The E. coli K-12 strains and plasmids used in this study are described in Table 1. Bacterial cultures were grown in aerobic conditions at 37°C in M9 minimal salt medium (Sigma-Aldrich) supplemented with 0.2% casamino acids, 0.2% glucose, 1 mM MgSO₄, and 1 mg/ml vitamin B1. Solid media contained 1.5% agar. For growth curves, culture aliquots were taken at different times and OD at 600 nm was measured. Anaerobic environments were generated in a Gaspak jar system using AnaeroGen sachets (OXOID, Sigma-Aldrich). When required, antibiotics (Kanamycin 50 μg/ml, Ampicilin 50 μg/ml) were added to media.

Plasmid DNA was isolated using the Wizard Miniprep DNA purification system (Promega) according to the manufacturer’s instructions. Transformation of competent cells using the CaCl₂ procedure was performed as described previously (Sambrook et al., 1989).

The β-Galactosidase activities were determined following the method described by Zhou and Gottesman (1998), with a few modifications. Strain carrying the entE::lacZY transcriptional fusion was grown for 6 h in M9 medium at 37°C with aeration in presence or not (control) of 25 and 50 μM of CuSO₄, FeCl₃ or a combination of both metals, and assayed for β-Galactosidase activities. For this, 600 μl of these cultures were permeabilized with 0.1% SDS (24 μl) and chloroform (48 μl) for 20 min. Then, 100 μl of permeabilized cells were placed on 96-well microtiter plate and 100 μl of a 1.32 mg/ml solution of 2-nitrophenyl-β-D-galactopyranoside in buffer Z was added. Finally, the absorbance at 420 nm was measured for 20 min in a SpectraMax 250 spectrophotometer. Specific activity was calculated by dividing the slope of the line over time by the corresponding OD_600nm and expressed as arbitrary units (AU).

In vitro copper reducing power of spent media obtained from different E. coli strains was measured using bathocuproine disulfonate (BCDS) (Sigma–Aldrich), following the method described by Volentini et al. (2011). This chelator forms a colored complex with cuprous ions, having a λmax at 480 nm (Poillon and Dawson, 1963). Cells were grown for 18 h at 37°C in M9 medium. Then, supernatants were filter sterilized and incubated with 100 μM CuSO₄ and 200 μM BCDS. The absorbance at 480 nm was determined and Cu⁺ concentrations were calculated by comparison to a standard curve obtained in M9 medium supplemented with different Cu²⁺ concentrations and 25 mM ascorbic acid as a reducing agent.

Minimal inhibitory concentrations (MIC) were determined in M9 medium supplemented with 0.2% casamino acids, 0.2% glucose, 1 mM MgSO4 and 1 μg/ml vitamin B1. 5 μl of double dilutions from a 30 mM CuSO₄ solution were spotted on M9 agar plates, dried and a lawn of the corresponding strain was overlaid. The plates were incubated at 37°C for 24 h under aerobic conditions or for 48 h under anaerobic conditions. MIC values were determined as the maximum dilution of CuSO₄ that showed a zone of clearing. Enterobactin (ENT), catalase (CAT) and FeCl₃ were used at concentrations of 1 μM, 1 mg/ml and 100 μM, respectively.

Cu-inhibition assays were performed using overnight cultures of the wild-type or the indicated mutant strains diluted 1:200 into fresh M9 medium supplemented with 0.2% casamino acids, 0.2% glucose, 1 mM MgSO₄, 1 mg/ml vitamin B1 and containing either 0, 25, 50, or 100 μM of CuSO₄. Experiments were done in a final volume of 10 ml. OD _600nm was followed over a 24 h-period of time for growth curves. For the evaluation of the impact of incremental concentrations of enterobactin on copper toxicity, overnight cultures of the entE strain were diluted 1:200 into fresh M9 medium supplemented with 0.2% casamino acids, 0.2% glucose, 1 mM MgSO4 and 1 mg/ml vitamin B1, containing 100 μM of CuSO₄ and 0, 10, 20, or 50 μM of pure enterobactin. Experiments were done in a final volume of 200 μl. Cultures were incubated at 37°C with shaking, and bacterial viability as CFU/ml in LBA was determined at different times (0, 3, 18, and 24 h). At 24 h, Cu⁺ concentration was determined in the supernatants using BCDS (Sigma–Aldrich), following the method described above.

Catechol siderophore production was estimated according to the colorimetric method described by Arnow (1937). Briefly, cells were grown for 20 h at 37°C in M9 medium and supernatants filter sterilized. Then, 1 ml 0.5 N HCl, 1 ml nitrite-molybdate (1 g sodium nitrite and 1 g sodium molybdate dissolved in 10 ml water), 1 ml 1 N NaOH and 1 ml H₂O were added to 1 ml of conditioned medium. Finally, the absorbance at 510 nm was determined and values were expressed as μM equivalents by comparison to a standard curve prepared using 2,3-dihydroxybenzoic acid (DHBA) (Sigma).

ROS levels were measured using the oxidation-sensitive fluorescent dye 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). DCFH-DA is a cell-permeable dye widely used for the detection of ROS. This probe is enzymatically hydrolyzed by the action of cellular esterases to DCFH and then oxidized by the action of intracellular ROS (species such as hydrogen peroxide, hydroxyl radical, peroxyl radicals and nitrogen radicals) into 2′,7′-dichlorofluorescein (DCF), a molecule highly fluorescent that can be easily detected at 530 nm when excited at 485 nm (Gomes et al., 2005). Cells grown in M9 medium with or without 25 μM CuSO₄ and 1 μM of pure enterobactin, were washed twice and resuspended in 50 mM sodium phosphate buffer, pH 7 at a final OD_600nm = 0.5. Then DCFH-DA was added at a final concentration of 10 μM and incubated for 30 min in darkness. After incubation, cells were washed, resuspended and sonicated in the same buffer. Fluorescence intensity was measured using a Perkin Elmer LS55 spectrofluorometer (excitation λ, 490 nm; emission λ, 519 nm) and results are expressed as relative fluorescence to that of the control.

Results are expressed as the mean ± standard deviation (SD). The statistical analysis of the data was performed using the InfoStat statistical software (InfoStat versión 2020, http://www.infostat.com.ar). Analysis of variance (ANOVA) using a general linear model was applied to determine the effects of the factors evaluated in each analysis and significant differences (p-value < 0.05) between mean values were determined by Tukey’s test.

To test copper sensitivity in a condition with no enterobactin present, we made use of an E. coli strain harboring a mutation in the siderophore synthesis operon entCEBA (E. coli entE). Comparison of copper toxicity in E. coli entE and in its isogenic wild-type strain showed that wild-type growth in M9 medium was practically unaffected by supplementation with up to 100 μM CuSO₄ (Figure 1A), while entE growth showed a dose-dependent inhibition response (Figure 1B). Interestingly, while iron addition restored entE growth to wild-type levels, it did not revert copper toxicity (Figure 1C). To rule out a pleiotropic effect caused by the mutation, we complemented entE strain with plasmid pNTR-entE and observed that the complemented strain showed an equivalent growth to that of the wild-type strain in all copper concentrations tested (25 and 50 µM is data not shown) (Figure 1A).

Next, we explored the possibility that the observed phenotype in the mutant strain was a consequence of an altered metabolic flux caused by the blockade in enterobactin biosynthesis pathway. Catechols found in wild-type strain supernatants are primarily enterobactin and biosynthetic intermediates in fewer amounts (Winkelmann et al., 1994). While disruption of enterobactin biosynthesis pathway in entE strain causes the absence of enterobactin and would favor the accumulation of the catechol precursor 2,3-dihydroxybenzoic acid (DHBA) (Cox et al., 1970; Pollack et al., 1970; Crosa and Walsh, 2002; Raymond et al., 2003; Ma and Payne, 2012), entA and entB mutants, by blocking enterobactin synthesis at an earlier biosynthesis step, produce no catecholate intermediates (Supplementary Figure S1). Thus, we analyzed whether there was a correlation between copper sensitivity and copper reduction capacity of supernatants having different catechol levels. Compared with wild-type, entE supernatant had higher catechol content and higher copper reducing ability (Figures 2A,B). As expected, entA and entB supernatants had negligible catechol content and consequently little copper reduction power which was about 5–7 fold lower compared to that of the entE strain (Figures 2A,B). Next, we evaluated copper sensitivity for the mutants (entA, entB, and entE) determining the copper minimal inhibitory concentration (MIC) for each strain. Table 2 shows that entA and entB strains had the same sensitivity to copper than entE strain which was significantly higher than that of the wild-type strain. Results thus far indicate that the increased sensitivity of mutants impaired in enterobactin synthesis stemmed from the lack of the siderophore and not from a pleiotropic effect or a metabolic shift due to the mutations.

To test the role of enterobactin in copper toxicity, we supplemented media with pure siderophore in a concentration of 1 μM which is in the range of the physiological concentration found in our experimental settings (Figure 2A). Table 2 shows that 1 μM of enterobactin added to entE, entA, and entB cultures rendered these strains equally sensitive to copper as the wild-type strain. Based on previous findings associating enterobactin with oxidative stress protection (Peralta et al., 2016) and the observation that iron supplementation did not reduce copper toxicity considerably (Table 2; Figure 1C), we assumed that enterobactin would protect against copper toxicity by reducing oxidative stress rather than facilitating iron uptake. Hence, we measured ROS levels in wild-type and entE strains in presence of copper and enterobactin. Equally to what we previously reported (Adler et al., 2014), entE strain grown in M9 with no copper or other stressor added, shows increased ROS levels compared with the isogenic strain (Figure 3 left panel). While medium supplementation with sublethal copper concentrations (25 μM of CuSO₄) further increased ROS levels in the mutant strain, ROS levels remained constant in the wild-type (Figure 3 central panel). Then, when medium was supplemented with 25 μM of CuSO₄ and 1 μM enterobactin, ROS levels dropped only for the entE mutant, reaching levels equal to those of the wild-type (Figure 3 right panel). Next, we tested the impact of medium supplementation with catalase on copper toxicity and found that the enzyme evenly reduced the sensitivity of entE and entB to CuSO₄ (Table 2). To further characterize the role of enterobactin, we studied the siderophore effect on copper toxicity under anaerobic conditions. We found that in these conditions the wild-type, entE and entB strains had the same sensitivity to copper and that the addition of 1 μM enterobactin to the medium did not protect entE mutant from copper toxicity (Table 2). Our observations imply enterobactin in reducing copper toxicity only under aerobic conditions, being this consistent with the proposed role as a ROS scavenger.

Previously, we demonstrated that in order to reduce the oxidative stress caused by pyochelin, paraquat or H₂O₂, enterobactin has to reach the cell cytoplasm and be hydrolyzed by the esterase Fes. Then, the hydroxyl groups in the catechol moieties are freed from iron coordination and are able to directly stabilize radicals (Peralta et al., 2016). We hypothesized that enterobactin, once linearized in the cell cytoplasm would also protect E. coli against copper-generated ROS. To address this hypothesis, we determined copper MIC for E. coli mutant strains in enterobactin uptake (fepG and fepB) and enterobactin hydrolysis (fes) in media supplemented with pure enterobactin. Table 2 shows that mutant strains fepG, fepB and fes were also significantly more sensitive to copper damage when compared to the isogenic wild-type strain. Unlike with strains impaired in enterobactin synthesis (entA, entB, and entE), addition of 1 μM enterobactin to the medium did not revert copper sensitivity of fepG, fepB, and fes mutants (Table 2). Consistently, addition of copper to fepG and fes cultures increased ROS levels and supplementation with 1 μM enterobactin did not reduce this increment (Supplementary Figure S2). As the increased copper toxicity of fepG, fepB, and fes mutants was correlated with augmented ROS and since both variables were unresponsive to enterobactin addition, the hydrolyzed form of the siderophore results as the candidate molecule responsible for ROS reduction.

Regulation of enterobactin synthesis by oxidative stress would be consistent with its proposed antioxidant role (Adler et al., 2014; Peralta et al., 2016). In fact, in this work we show that the entE::lacZY chromosomal fusion activity (Figure 4A) and the catechol content in supernatants of the wild-type strain (Figure 4B), were significantly higher in aerobic culture conditions compared with anaerobic ones. Next, we wondered whether copper would also be able to induce enterobactin expression. For this, we used the same strain with the chromosomal entE::lacZY transcriptional fusion and grew it in M9 medium with the addition of CuSO₄ (see materials and methods). Compared with the control with no copper added, medium supplementation with 25 μM or 50 μM of CuSO₄ significantly increased entE transcriptional expression in a dose-dependent manner (Figure 5). As expected, addition of 25 μM or 50 μM of FeCl₃ with no copper added, highly repressed the expression of enterobactin (Figure 5). However, when medium was supplemented with both metals (copper and iron), entE transcription was only repressed partially (Figure 5). By measuring the activity of the Fur regulated promoter ryhB in the entE strain, we proved that iron availability was not affected by the addition of copper to the medium (data not shown). Evidence showed that aerobic culture conditions and copper, triggered enterobactin transcription thus further supporting the involvement of the siderophore in oxidative stress protection.

After establishing that physiological concentrations of enterobactin, at least those found in our experimental conditions (Figure 2), protected against oxidative stress (Table 2; Figure 3), we decided to perform a dose-response curve for enterobactin concentrations in presence of copper. For that, we measured CFU/mL at different time points of entE cultures supplemented with 100 μM CuSO₄ and with increasing enterobactin concentrations (1, 10, 20, and 50 μM enterobactin). As controls, we used entE cultures with no enterobactin added in the presence and absence of copper.

As observed before (Figure 1), entE mutant with no enterobactin added showed higher copper sensitivity (log CFU/mL at 24 h of 11.89 vs. 8.69 for entE and entE + 100 μM CuSO₄, respectively) (Figure 6A). When enterobactin was added in relatively low concentrations (1–10 μM), copper toxicity was clearly reduced at all time points measured. Medium enterobactin concentration (20 μM) still protected from copper toxicity at 18 and 24 h but to a lesser extent compared with 1 and 10 μM enterobactin. Finally, a high enterobactin concentration (50 μM) resulted radically harmful for E. coli cells (Figure 6A). As a control for this condition, we supplemented media with 50 µM enterobactin with no addition of copper and found an entE strain growth of 11.75 log CFU/ml at 24 h. Analysis of Cu⁺ levels in supernatants of 24 h old cultures, revealed a substantial increment of Cu⁺ in the condition having 50 μM of enterobactin compared with no enterobactin or 1, 10, or 20 μM enterobactin (Figure 6B). It is likely that low enterobactin concentrations protected cells from copper toxicity by reducing ROS and that higher concentrations of the siderophore were detrimental to cells because of the Cu²⁺ to Cu⁺ reduction which increases copper toxicity.