

1.0

PMS PQ MDs 0

19.1 ± 18.6

7.5 4.7 ± 6.8 ± 5.8

PMS PQ MDs

21.3 ± 6.7

1.0

1.1 ± 0.1

PMS

1200 1000

PQ

11.3 5.2

3.5 3.2

800

200

MDs

6.7

600 400

0.9 ± 0.1

Non-treated

6.1

1400

activity (MU)

b-galactosidase

The change in the redox potential is due to the change in the free energy difference between the reduced and the oxidized states, caused by the mutations. Because the most important difference between the reduced and the oxidized states is the electric charge of the [2Fe-2S] cluster (+1 for the reduced state and +2 for the oxidized state), we focused on the change in the electrostatic effect. To investigate the change in electrostatic character around the [2Fe-2S] cluster by the key mutations of SoxR, explicit-water molecular dynamics simulations were per-

S. coelicolor D soxR + pSET-EcSoxR

A

B

Simulation analysis of changes in structure and charge distribution around [2Fe-2S] cluster in EcSoxR and ScSoxR by point mutations

1 1

2.3

4.0 16.5

1.6 1 1

1.5

4.5

0

WT

R127L

P131V

E. coli D soxR + pTac-EcSoxR

R127L P131V

Fig. 6. Activation profile of EcSoxR variants in S. coelicolor and in E. coli. A. Activation profile of EcSoxR mutants in S. coelicolor. Amino acid substitution mutations (R127L, P131V or R127L/P131V double) were introduced to EcSoxR. The mutated E. coli soxR genes were cloned in pSET162 and introduced into the chromosome of S. coelicolor ΔsoxR. Activation assay was done by S1 mapping as described in Fig. 4A. Relative expression levels obtained from at least three independent experiments were presented at the bottom of the representative S1 mapping data. B. Activation profile in E. coli. The same E. coli soxR mutant genes were cloned in pTac4-derived plasmid and introduced into E. coli ΔsoxR strain that contain soxSp-lacZ gene. Activation assay was done as described in Fig. 4B.

© 2015 John Wiley & Sons Ltd, Molecular Microbiology, 97, 808–821

SoxR activation by redox-active compounds 815

Pro has no such hydrogen because of the side-chain ring structure (Fig. S4C). The L126R mutation in ScSoxR removes the broad positive peaks around 9–14 Å (Fig. 8B), and the V130P mutation also removes a small positive peak at 3.8 Å (Fig. 8D). Therefore, Ec-mimicking mutations in ScSoxR make the environment of the [2Fe2S] cluster more negative, consistent with the observed change in redox potential.

Discussion

Fig. 7. Redox titration of purified SoxR variants. ScSoxRΔCL126R/V130P (open circle) and EcSoxRR127L/P131V (filled circle) proteins anaerobically purified from E. coli were re-suspended to 20 μM each in 20 mM Tris-HCl (pH 7.8) containing 500 mM NaCl, 10% glycerol and 1 mM DTT in a stoppered cuvette. The [2Fe-2S] cluster of the proteins were reduced in the presence of the redox mediator safranin-O (5 μM) at 25°C by adding increasing amounts of sodium dithionite in the anaerobic chamber. The redox potential of the solution was measured with a platinum and Ag/AgCl electrode (HACH-MTC101-1), and the amount of oxidized SoxR in the same solution was measured by taking spectrophotometric absorbance at 415 nm as described in Experimental procedures. Percent fraction of oxidized SoxR (y-axis) was plotted against redox potential (Eh in mV) of the solution. Data points from three independent measurements were presented, with calculated mid-point potential from fitted curves. Previously determined redox potential values of wild-type EcSoxR and ScSoxR proteins were also presented (Singh et al., 2013), along with the value for ScSoxRΔC measured in this study (Supplementary Fig. S3).

formed for eight wild-type and mutant EcSoxR and ScSoxR sequences, as described in Experimental procedures. The total net charge of the simulation system was adjusted to be neutral by adding either Na+ or Cl−. Change in electric charge distribution was shown in Fig. 8 as a function of the distance from the [2Fe-2S] cluster for each of the two positions: the Arg/Leu position (A and B; R127 of EcSoxR and L126 of ScSoxR) and the Pro/Val position (C and D; P131 of EcSoxR and V130 of ScSoxR). The R127L mutation in EcSoxR removes the large negative peak at 11 Å (Fig. 8A), and the P131V mutation creates a small positive peak at 3.5 Å (Fig. 8C). Hence, both Sc-mimicking mutations make the environment of the [2Fe-2S] cluster in EcSoxR more electropositive. It is interesting that the neutral amino acid Leu makes the environment more electropositive than the positively charged Arg. This is because Arg can recruit an additional chloride ion closer to the [2Fe-2S] cluster (12 Å from the cluster), whereas Leu does not play such a role, with the closest chloride ion at ∼ 19 Å (Supplementary Fig. S4A). The increase in electropositivity by the Pro to Val mutation is due to the existence of the backbone amide hydrogen in Val that has partially electropositive character, whereas © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 97, 808–821

Our previous study proposed that the different sensitivities of ScSoxR and EcSoxR to RACs could be determined by the redox property of their [2Fe-2S] cluster (Singh et al., 2013). Insensitivity of ScSoxR to PQ can be explained by its high redox potential. In this study, we analyzed the contribution of two critical residues around the [2Fe-2S] cluster that could determine the sensitivity behavior by changing its redox potential. Among the residues around the conserved cysteine motif (CX2CXCX5C) in SoxR, enterobacteria-specific RSD residues, especially the R127 preceding the fourth cysteine (C130) in EcSoxR, has been noted to alter sensitivity of E. coli SoxR toward PQ (Chander and Demple, 2004). The same site mutations in P. aeruginosa (Pa) SoxR (L125R or L125A) were reported to constitutively activate soxS expression in E. coli (Sheplock et al., 2013). In our study, we demonstrated that EcSoxR acquired ScSoxR-like sensitivity profile by R127L and P131V mutations. The change in selectivity behavior was accompanied with 95 mV increase in redox potential from −287 to −192 mV, approaching that of ScSoxR (−187 mV). Insensitivity to PQ is explainable by this shift in redox potential. Molecular dynamics simulations revealed that both mutations made the electric charge distribution around [2Fe-2S] more positive than in the wild type. Because the [2Fe-2S] cluster of the oxidized state of SoxR is more highly charged (+2) than that of the reduced state (+1), electropositive environment is expected to destabilize the oxidized state more than the reduced state, thus increasing the redox potential and decreasing the sensitivity to PQ. Shifts in redox potential by amino acid changes near [FeS] cluster in a protein were mostly studied in ferredoxins, as exemplified by Anabaena [2Fe-2S] ferredoxin (Hurley et al., 1997) and [4Fe-4S] ferredoxins (Perrin and Ichiye, 2013), which interact with partner proteins for electron transfer. Our study demonstrates an example of a chemical sensor with [2Fe-2S], whose reactivity could be modulated by amino acid changes, through shifting its redox potential. One of the constitutively active mutations in EcSoxR (S95L mutation) was reported to decrease reduction potential by 65 mV (Hidalgo et al., 1997), consistent with the correlation between PQ reactivity and redox potential that we observed in this study.

816 K-L. Lee et al. ■

Fig. 8. Charge distribution around [2Fe-2S] cluster of the wild-type and mutant SoxRs. Electric charge distribution obtained from molecular simulation analyses was presented as a function of distance from the [2Fe-2S] cluster for eight SoxR proteins: the wild-type (Arg-127) and Leu-127 mutant EcSoxRs (A), the wild-type (Leu-126) and Arg-126 mutant of ScSoxRs (B), the wild-type (Pro-131) and Val-131 mutant EcSoxRs (C), and the wild-type (Val-130) and Pro-130 mutant ScSoxRs (D).

Streptomyces coelicolor SoxRL126R is somewhat relaxed in its sensitivity pattern, showing partially constitutive activation behavior, but it can be further activated by PMS, PQ and MDs (Fig. 5A). ScSoxRL126R/V130P double mutant is fully constitutive in activation. Considering the significant decrease in redox potential by the double mutation, it can be hypothesized that constitutive activation results from nearly full oxidation of SoxR in the intracellular environment. This could happen if the mid-point redox potential is lower than the local intracellular environment. However, the redox potential of ScSoxRL126R/V130P (Eh −273 mV) was estimated to be similar to or slightly higher than that of EcSoxR (Eh −287 mV). As the heterologously expressed EcSoxR is not constitutively activated in S. coelicolor cells, the change in redox potential alone cannot explain the constitutively activated phenotype of the L126R/V130P mutant ScSoxR. The double mutation, in addition to shifting redox potential to lower level, somehow could have created some conformational changes that favor the orientation of the DNA-binding domain in an activated conformation. The observation that the Swap1 mutation that replaces the C-terminal tail of ScSoxR from L126 with EcSoxR tail from R127 also demonstrated partially constitutive phenotype (Fig. 4A), in comparison with Swap2, suggests contribution of the R residue in triggering activated conformation, possibly through interaction with the N-terminally located residue(s) in ScSoxR. In EcSoxR

structure, the positively charged side-chain of R127 is located closely to the negatively charged carbonyl oxygen atom of the conserved R20 on Helix 1 of the other subunit, independent of DNA binding (Watanabe et al., 2008). Therefore, the substituted R residue in ScSoxR could have elicited changes in the interaction between the DNA binding domain and the target promoter to favor the formation of promoter open complex. The molecular dynamics simulation of ScSoxR mutants showed that L126R substitution created subtle changes in the orientation of helix 3 in the DNA-binding domain, which could have facilitated the active conformation of DNA-bound SoxR (Fig. S4B). This contrasts with little structural change around [2Fe-2S] in EcSoxR by the corresponding mutation (Fig. S4A). The Pro to Val in EcSoxR or Val to Pro mutation in ScSoxR appears not to create any structural changes (Fig. S4C and D), partly predictable from the presence of conserved flanking residues (C and hydrophobic residue) that provide relatively stable structural environment. Indications of the influence of L126R/V130P mutations in ScSoxR on protein conformation could also be found in the extremely labile nature of the mutant protein, being easily demetallated and aggregated upon air exposure, unlike other SoxR proteins examined in this study. This led us to prepare the protein and measure redox potential under anaerobic conditions. When we monitored the in vivo electron paramagnetic resonance (EPR) signal from E. coli cells that overex© 2015 John Wiley & Sons Ltd, Molecular Microbiology, 97, 808–821

SoxR activation by redox-active compounds 817

pressed ScSoxRΔCL126R/V130P following 12 h of IPTG induction in aerobic shaking culture, we also found that the protein existed mostly as apo-forms, unlike ScSoxRΔC, which existed mostly as a reduced form (supplementary Fig. S5). This behavior resembles that of some constitutive EcSoxR mutants, which maintain similar redox potential as the wild type but are sensitive to oxidation (Hidalgo et al., 1997). The contribution of SoxR in protecting bacteria from the toxicity of exogenous RACs has been well demonstrated in E. coli and S. coelicolor (Greenberg et al., 1990; Singh et al., 2013). S. coelicolor, which inhabits in soil, has a need to respond to a variety of chemicals produced by itself (endogenously) and by other organisms (exogenously). Our study demonstrated that it does respond to both endogenous and exogenous compounds but does not respond to chemicals that do not inhibit its growth, to the extent we examined. Therefore, the development of differential sensitivity appears to go hand in hand with the versatile physiological necessity. Even though the nontoxic PQ and menadione bisulfite are capable of penetrating into the S. coelicolor cells, retaining the ability to activate heterologously expressed EcSoxR, they do not activate ScSoxR and thus save the cost of unnecessary gene expression. We found that these two compounds do not generate detectable amount of ROS nor redox-cycle inside S. coelicolor cells. This could possibly reflect the lack of reducers available for these RACs. The finding that PQ and MDs still activates EcSoxR in S. coelicolor, in the absence of detectable ROS generation, further supports the idea that EcSoxR is not effectively activated by superoxide. Even though superoxide can oxidize EcSoxR in vitro (Fujikawa et al., 2012; Kobayashi et al., 2014), it may do so inefficiently in vivo as demonstrated in superoxide dismutase (SOD)-overexpressing or depleted cells (Gu and Imlay, 2011). The inability of ScSoxR or Sc-mimicking EcSoxR double mutant to be activated by PQ inside E. coli, where PQ can produce superoxide and ROS, is an additional piece of evidence that superoxide is not an effective oxidant for SoxRs in vivo. Whether the correlation of toxicity and SoxR-activating ability of RACs is a general phenomenon needs further verification in other bacteria through careful pursuit of viability assays, as well as examining the effect of more RACs.

Experimental procedures Bacterial strains, plasmids and culture conditions Bacterial strains and plasmids used in this study were listed in Table 1. S. coelicolor A3(2) M145 (wild-type) and its mutants were grown in YEME liquid medium containing 5 mM MgCl2·6H2O and 10.3% sucrose at 30°C by inoculating spore suspension (Kieser et al., 2000). Cells were grown to midexponential phase (OD600 of 0.3 to 0.5) and treated with © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 97, 808–821

chemicals as indicated. E. coli cells were grown in Luria– Bertani (LB) medium at 37°C. To examine the effect of various RACs on the growth of S. coelicolor, 108 spores were inoculated into 100 ml of YEME in a 1 L baffled flask, incubated at 30°C with shaking at 180 r.p.m. until the culture reached an OD600 of about 0.2–0.3 at 15 h after inoculation. To each flask, methyl viologen (PQ; Sigma), MDs (Sigma), γ-ACT (Shin et al., 2011), MD (Sigma), PL (Sigma) or PMS (Sigma) were added at various concentrations, and OD600 was taken at every 90 min for 8 h.

Fluorescence measurement of intracellular peroxides Exponentially grown S. coelicolor cells (OD600 ∼ 0.2–0.3) were treated with indicated amounts of RACs for 30 min, followed by cell harvest by centrifugation for 3 min at 5000 g at 4°C. Cell pellets were briefly washed twice with ice-cold P buffer [sucrose 103 mg l−1, potassium sulfate 0.25 mg l−1, trace element solution 2 ml, KH2PO4 0.05 mg l−1, MgCl2·6H2O 2.03 mg l−1, CaCl2·2H2O 3.68 mg l−1 and 0.25 M TES buffer (pH 7.2) 100 ml]. Trace element solution contains 40 mg l−1 ZnCl2, 200 mg ml−1 FeCl3·6H2O, 10 mg ml−1 CuCl2·2H2O, 10 mg ml−1 MnCl·4H2O, 10 mg l−1 Na2B4O7·10H2O, 10 mg l−1 (NH4)6Mo7O24·4H2O (Kieser et al., 2000). Cells were resuspended in pre-warmed P buffer containing 5 μM DCFH-DA (2′-7′-dichlorofluorescein diacetate; Sigma) and incubated for 30 min at 30°C in the dark. Intracellular conversion of DCFH-DA by esterase in the cell produces non-fluorescent DCFH, which can then be oxidized by peroxides, to produce fluorescent DCF (Keston and Brandt, 1965; Bass et al., 1983). The DCF fluorescence was measured in a fluorometric plate reader (EnVision Multilabel Plate Readers, Perkin-Elmer) with excitation and emission at 492 and 535 nm respectively. Optical density of each sample was measured at 600 nm in a spectrophotometric plate reader (PowerWave X, BioTek), to normalize fluorescence by the amount of cells examined.

Construction of recombinant plasmids Swap constructs of a ScSoxR N-terminal region linked with an EcSoxR C-terminal region were produced via gene splicing by overlap extension (Horton et al., 1989). ScSoxR N-terminals were amplified from S. coelicolor genome DNA by PCR using pairs of upstream primer, ScSoxR-F (5′-GGT TCG AGC ATA TGC CTC AGA TTC-3′; NdeI site underlined) and downstream primers, ScEcSwap1-R (5′-GCA GCC GCA GCC GAT GCA GTC GGT GAG GTG GTC GCG CAG-3′) or ScEcSwap2-R (5′-GCA GGT TTC CAG GGA CAG GCA GCC GCA GCC GAT GCA GTC-3′). EcSoxR C-terminals were amplified from E. coli genome DNA by PCR using pairs of upstream primers, ScEcSwap1-F (5′-CTG CGC GAC CAC CTC ACC GAC TGT ATT GGT TGT GGC TGC CTT-3′; EcSoxR sequence underlined) or ScEcSwap2-F (5′-GCC TGT CCC TGG AAA CCT GCC CGT TGC GTA ACC CGG GCG A-3′; EcSoxR sequence underlined), and downstream primer, EcSoxR-R (5′-GCG CCC TGG ATC CGC TTT AGT TTT-3′; BamHI site underlined). PCR products were separated with agarose gel electrophoresis, and the second PCR to construct hybrid ORFs were done by using ScSoxR-F and EcSoxR-R, generating Swap1 or Swap2 mutants of ScSoxR

818 K-L. Lee et al. ■

Table 1. Bacterial strains and plasmids used in this study. Strains/plasmids S. coelicolor M145 ΔsoxR ΔsoxR::ScSoxRΔC ΔsoxR::ScSoxRΔCL126R ΔsoxR::ScSoxRΔCV130P ΔsoxR::ScSoxRΔCL126R/V130P ΔsoxR::EcSoxRR127L ΔsoxR::EcSoxRP131V ΔsoxR::EcSoxRR127L/P131V E. coli BL21 (λDE3) pLysS MS1343 ΔsoxR ET12567 Plasmids pSET162 pSET162-soxRp-ScSoxR pSET162-soxRp-EcSoxR pTac4 pTac4-ScSoxRΔC pTac4-ScSoxRΔCL126R pTac4-ScSoxRΔCV130P pTac4-ScSoxRΔCL126R/V130P pTac4-EcSoxRR127L pTac4-EcSoxRP131V pTac4-EcSoxRR127L/P131V pET15b pET15b-ScSoxRΔCL126R/V130P pET15b-EcSoxRR127L/P131V pET15b-EcSoxRR127L pET15b-ScSoxRΔC

Relevant genotypes and characteristics

Sources

Wild type strain, SCP1− SCP2− M145 with a deletion of soxR gene M145 ΔsoxR::pSET162-soxRp-ScSoxRΔC M145 ΔsoxR::pSET162-soxRp-ScSoxRΔCL126R M145 ΔsoxR::pSET162-soxRp-ScSoxRΔCV130P M145 ΔsoxR::pSET162-soxRp-ScSoxRΔCL126R/V130P M145 ΔsoxR::pSET162-soxRp-EcSoxRR127L M145 ΔsoxR::pSET162-soxRp-EcSoxRP131V M145 ΔsoxR::pSET162-soxRp-EcSoxRR127L/P131V

Kieser et al. (2000) Shin et al. (2011) Singh et al. (2013) This study This study This study This study This study This study

fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS λ DE3 = λ sBamHIo ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 Δnin5 GC4468, soxSp::lacZ, Ampr ΔsoxR::Kanr F′ dam13::Tn9 dcm6 hsdM hsdR recF143::Tn10 galK2 galT22 ara-14 lacY1 xyl-5 leuB6 thi-1 tonA31 rpsL hisG4 tsx-78 mtl-1 glnV44

Lab culture stock

Streptomyces integration plasmid pSET152 (Apramycinr lacZa MCS reppUC) modified to contain a thiostrepton resistance marker Recombinant plasmid expressing ScSoxR from its own promoter Recombinant plasmid expression EcSoxR from ScSoxR promoter E. coli expression vector, modified from pTac1 by inserting Chlr instead of Ampr pTac4-based recombinant expressing ScSoxRΔC Recombinant expressing ScSoxRΔCL126R Recombinant expressing ScSoxRΔCV130P Recombinant expressing ScSoxRΔCL126R/V130P Recombinant expressing EcSoxRR127L Recombinant expressing EcSoxRP131V Recombinant expressing EcSoxRR127L/P131V N-terminally histidine-tagging expression vector Recombinant plasmid for overproducing ScSoxRΔCL126R/V130P protein in E. coli Recombinant plasmid for overproducing EcSoxRR127L/P131V in E. coli Recombinant plasmid for overproducing EcSoxRR127L Recombinant plasmid for overproducing ScSoxRΔC

(Fig. 3B). The coding regions of ScSoxR with C-terminal truncation (ScSoxRΔC; G158 to D175 deleted; Fig. 3B) and the entire EcSoxR were amplified from the corresponding genomic DNAs using mutagenic primers: ScSoxR-F and ScSoxRDC-R (5′-GCG GCT CGG GAT CCG GCC TCA CCT GGC GGT G-3′; BamHI site underlined, stop codon in italic), EcSoxR-F (5′-GAG GTG GAT CCA CAT ATG GAA AAG-3′; NdeI site underlined) and EcSoxR-R. PCR products were digested with NdeI/BamHI and cloned into pTac4 vector using NdeI/BamHI restriction site. Site-specific mutagenesis of EcSoxR, ScSoxRΔC and ScSoxR-Swap2 was done with QuikChange Site-Directed Mutagenesis Kit (Stratagene). Each mutant constructs were subcloned into Streptomyces integration vector containing the ScSoxR promoter (pSET162-pScSoxR vector) using the NdeI/BamHI site.

Koo et al. (2003) MacNeil et al. (1992)

Kim et al. (2006) This study This study Singh et al. (2013) This study This study This study This study This study This study This study Novagen This study This study This study This study

genes from S. coelicolor were amplified by PCR and cloned into the pGEM-T Easy plasmid (Promega). The fragments containing the soxRp-soxR region were cut out with EcoRI and BamHI restriction enzymes and cloned into pSET162, which is a derivative of integration vector pSET152 with a thiostrepton resistance marker (Bierman et al., 1992). The pSET162-based recombinant plasmids were introduced into methylation-negative, conjugal host strain E. coli ET12567 and then transferred to the ΔsoxR mutant by bacterial conjugation. The proper chromosomal integration through the att site in exoconjugants that showed apramycinR and thiostreptonR phenotypes were verified by genomic PCR analysis. For expression studies in E. coli, the ΔsoxR mutant of MS1343 strain (Koo et al., 2003; Table 1) was transformed with pTac4based recombinant plasmids containing ORFs for the wild type and mutant ScSoxR or EcSoxR.

Construction of ΔsoxR strains expressing ScSoxR and EcSoxR variants

S1 nuclease mapping analysis

The ΔsoxR mutant of S. coelicolor (Shin et al., 2011) was transformed with pSET162-based recombinant plasmids containing ORFs for the wild type and mutant ScSoxR or EcSoxR. To construct the recombinant plasmids, DNA fragments containing the promoter of the S. coelicolor soxR gene (soxRp) and the coding sequences of the mutated soxR

To prepare RNAs, S. coelicolor cells were grown in liquid YEME media to OD600 of 0.4–0.5. RNAs were isolated by acidic phenol extraction, following fixation of cells with RNAprotect® bacterial reagent (Qiagen). To prepare RNAs from E. coli, cells were grown in LB to OD600 of 0.4–0.5 before treatment with chemicals. Gene-specific S1 probes for © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 97, 808–821

SoxR activation by redox-active compounds 819

SCO2478 were generated by PCR using S. coelicolor M145 genomic DNA as a template. The probe for soxS RNA was generated by PCR using E. coli genomic DNA as templates. The amplified probe encompassed sequences for SCO2478 from −177 to +100 and for soxS −84 to +88 nt relative to the start codon. Each RNA sample (50 to 100 μg) was hybridized at 50°C with gene-specific probes labeled with [γ-32P]-ATP. Hybridization and S1 nuclease mapping were carried out according to standard procedures (Kieser et al., 2000). Following S1 nuclease treatment, the protected DNA probes were loaded on 6% polyacrylamide gel containing 7 M urea. The signal was detected and quantified by BAS-2500 (Fuji).

β-galactosidase assay Escherichia coli cells were grown in LB medium to an OD600 of 0.2 and was left either untreated or treated with 50 μM PMS, 500 μM MDs or 200 μM PQ for 1 h at 37°C respectively. β-galactosidase activity was assayed by adding o-nitrophenyl-β-D-galactopyranoside (ONPG) after permeabilization of the cell with SDS-chloroform (Miller, 1972).

Overproduction and purification of SoxR proteins The pET15b-based recombinant plasmids were transformed into E. coli BL21 (λDE3) pLysS. To purify SoxR proteins, transformants were grown in LB at 37°C to OD600 of 0.5 and induced with 0.1 mM (final concentration) isopropyl-β-Dthiogalactopyranoside (IPTG) for 3 h at 25°C. Harvested Cells were resuspended in N2-purged TN500 (20 mM TrisHCl, pH 7.8, 500 mM NaCl) and ruptured with EmulsiFlex-C3 homogenizer (Avestin) for 3 min. Remaining cells and cell debris were removed by centrifugation at 15 000 g for 15 min at 4°C. His-tagged SoxR proteins were purified through nickel-charged NTA column (Novagen) as recommended by the manufacturer in the anaerobic chamber (Coy). Purified proteins were dialyzed against TGDN500 buffer (20 mM TrisHCl, pH 7.8, 500 mM NaCl, 10% glycerol and 1 mM DTT) and kept in the anaerobic chamber at 4°C until redox titration. The purity of the protein preparation was estimated through SDS-PAGE with Coomassie brilliant blue staining, and the final concentration was determined by Bradford assay.

EcSoxR-WT, R127L, P131V and R127L + P131V, and ScSoxR-WT, L126R, V130P and L126R + V130P. Initial structure for each molecular dynamics simulation was prepared from the crystal structure for EcSoxR-WT (Watanabe et al., 2008) (PDB ID: 2ZHH and 2ZHG) or by building a homology model using the GalaxyTBM (Ko et al., 2012) template-based modeling program and refining the model using the GalaxyRefine (Heo et al., 2013) model refinement program for all other sequences. The crystal structure for EcSoxR was used as a template. Dimer configurations and positions of the [2Fe-2S] clusters were also transferred from the crystal structures. For molecular dynamics simulations, the AMBER10 package (D.A. Case et al., 2008) was used with Amber99SB force field (Hornak et al., 2006) for protein and with the force field from Carvalho et al. (Carvalho et al., 2013) for the [2Fe-2S] cluster. Each protein was solvated in a cubic box filled with TIP3P water molecules with at least 10 Å margin from the protein surface in each dimension. The system was neutralized with sodium or chloride ions. Each simulation system was prepared by heating up from 0 K to 200 K for 50 ps with restraint of 0.1 kcal mol−1 Å2−1 applied to all Cα atoms and the [2Fe-2S] clusters and from 200 K to 300 K for additional 50 ps without restraint, and then by equilibrating for 100 ps at 300 K. A production simulation was then run for 200 ps, and 1000 snapshots were collected from the production run for analysis of the electric charge distribution. The charge distribution was obtained by averaging over the simulation trajectories for wild-type and mutant proteins with the corresponding amino acid at the key position. Only the charges of varying atoms (atoms of Arg/Leu or Pro/Val and Na+/Cl− ions) were considered.

Acknowledgements This work was supported by a grant for Intelligent Synthetic Biology Center of Global Frontier Project by MEST for Laboratory of Molecular Microbiology to JH Roe (2011-0031960) and a grant from National Research Foundation of Korea to C Seok (NRF-2013R1A2A1A09012229). KL Lee was supported by BK21 fellowship for Life Sciences at SNU. Atul Kumar Singh was supported as a Korean Government Scholarship Grantee by Ministry of Education, Science and Technology.

Redox titration of SoxR Purified SoxR protein was diluted to 10 μM in TGDN500 buffer containing redox mediator safranin-O (5 μM) in a stoppered cuvette of 1 mm path length. The amount of oxidized SoxR was estimated by measuring absorption at 415 nm in a UV-1650PC spectrophotometer (Shimadzu). Redox titration was done by adding different amount of sodium dithionite at 25°C. The redox potential of the solution at each addition of sodium dithionite was measured with a combined platinum and Ag/AgCl electrode (HACH-MTC101-1) in an anaerobic chamber. The fraction of oxidized SoxR in each redox condition was calculated as described previously (Kobayashi and Tagawa, 2004).

Simulation Molecular dynamics simulations were applied to the following eight wild-type and mutant EcSoxR and ScSoxR proteins: © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 97, 808–821

References Bass, D.A., Parce, J.W., Dechatelet, L.R., Szejda, P., Seeds, M.C., and Thomas, M. (1983) Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol 130: 1910– 1917. Bierman, M., Logan, R., O’Brien, K., Seno, E.T., Rao, R.N., and Schoner, B.E. (1992) Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116: 43–49. Carvalho, A.T., Teixeira, A.F., and Ramos, M.J. (2013) Parameters for molecular dynamics simulations of ironsulfur proteins. J Comput Chem 34: 1540–1548. Case, D.A., Darden, T.A., Cheatham, I.T.E., Simmerling, C.L., Wang, J., Duke, R.E., et al. (2008) AMBER 10 User Manual. San Francisco: University of California, San Francisco.

820 K-L. Lee et al. ■

Chander, M., and Demple, B. (2004) Functional analysis of SoxR residues affecting transduction of oxidative stress signals into gene expression. J Biol Chem 279: 41603– 41610. Dietrich, L.E., Teal, T.K., Price-Whelan, A., and Newman, D.K. (2008) Redox-active antibiotics control gene expression and community behavior in divergent bacteria. Science 321: 1203–1206. Ding, H., and Demple, B. (1996) Glutathione-mediated destabilization in vitro of [2Fe-2S] centers in the SoxR regulatory protein. Proc Natl Acad Sci USA 93: 9449–9453. Ding, H., and Demple, B. (2000) Direct nitric oxide signal transduction via nitrosylation of iron-sulfur centers in the SoxR transcription activator. Proc Natl Acad Sci USA 97: 5146–5150. Fujikawa, M., Kobayashi, K., and Kozawa, T. (2012) Direct oxidation of the [2Fe-2S] cluster in SoxR protein by superoxide: distinct differential sensitivity to superoxidemediated signal transduction. J Biol Chem 287: 35702– 35708. Gaudu, P., and Weiss, B. (1996) SoxR, a [2Fe-2S] transcription factor, is active only in its oxidized form. Proc Natl Acad Sci USA 93: 10094–10098. Greenberg, J.T., Monach, P., Chou, J.H., Josephy, P.D., and Demple, B. (1990) Positive control of a global antioxidant defense regulon activated by superoxide-generating agents in Escherichia coli. Proc Natl Acad Sci USA 87: 6181–6185. Gu, M., and Imlay, J.A. (2011) The SoxRS response of Escherichia coli is directly activated by redox-cycling drugs rather than by superoxide. Mol Microbiol 79: 1136– 1150. Hassan, H.M., and Fridovich, I. (1979) Paraquat and Escherichia coli. Mechanism of production of extracellular superoxide radical. J Biol Chem 254: 10846–10852. Heo, L., Park, H., and Seok, C. (2013) GalaxyRefine: protein structure refinement driven by side-chain repacking. Nucleic Acids Res 41: W384–W388. Hidalgo, E., Ding, H., and Demple, B. (1997) Redox signal transduction: mutations shifting [2Fe-2S] centers of the SoxR sensor-regulator to the oxidized form. Cell 88: 121– 129. Hornak, V., Abel, R., Okur, A., Strockbine, B., Roitberg, A., and Simmerling, C. (2006) Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins 65: 712–725. Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K., and Pease, L.R. (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77: 61–68. Hurley, J.K., Weber-Main, A.M., Stankovich, M.T., Benning, M.M., Thoden, J.B., Vanhooke, J.L., et al. (1997) Structurefunction relationships in Anabaena ferredoxin: correlations between X-ray crystal structures, reduction potentials, and rate constants of electron transfer to ferredoxin: NADP+ reductase for site-specific ferredoxin mutants. Biochemistry 36: 11100–11117. Imlay, J.A. (2013) The molecular mechanisms and physiological consequences of oxidative stress: lessons from a model bacterium. Nat Rev Microbiol 11: 443–454. Keston, A.S., and Brandt, R. (1965) The fluorometric analysis

of ultramicro quantities of hydrogen peroxide. Anal Biochem 11: 1–5. Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F., and Hopwood, D.A. (2000) Practical Streptomyces Genetics. Norwich: John Innes Foundation. Kim, I.K., Lee, C.J., Kim, M.K., Kim, J.M., Kim, J.H., Yim, H.S., et al. (2006) Crystal structure of the DNA-binding domain of BldD, a central regulator of aerial mycelium formation in Streptomyces coelicolor A3(2). Mol Microbiol 60: 1179–1193. Ko, J., Park, H., and Seok, C. (2012) GalaxyTBM: templatebased modeling by building a reliable core and refining unreliable local regions. BMC Bioinformatics 13: 198. Kobayashi, K., and Tagawa, S. (2004) Activation of SoxRdependent transcription in Pseudomonas aeruginosa. J Biochem 136: 607–615. Kobayashi, K., Fujikawa, M., and Kozawa, T. (2014) Oxidative stress sensing by the iron-sulfur cluster in the transcription factor, SoxR. J Inorg Biochem 133: 87–91. Koo, M.S., Lee, J.H., Rah, S.Y., Yeo, W.S., Lee, J.W., Lee, K.L., et al. (2003) A reducing system of the superoxide sensor SoxR in Escherichia coli. EMBO J 22: 2614–2622. Lee, J.H., Lee, K.L., Yeo, W.S., Park, S.J., and Roe, J.H. (2009) SoxRS-mediated lipopolysaccharide modification enhances resistance against multiple drugs in Escherichia coli. J Bacteriol 191: 4441–4450. Ma, D., Cook, D.N., Alberti, M., Pon, N.G., Nikaido, H., and Hearst, J.E. (1995) Genes acrA and acrB encode a stressinduced efflux system of Escherichia coli. Mol Microbiol 16: 45–55. MacNeil, D.J., Gewain, K.M., Ruby, C.L., Dezeny, G., Gibbons, P.H., and MacNeil, T. (1992) Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111: 61– 68. Miller, J.H. (1972) A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Naseer, N., Shapiro, J.A., and Chander, M. (2014) RNA-Seq analysis reveals a six-gene SoxR regulon in Streptomyces coelicolor. PLoS ONE 9: e106181. Nunoshiba, T., Hidalgo, E., Amabile Cuevas, C.F., and Demple, B. (1992) Two-stage control of an oxidative stress regulon: the Escherichia coli SoxR protein triggers redoxinducible expression of the soxS regulatory gene. J Bacteriol 174: 6054–6060. Okegbe, C., Sakhtah, H., Sekedat, M.D., Price-Whelan, A., and Dietrich, L.E. (2012) Redox eustress: roles for redoxactive metabolites in bacterial signaling and behavior. Antioxid Redox Signal 16: 658–667. Palma, M., Zurita, J., Ferreras, J.A., Worgall, S., Larone, D.H., Shi, L., et al. (2005) Pseudomonas aeruginosa SoxR does not conform to the archetypal paradigm for SoxRdependent regulation of the bacterial oxidative stress adaptive response. Infect Immun 73: 2958–2966. Perrin, B.S., Jr, and Ichiye, T. (2013) Identifying sequence determinants of reduction potentials of metalloproteins. J Biol Inorg Chem 18: 599–608. Pierson, L.S., 3rd, and Pierson, E.A. (2010) Metabolism and function of phenazines in bacteria: impacts on the behavior © 2015 John Wiley & Sons Ltd, Molecular Microbiology, 97, 808–821

SoxR activation by redox-active compounds 821

of bacteria in the environment and biotechnological processes. Appl Microbiol Biotechnol 86: 1659–1670. Pomposiello, P.J., Bennik, M.H., and Demple, B. (2001) Genome-wide transcriptional profiling of the Escherichia coli responses to superoxide stress and sodium salicylate. J Bacteriol 183: 3890–3902. Sheplock, R., Recinos, D.A., Mackow, N., Dietrich, L.E., and Chander, M. (2013) Species-specific residues calibrate SoxR sensitivity to redox-active molecules. Mol Microbiol 87: 368–381. Shin, J.H., Singh, A.K., Cheon, D.J., and Roe, J.H. (2011) Activation of the SoxR regulon in Streptomyces coelicolor by the extracellular form of the pigmented antibiotic actinorhodin. J Bacteriol 193: 75–81. Singh, A.K., Shin, J.H., Lee, K.L., Imlay, J.A., and Roe, J.H. (2013) Comparative study of SoxR activation by redoxactive compounds. Mol Microbiol 90: 983–996. Steckhan, E., and Kuwana, T. (1974) Spectrochemical study of mediators. I bipyridylium salts and their electron transfer rates to cytochrome c. Ber Bunsenges Phys Chem 78: 253–259. Thompson, J.D., Higgins, D.G., and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting,

© 2015 John Wiley & Sons Ltd, Molecular Microbiology, 97, 808–821

position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673–4680. Tsaneva, I.R., and Weiss, B. (1990) soxR, a locus governing a superoxide response regulon in Escherichia coli K-12. J Bacteriol 172: 4197–4205. Watanabe, S., Kita, A., Kobayashi, K., and Miki, K. (2008) Crystal structure of the [2Fe-2S] oxidative-stress sensor SoxR bound to DNA. Proc Natl Acad Sci USA 105: 4121– 4126. Wheeler, T.J., Clements, J., and Finn, R.D. (2014) Skylign: a tool for creating informative, interactive logos representing sequence alignments and profile hidden Markov models. BMC Bioinformatics 15: 7. Wu, J., and Weiss, B. (1991) Two divergently transcribed genes, soxR and soxS, control a superoxide response regulon of Escherichia coli. J Bacteriol 173: 2864–2871. Wu, J., and Weiss, B. (1992) Two-stage induction of the soxRS (superoxide response) regulon of Escherichia coli. J Bacteriol 174: 3915–3920.

Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web-site.