Structural and Functional Characterization of ScsC,a Periplasmic Thioredoxin-Like Protein from Salmonella enterica Serovar Typhimurium

Three-dimensional architecture of ScsC: ScsC is a soluble TRX-like protein

StScsC was crystallized in the monoclinic space group P2₁, the crystals diffracted to 2.04 Å and contained four molecules in the asymmetric unit. The structure was solved by molecular replacement using the TRX-like protein from Silicibacter pomeroyi (PDB: 3GYK) as a model. After subsequent building and refinement, the structure of StScsC corresponding to residues 9–186 of the processed peptide (178 residues) was refined to a final R _factor of 19.9 (R _free=25.3) (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/ars). Pairwise comparison of the four symmetrically independent monomers showed no significant differences between molecules in the asymmetric unit; all following structural comparisons were performed with monomer A.

The architecture of StScsC comprises all structural hallmarks that characterize canonical thiol oxidases like StDsbA [PDB: 3L9S (25)]; it consists of a TRX domain (residues 38–75 form the βαβ motif and residues 141 to 189 form the connecting helix and the ββα motif) and a long helical insertion (residues 75–140) (Fig. 1A, B). Despite the overall structural similarity, superposition of these two proteins is poor with an r.m.s.d. value of 3.3 Å for 128 of the 189 Cα aligned. This poor match is primarily due to the different topology of StScsC, where the core β-strands (β2–β5) are structurally equivalent to those of StDsbA, but the strand β1 is hydrogen bonded to the opposite edge of the central β-sheet in the TRX domain (Figs. 1B & 2A). Another notable discrepancy between StScsC and StDsbA resides in the helical domain inserted in the TRX fold. In StDsbA, this region consists of a three helical bundle (α2–α4), plus an additional helix (α5) and an extension of the connecting helix α6, while in StScsC, a deletion in this section results in the loss of the α5 helix, which is substituted by a comparatively short loop (Fig. 1A, B). Additionally, superposition of StScsC with StDsbA (Fig. 2A) highlights a shorter loop linking β5 to α7 and a shorter α7 helix (by 1.5 turns) in StScsC compared to StDsbA. Moreover, unlike StDsbA, the StScsC β5 strand and β5-α7 loop map near the active site, a position that is stabilized by hydrogen bonds between Ile¹⁶⁶ (O and NH) and Thr¹⁵⁹ (NH and O) and Gly¹⁶⁸ (NH) and cis-Pro¹⁵⁷ (Supplementary Fig. S1). The latter modifications in StScsC alter the size and shape of the groove adjacent to the active site, which in prototypical DsbAs is defined as the hydrophobic peptide-binding groove, which binds to the redox partner DsbB (31). Other minor differences between StScsC and StDsbA include a less pronounced kink in α1 of StScsC, and longer loops linking α2, α3, and α4 in StDsbA.

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FIG. 1.

Secondary structural elements of ScsC protein of Salmonella Typhimurium (StScsC) and structural homologues. (A) Structure-based sequence alignment of StScsC, BdbD from Bacillus subtilis (BsBsbD), DsbG from Escherichia coli (EcDsbG), and DsbA from S. Typhimurium (StDsbA). Cleavage site is depicted by a gray triangle, and conserved active site residues are highlighted in yellow and green. (B) Three-dimensional structures of StScsC (PDB: 4GXZ), BsBsbD [PDB: 3GHA (11)], the thioredoxin (TRX) domain of EcDsbG [PDB: 1V58 (23)], and StDsbA [PDB: 3L9S (25)]. The TRX-fold and N-terminus are shown in tan and black, respectively. Secondary structural features are labeled with the active site sulfur atoms shown as yellow spheres.

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FIG. 2.

Superpositions of StScsC and structural homologues. α-carbon trace stereo views of StScsC (black) superposed with (A) StDsbA (cyan), (B) BsBdbD (blue), and (C) the TRX domain of EcDsbG (orange). α5 indicates the position of the fifth alpha helix in the inserted helical domain of DsbA-like proteins StDsbA and BsBdbD. Structures in the left panel are in the same orientation as those in Fig 1B, and in the right panel, all three structures are rotated clockwise 90° around the vertical axis, highlighting the extended loop between α3 and α4 in BsBdbD (indicated with blue asterisk).

The Dali search (29) identified 3GYK, BdbD (PDB code 3GHA), and DsbG (PDB code 1V58) as the closest structural homologues to StScsC. 3GYK and StScsC exhibit high structural similarity (r.m.s.d. value of 1.5 Å over 165 Cα aligned); this protein is a DsbA-like homologue from Silicibacter pomeroyi although no functional characterization or publication is available. Structural superposition of StScsC with Bacillus subtilis DsbA protein BsBdbD (11) (r.m.s.d. value of 2.0 Å for 148 Cα aligned) highlights features of StScsC that deviate from this close structural homologue. For example, although BsBdbD and StScsC share a similar topology (β1 interacts with β3 instead of β5), the α5 helix is present in the helical domain of BsBdbD (Fig. 1B, 2B). Rotation of these structures by 90^o also shows a longer loop connecting BsBdbD α3 and α4, which protrudes into the catalytic site (Fig. 2B). StScsC also shows structural similarity with DsbG, a disulfide isomerase from E. coli (23), which has also been shown to control cysteine sulfenylation in the oxidizing periplasm (13). EcDsbG is a homodimer, where each monomer incorporates a dimerization domain and a TRX domain linked by a connecting helix. StScsC superimposes unexpectedly well with the TRX domain of EcDsbG (2.3 Å for 134 Cα aligned, Fig. 2C). Furthermore, the lack of α5 in StScsC appears to emulate the shorter insertion present in this reductase, which only contains two helices and a long loop embedded in the TRX fold (23).

Analysis of surface electrostatics of StScsC

The electrostatic surface of StScsC partially resembles that of the thiol oxidase StDsbA, displaying a hydrophobic peptide-binding groove running along the protein surface and a hydrophobic patch, both adjacent to the CPYC catalytic motif (Fig. 3A). However, compared with StDsbA (Fig. 3B), the hydrophobic groove in StScsC is truncated. Moreover, next to the hydrophobic patch, StScsC displays a positively charged ridge above the exposed cysteine, comprising Lys⁵² on α1, Lys⁷⁸ on the loop between β3 and α2 and Arg¹¹¹ in the α3-α4 loop that overhangs the entrance to the active site cysteines (Fig. 3A). This cluster of positive residues surrounding the catalytic site is likely to stabilize the thiolate form of the nucleophilic cysteine Cys⁴⁸. Interestingly, EcDsbG also has a positively charged ridge above the active site, with Lys¹³⁹ on the loop between β3 and α2 directly above the exposed cysteine, although not as close as the Lys⁷⁸ of StScsC. Lys¹⁶⁸ (loop between α3 and α6) and Lys¹¹³ (α1) provide the additional positive charge to the right of Lys¹³⁹ of EcDsbG (Fig. 3C).

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FIG. 3.

Electrostatic surfaces of StScsC and structural homologues. Positive (blue), negative (red), and hydrophobic (white) surfaces (saturation at 20 kT/e) of (A) ScsC from S. Typhimurium, (B) DsbA from S. Typhimurium, (C) the TRX domain of DsbG form E. coli, and (D) BdbD from B. subtilis. Surface is omitted from the exposed active site cysteine residues to reveal the S atoms (shown as yellow spheres). Structures in the left panel are in the same orientation as those in Fig 1B.

Although BsBdbD is the closest structural homologue for StScsC, these two proteins are not closely related (24% sequence identity), which results in discrepancies in their surface properties. Overall, BsBdbD displays a more acidic surface, primarily in areas surrounding the catalytic CPSC motif (Fig. 3A–D). The long loop between α3 and α4 is lined with negatively charged residues that map on top of the active site (11) (Fig. 3D). Despite their different surface properties, both StScsC and BdBdbD have acidic patches in the groove between the TRX and the helical domain, which map at the opposite side of their active sites, (Fig. 3, right panels). This acidic patch is conserved in other DsbA homologues although its exact role remains unclear (40).

The StScsC redox active site

The catalytic active site of StScsC resembles that of the disulfide isomerase EcDsbG rather than the oxidase StDsbA, it consists of a ⁴⁸CPYC⁵¹ motif located at the N-terminus of helix α1 and a cis-Pro loop (¹⁵⁶Thr-¹⁵⁷ cisPro) in the α6-β4 connecting loop (Fig. 4A). StScsC was crystallized in its reduced form, as cleavage of the His-tag with tobacco etch virus (TEV) protease before crystallization involved incubation with 1 mM dithiothreitol (DTT). As shown in Figure 4B, the StScsC cysteine sulfur atoms are separated by a distance of 3.2 Å, which clearly indicates that these residues remained in their free thiol/thiolate forms upon crystallization. Several interactions may stabilize the reduced form of this protein; the nucleophilic N-terminal Cys⁴⁸ Sγ is within hydrogen bond distance of the main chain nitrogen of the second cysteine (3.5 Å) and the side chain hydroxyl of Thr¹⁵⁶ (3.5 Å). Furthermore, the protruding lysine residue (Lys⁷⁸) forms a positively charged lip that overhangs the access route to the Cys⁴⁸ residue. The close proximity between Lys⁷⁸ and Cys⁴⁸ could promote stabilization of the Cys⁴⁸ thiolate, following a conformational rearrangement in this region; the Cδ atom of Lys⁷⁸ is 3.9 Å away from the Cα atom of Cys⁴⁸, but the Nζ of Lys⁷⁸ is 6.6 Å from the Sγ of Cys⁴⁸, so a conformational rearrangement would be required for this interaction to provide a significant stabilizing effect (Fig. 4A). The catalytic sites of BsBdbD and EcDsbG also reveal hydrogen bonds between the Sγ of the first cysteine and two acceptor groups: the second cysteine mainchain NH and the hydroxyl group of the Thr preceding the cis-Pro (Fig. 4C). The residues corresponding to StScsC Lys⁷⁸ in BsBdbD and EcDsbG are His¹⁰³ and Lys¹³⁹, respectively. In BsBdbD, the Nδ2 of His¹⁰³ is 4.8 Å away from the Sγ of Cys⁶⁹, although a histidine residue is less likely to be cationic at physiological pH compared to a lysine residue, and is therefore less able to stabilize a cysteine thiolate. In EcDsbG, Lys¹³⁹ generates a basic patch near the active site, which may also contribute to stabilizing the thiolate form of the cysteine (Fig. 4C).

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FIG. 4.

Active site regions of StScsC and structural homologues. (A) Active sites of StScsC showing the catalytic Cys-Pro-Tyr-Cys motif, the adjacent cisPro loop (Thr¹⁵⁶-cisPro¹⁵⁷) and the sidechain of Lys⁷⁸, which protrudes into the solvent to form a positively charged lip over the active site. The 2F_o-F_c map is also shown contoured at 1.3 σ. (B) Close-up view of the StScsC active site, refined in the reduced form (2Fo-Fc maps contoured at 1.3 σ). (C) Active site regions of StScsC, BsBdbD, EcDsbG, and StDsbA showing the hydrogen bond interactions stabilizing the side chain of the nucleophilic cysteine in each active site. Hydrogen bonds are depicted by dashed lines.

Biochemical characterization of StScsC

The biochemical properties of StScsC were characterized to gain further insights into its potential function. The oxidizing/reducing power of StScsC was quantified by measuring the equilibrium constant (K _eq) for the disulfide exchange reaction with glutathione via monitoring changes in NMR spectra (Fig. 5A). The measured constants for StScsC and DsbA protein of E. coli (EcDsbA) were 21.7±2.6×10⁻⁵ M and 12.9±2.1×10⁻⁵ M, respectively. These K _eq values convert into intrinsic redox potential values of −132 mV and −125 mV, respectively (Table 1). The redox midpoint of our EcDsbA-positive control is in close agreement with previously measured values for this protein (3). In addition, control spectra were recorded for the fully oxidized protein (using 5 mM oxidized glutathione [GSSG]) and for the fully reduced protein (using 5 mM DTT), providing confidence for the choice of peaks used to monitor the redox transition.

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FIG. 5.

Biochemical characterization of StScsC. (A) Redox equilibria of EcDsbA and StScsC with glutathione. The fraction of oxidized and reduced EcDsbA and StScsC was determined using NMR spectroscopy and these data were used to calculate K _eq and redox potential (E^o′) as previously described (42). K _eq(StScsC)=21.7±2.6×10⁻⁵ M, E^o′(StScsC)=−132 mV, K _eq(DsbA)=12.9±2.1×10⁻⁵ M, E^o′(DsbA)=−125 mV. (B) Determination of the pK _a of the nucleophilic cysteine in StScsC. The pH dependence of the thiolate-specific absorbance signal (S=(A240/A280) reduced/(A240/A280) oxidized) was fitted according to the Henderson–Hasselbalch equation (the oxidized proteins were used as a reference). pK _a (StScsC)=3.38±0.03. (C) Catalyzed reduction of insulin. Reduction of insulin (131 μM) was measured in a 0.1 M phosphate buffer, pH 7.0, 2 mM EDTA. The assay was performed in the absence or presence of 10 μM StScsC and 10 μM EcDsbA was used as a positive control. At time zero, DTT was added to each reaction to a final concentration of 0.35 mM, and the catalyzed reduction of insulin was measured as an increase in absorbance at 650 nm. The maximal rates have been fitted using linear regression. (D) Determination of the in vivo redox state of StScsC. (i) The fully reduced (R) and fully oxidized (O) purified StScsC is shown in lanes 1 and 2, respectively. (ii) Samples prepared from SL1344. Samples were incubated with 4-acetamino-4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) or left untreated as indicated. The shift in the migration of StScsC following treatment with AMS demonstrates it exists in a reduced form in vivo. (iii) Samples prepared from SL1344scsC pScs. Samples were incubated with AMS or left untreated as indicated. The use of a plasmid containing the scsC gene resulted in increased expression of StScsC. As observed in the wild-type strain, shift in the migration of StScsC following treatment with AMS demonstrates it exists in a reduced form in vivo. Lane M on panels A, B, and C correspond to the 30 and 20 kDa bands of the ladder Novex Sharp protein standard (Life Technologies).

The first of the two cysteines in the CXXC motif of TRX-like proteins is a solvent-exposed, reactive nucleophilic cysteine. This reactivity is partly due to a lower pK _a (∼3.5–5) compared with most cysteines (∼8.5) (43). The pK _a of StScsC was measured as 3.4 (Fig. 5B), an unusually low value for a CXXC motif with such reducing power. Indeed, this is comparable to that of the oxidizing exposed thiol of EcDsbA [pK _a=3.5 (43), Table 1].

Given the redox potential exhibited by StScsC is similar to that of disulfide isomerases (Table 1) and its low pK _a, which is also common in highly oxidizing proteins, we investigated the in vitro oxidoreductase activity using the classic insulin reduction assay: most TRX-like oxidoreductases, including DsbC and DsbA, catalyze DTT-induced insulin reduction. StScsC catalyzed the reduction of insulin more slowly than EcDsbA, and showed an activity ∼71% of the maximal rate of EcDsbA (Fig. 5C). To further understand the function of StScsC, we also investigated its ability to catalyze Dsb isomerization using the scrambled RNaseA (scRNaseA) refolding assay (27). Under our conditions, StScsC did not show any activity in this assay (Supplementary Fig. S2). In addition, to experimentally verify that StScsC is able to perform disulfide reduction in the native host, the in vivo redox status was measured using the thiol modification reagent 4-acetamino-4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) (Fig. 5D). Alkylation assays clearly indicate that StScsC is predominantly in the reduced state in the periplasm of S. Typhimurium.

Loss of StScsC elicits copper sensitivity in S. Typhimurium

To examine the role of StScsC in S. Typhimurium, we constructed a scsC mutant in SL1344 (referred to as SL1344scsC). Loss of StScsC expression in SL1344scsC was confirmed by Western blot analysis with a StScsC-specific antibody (data not shown). Wild-type SL1344 and SL1344scsC were assessed for copper sensitivity using disk diffusion assays. In these assays, SL1344scsC displayed enhanced sensitivity to copper, as evidenced by the larger zones of growth inhibition observed in the presence of copper (Fig. 6A). Reintroduction of the scs genes on plasmid pScs restored the wild-type phenotype, confirming a role for StScsC in resistance to copper. To confirm this role for StScsC in S. Typhimurium and to more accurately assess the concentration of copper that inhibited growth of SL1344scsC, wild-type SL1344 and SL1344scsC strains were assessed for copper sensitivity by spotting a series of 10-fold dilutions of overnight cultures onto agar plates containing a range of different copper concentrations. In these assays, wild-type SL1344 and SL1344scsC displayed significantly different sensitivity to 2.8 mM copper (Fig. 6B). Reintroduction of the scs genes on plasmid pScs restored the wild-type phenotype, confirming a role for StScsC in resistance to copper.

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FIG. 6.

ScsC is required for S . Typhimurium copper resistance in vitro . (A) Disk diffusion assays of wild-type S. Typhimurium SL1344, SL1344scsC and SL1344scsC complemented with plasmid pScs. Sterile filter disks were placed on bacterial lawns and treated with 5 μl CuSO₄ (1 M). The data presented are representative of five independent repeats. (B) Copper sensitivity and complementation of S. Typhimurium SL1344 scsC mutant was tested by spotting a series of 10-fold dilutions of cultures of the same density onto Tris agar plates containing increasing amounts of copper. Pictures were taken after a 24-h incubation at 37°C in the presence (right panel) or absence (left panel) of 2.8 mM CuSO₄. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Production of purified proteins

Native ScScsC was expressed using a method similar to the one descried previously for other S. Typhimurium DsbA-like proteins (32). Briefly, the coding DNA sequence for ScScsC lacking the signal peptide (residues 19–189), was cloned into a pET21a-based LIC vector (for cloning primers, see Supplementary Table S1). The recombinant protein containing an N-terminal hexa-histidine tag was expressed using autoinduction (54), and then purified by Ni²⁺-affinity chromatography and gel filtration chromatography (in 50 mM HEPES pH 7.0, 150 mM NaCl). Upon removal of the tagged signal peptide by cleavage with TEV protease (in a gel filtration buffer supplemented with 1 mM DTT), uncleaved protein was removed using cobalt affinity (Talon resin) chromatography. Histidine-tagged E. coli DsbA (EcDsbA) was purified as previously described (48).

Crystallization and diffraction data collection

Purified StScsC (in 50 mM HEPES pH 7.0, 150 mM NaCl) was stored in aliquots frozen at −80°C until needed. StScsC was concentrated using a 3K Amicon centrifugal concentrator up to 26 mg·ml⁻¹. High-throughput crystallization experiments were performed with a Mosquito robot using commercial screening kits. Initial condition screening was performed in the hanging-drop format in 96-well plates, StScsC was at 26 mg·ml⁻¹ and the commercial screens JCSG (Molecular Dimensions), Index (Hampton), and PegRx1/PegRx2 (Hampton) were used. Drops were set using the Mosquito^® robot (TTP LabTech Ltd.) by mixing 100 nL of protein with 100 nL of well condition. The plates were incubated at 20°C and monitored and scored using the RockImager and Rockmaker system (Formulatrix, Inc.). Crystals were observed in three PegRx conditions (PegRx1 #33, 0.1 M Tris pH 8.0, 28% w/v PEG 4000; PegRx2 #28, 18% v/v 2-Propanol, 0.1 M sodium citrate tribasic dihydrate pH 5.5, 20% w/v PEG 4000; PegRx2 #29, 6% v/v Tacsimate pH 6.0, 0.1 M 2-(N-morpholino)ethanesulfonic acid monohydrate pH 6.0, 25% w/v PEG 4000). Optimization using random and grid screening strategies produced crystals that diffracted to 2.04 Å. These crystals were grown in 96-well plates in gradient screens of 18%–27% (w/v) PEG 4000 and 0.1 M Tris buffer pH 8.2–9.2. A further round of optimization designed to grow larger crystals free of precipitate was performed in 24-well plates using 18%–27% (w/v) PEG 4000 with 0.1 M Tris buffer pH 8.2 with protein at 9 mg·ml⁻¹ using microseeding. The hanging drops were prepared by adding 1 μl of protein (9 mg·ml⁻¹) to 1 μl of well condition and were microseeded within 30 min of setup with a seed stock prepared from small crystals grown in the same condition. Diffraction data were collected on a crystal (∼300×50×50 μm) grown by this method (Supplementary Fig. S4) with a well condition of 22% (w/v) PEG 4000 in 0.1 M Tris buffer pH 8.2. The crystal was cryoprotected by the well solution supplemented with 25% ethylene glycol and flash cooled in the cryostream. Data were collected using a Rigaku FR-E Superbright X-ray generator with Osmic HiRes2 optics and a Rigaku R-Axis IV++ Image Plate detector. CrystalClear 2.0 was used to record the diffraction and indexing and scaling was performed using HKL2000 (45).

Structure determination

The crystal structure of StScsC was solved by molecular replacement using BALBES (39) with a final search model based on the crystal structure of a TRX-like oxidoreductase from Silicibacter pomeroyi (PDB: 3GYK): Model building and refinement were carried out iteratively using COOT (16) and Phenix (2). Structure validation was performed using Molprobity (8). Supplementary Table S1 provides the statistics for the X-ray data collection and final refined model. Superposition of molecules and generation of molecular figures (including electrostatic potential) were carried out using the CCP4 Molecular Graphics program (47).

Bioinformatic analyses

Pairwise sequence alignments and calculation of sequence identities were performed using Biolign (21). Structural alignments were performed using MUSTANG (34), and the secondary structure was assigned using PDBsum.

Determination of the equilibrium constants with glutathione

The redox equilibrium of StScsC with glutathione was determined alongside that of EcDsbA as a positive control, using a recently developed NMR approach (56) (using his-tagged proteins). To determine the reduction potentials, different ratios of [reduced glutathione (GSH)]/[GSSG] were used, where [GSH]+[GSSG] equalled 5 mM. All NMR data were obtained at 298 K using a 14.1 T (600 MHz ¹H) Bruker Avance III NMR spectrometer equipped with a QCI-F cryoprobe. All NMR samples were 330 μL within a Shigemi NMR tube and contained 100 μM protein in a 20 mM sodium phosphate buffer at pH 6.5 containing 50 mM sodium chloride and 5% (v/v) deuterium oxide. All buffers had nitrogen bubbled through to ensure the removal of any oxygen. We found that bubbling for 30–60 min was sufficient to remove all effects of dissolved oxygen and the redox equilibrium was complete in all samples within 5 min of equilibration of buffers. All ¹⁵N-¹H HSQC spectra were acquired with 2048 points in the direct F2 dimension (¹H) and 256 points in the F1 dimension (¹⁵N). NMR data processing was completed using NMRpipe. NMR backbone assignments have previously been completed for EcDsbA (10) and were used to confirm analysis. Two control spectra were recorded for each protein (Supplementary Fig. S5), the fully oxidized protein (using 5 mM GSSG) and the fully reduced protein (using 5 mM DTT). To determine the reduction potential of each protein, the fraction reduced was calculated for four and six resonances for StScsC and EcDsbA, respectively. The equilibrium constant K _eq and redox midpoint potential were determined from standard thermodynamic equations (42). A redox midpoint potential of −240 mV was used for the glutathione couple (49).

Determination of pK_a values

The pK_a of the nucleophilic cysteine in StScsC was determined by monitoring the specific absorbance of the thiolate anion at 240 nm (43). Measurements were carried out at room temperature in a buffer system consisting of 10 mM K₂HPO₄, 10 mM boric acid, 10 mM sodium succinate, 1 mM EDTA, and 200 mM KCl, pH 7.5. Oxidized and reduced protein samples (final concentration of 20 μM) were prepared by incubating the proteins with 20 mM GSSG or 10 mM DTT, respectively. Upon removal of the oxidizing and reducing agents using a PD10 column (GE Healthcare), the absorbance of the samples at 240 and 280 nm was recorded as the pH of the protein solution was lowered to 2.5 by the stepwise addition of aliquots of 0.2 M HCl. The pH dependence of the thiolate-specific absorbance signal (S=(A ₂₄₀/A ₂₈₀)_reduced/(A ₂₄₀/A ₂₈₀)_oxidized) was fitted according to the Henderson–Hasselbalch equation as previously described (42). The oxidation state of StScsC samples was assessed using the DTNB assay with an extinction coefficient of ɛ₄₁₂₌ 13.8 mM ⁻¹ cm⁻¹ (17).

Insulin reduction and refolding of scRNaseA assays

The disulfide oxidoreductase activity of StScsC and EcDsbA was determined by measuring the ability to catalyze insulin reduction in the presence of DTT as described previously (37). The in vitro isomerase activity of StScsC, EcDsbC, and EcDsbA was assessed using the refolding of scRNaseA assay essentially as described by Hillson et al. (27).

Bacterial strains, plasmids, and culture conditions

S. Typhimurium SL1344 strains (wild-type and deletion mutants) and E. coli strains (BL21[DE3] and DH5α) were routinely cultured at 37°C on the solid or in liquid Luria Bertani (LB) medium supplemented with the appropriate antibiotics; kanamycin (Km, 50 μg·ml⁻¹), ampicillin (Ap, 100 μg·ml⁻¹ or chloramphenicol (Cm, 30 μg·ml⁻¹). Culture media were supplemented with 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) to induce expression of StScsABCD from plasmid pScs. Plasmid pScs was generated by PCR amplification of the scsABCD operon (primers scsA-DF, scsA-DR) from the chromosome of SL1344 and subsequent cloning into EcoRI-XhoI digested pWSK29 (61). Expression of cloned genes was controlled by the inducible lac promoter. All primers used in this section are shown in Supplementary Table S2.

Construction of S. Typhimurium SL1344 mutants

To delete the scsC gene, a three-step PCR procedure was employed to generate an amplification product containing the Km cassette from pKD4 (12) flanked on both sides by ∼500 bp of DNA sequence homologous to the target gene to be modified. The following sets of primers were used for scsC: scsCP1F, scsCP2R, scsCP5F, and scsCP6R (Supplementary Table S2). These primers were used in combination with the Km cassette amplification primers KanP3 and KanP4 (Supplementary Table S2). The final three-step PCR product for each target gene was gel-purified and cloned into pT7Blue (Merck) according to the manufacturer's instructions, to generate a donor plasmid. SL1344 deletion mutants were constructed using the gene gorging method as previously described (26). Briefly, SL1344 cells were electrotransformed with a donor plasmid [pT7Blue::scsC::kan, and a mutagenesis plasmid (pACBSR, (26)]. Cells containing both plasmids were cultured for 9 h in the LB broth supplemented with 25 μg·ml⁻¹ Cm and 0.2% (w/v) L-arabinose to induce the λ-Red and I-SceI genes on pACBSR. Induction of these genes leads to linearization of the donor plasmid and a double recombination event with the regions flanking scsC, resulting in disruption of the respective gene. Clones sensitive to Ap and Cm (but resistant to Km) were selected and deletion of scsC was confirmed by PCR and subsequent DNA sequencing.

Copper sensitivity assays

Copper sensitivity was assessed via growth on solid media. For disk diffusion assays, strains were grown at 37°C to mid-exponential phase, adjusted to the same cell density and plated using the agar overlay technique on LB supplemented with 1.5% agar. Sterile filter disks soaked with 5 μL of 1 M CuSO₄ were placed on the center of each plate and the zones of inhibition were measured after an overnight incubation at 37°C. The data presented are representative of three independent experiments. For colony spot assays, serial 10-fold dilutions of cultures of the same density were prepared and 5 μl of each dilution was spotted on the Tris agar medium supplemented with 1.4–3.6 mM CuSO₄ (at 0.2 mM increments) or left untreated. Pictures were taken after 24 h of incubation at 37°C. The data presented are representative of three independent experiments comparing growth in the presence or absence of 2.8 mM CuSO₄.

Determination of the in vivo redox state of StScsC

The periplasmic protein fraction of SL1344 and SL1344scsC pScs was prepared by cold osmotic shock. Bacteria grown overnight at 37°C were washed in an ice-cold buffer containing 20% surcrose, 200 mM Tris HCl, pH8, and cells were harvested by centrifugation and resuspended in ice-cold 10 mM Tris HCl, pH8. After centrifugation, the supernatant was retained for analysis. The redox state of ScsC was measured by thiol alkylation using AMS and analysis of the molecular mass of proteins following sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting as previously described (30). ScsC proteins were detected using a StScsC-specific antibody (1:100) and an anti-rabbit IgG coupled with alkaline phosphatase (1:10000; Sigma) as the secondary antibody. Blots were stained with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium.