IscA/SufA paralogues are the members of the iron-sulfur cluster assembly machinery in Escherichia coli. Whereas deletion of either IscA or SufA has only a mild effect on cell growth, deletion of both IscA and SufA results in a null-growth phenotype in minimal medium under aerobic growth conditions. Here we report that cell growth of the iscA/sufA double mutant (E. coli strain in which both iscA and sufA had been in-frame-deleted) can be partially restored by supplementing with BCAAs (branched-chain amino acids) and thiamin. We further demonstrate that deletion of IscA/SufA paralogues blocks the [4Fe-4S] cluster assembly in IlvD (dihydroxyacid dehydratase) of the BCAA biosynthetic pathway in E. coli cells under aerobic conditions and that addition of the iron-bound IscA/SufA efficiently promotes the [4Fe-4S] cluster assembly in IlvD and restores the enzyme activity in vitro, suggesting that IscA/SufA may act as an iron donor for the [4Fe-4S] cluster assembly under aerobic conditions. Additional studies reveal that IscA/SufA are also required for the [4Fe-4S] cluster assembly in enzyme ThiC of the thiamin-biosynthetic pathway, aconitase B of the citrate acid cycle and endonuclease III of the DNA-base-excision-repair pathway in E. coli under aerobic conditions. Nevertheless, deletion of IscA/SufA does not significantly affect the [2Fe-2S] cluster assembly in the redox transcription factor SoxR, ferredoxin and the siderophore-iron reductase FhuF. The results suggest that the biogenesis of the [4Fe-4S] clusters and the [2Fe-2S] clusters may have distinct pathways and that IscA/SufA paralogues are essential for the [4Fe-4S] cluster assembly, but are dispensable for the [2Fe-2S] cluster assembly in E. coli under aerobic conditions.

INTRODUCTION

Iron-sulfur clusters are one of the most ancient and ubiquitous redox centres in biology. They are involved in diverse physiological processes, including respiratory electron transfer, nitrogen fixation, photosynthesis, biosynthesis of amino acids, thiamin, haem, biotin, and lipoic acid, DNA synthesis and repair, RNA modification and the regulation of gene expression [1,2]. However, the mechanism underlying the iron-sulfur-cluster assembly is still not fully understood [3]. The discovery of NifS (cysteine desulfurase) in Azotobacter vinelandii by Dean's group in the 1990s [4] led to identification of a highly conserved iron-sulfur cluster assembly gene cluster iscRSUAhscBAfdx in Escherichia coli and many other bacteria [5,6] (hsc refers to heat-shock cognate proteins A and B and fdx refers to ferrodoxin). IscR is a repressor that contains a [2Fe-2S] cluster [7]. Disassembly of the [2Fe-2S] cluster in IscR de-activates its function as a repressor and switches on expression of the gene cluster iscRSUAhscBAfdx [8,9]. IscS is a cysteine desulfurase that catalyses desulfurization of L-cysteine and transfers sulfane sulfur to a proposed iron-sulfur cluster assembly scaffold IscU [1015]. HscB and HscA specifically interact with IscU [16] and promote transfer of assembled clusters from IscU to apo-ferredoxin in an ATP-dependent reaction [17,18]. Ferredoxin, encoded by the gene fdx, contains a stable [2Fe-2S] cluster. Recent work indicated that ferredoxin could be involved in the [4Fe-4S] cluster formation in IscU [19]. For the function of IscA, at least three hypotheses have been proposed. The first hypothesis stated that IscA may act as an alternative iron-sulfur-cluster assembly scaffold, since IscA, like IscU, can host an iron-sulfur cluster and transfer the cluster to target proteins in vitro [2023]. The second hypothesis suggested that IscA may act as a regulatory protein for iron homoeostasis and redox stress responses in cyanobacterium Synechococcus sp. strain PCC 7002, as deletion of IscA and its paralogue promotes cell growth in A+ medium under iron-limitation conditions [24]. The third hypothesis stated that IscA may act as an iron donor for iron-sulfur-cluster assembly [2529]. Purified IscA from E. coli binds ferrous iron (ferrous ammonium sulfate) in the presence of dithiothreitol or the thioredoxin/thioredoxin reductase system with an iron association constant of 2.0×1019 M−1, and the iron centre in IscA can be mobilized specifically by L-cysteine [29] for the iron-sulfur cluster assembly in IscU in vitro [27,29]. Furthermore, IscA is able to recruit iron from the iron-storage protein ferritin A and transfer the iron for the iron-sulfur cluster assembly in IscU [30]. These results led us to propose that the primary function of IscA is to recruit intracellular iron and deliver iron for the biogenesis of iron-sulfur clusters [27].

IscA is highly conserved from bacteria to yeast [25], plants [31] and humans [32]. It has been reported that depletion of IscA in A. vinelandii resulted in a null-growth phenotype under elevated oxygen conditions [33]. In baker's yeast (Saccharomyces cerevisiae), deletion of the IscA homologues led to accumulation of iron in mitochondria and a dependency on lysine and glutamate for cell growth under aerobic conditions [25]. However, the mechanism underlying the IscA-deletion phenotypes has not been fully explored. In E. coli, IscA has two additional paralogues, namely ErpA and SufA. ErpA, which maps at a distance from any iron-sulfur-cluster-assembly-related genes, has been characterized as a dedicated scaffold for maturation of the key iron-sulfur enzymes in the isoprenoid-biosynthetic pathway [34]. SufA, on the other hand, is a member of the second iron-sulfur cluster assembly gene cluster sufABCDSE [35]. Purified SufA, like IscA, can bind ferrous iron (ferrous ammonium sulfate) and subsequently provide iron for the iron-sulfur cluster assembly in IscU in vitro [28]. Whereas deletion of either IscA or SufA only has a mild effect on cell growth of E. coli, deletion of both IscA and SufA results in a null-growth phenotype in minimal medium under aerobic conditions [28]. Cell growth of the iscA/sufA double mutant is fully restored when either IscA or SufA is re-introduced into the iscA/sufA double mutant, further indicating that IscA and SufA have complementary roles for the biogenesis of iron-sulfur clusters [28].

Here we report that cell growth of the E. coli iscA/sufA double mutant can be partially restored by supplementing with BCAAs (branched-chain amino acids) and thiamin in growth medium under aerobic conditions. We further demonstrate that IscA/SufA paralogues are required for the [4Fe-4S] cluster assembly in the enzyme IlvD (dihydroxyacid dehydratase) of the BCAA-biosynthetic pathway [36], enzyme ThiC of the thiamin-biosynthetic pathway [37], aconitase B of the citrate acid cycle [38] and endonuclease III of the DNA-base-excision-repair pathway [39] in E. coli cells under aerobic growth conditions. By contrast, deletion of IscA/SufA paralogues does not significantly affect the [2Fe-2S] cluster assembly in the redox transcription factor SoxR [40], ferredoxin [41] and the siderophore-iron reductase FhuF [42]. The results suggest that IscA/SufA paralogues are required for the [4Fe-4S] cluster assembly, but are dispensable for the [2Fe-2S] cluster assembly in E. coli cells under aerobic conditions, and that the biogenesis of the [4Fe-4S] clusters and the [2Fe-2S] clusters may have very distinct pathways in cells.

EXPERIMENTAL

Mutant strains and cell growth

The E. coli deletion mutants in which iscA and sufA were in-frame-deleted were constructed as previously described [28]. Each deletion in E. coli cells was confirmed by PCR, also as described in [28]. For cell growth analysis, overnight cell cultures grown in LB (Luria–Bertani) medium were washed twice with minimal medium containing glucose (0.2%) before being inoculated on minimal medium plates or in liquid minimal medium at 37 °C with aeration (agitation at 250 rev./min). Cell growth was recorded by measuring the attenuance (D) of the cell culture at 600 nm. When indicated, minimal medium was supplemented with the three BCAAs (final concn. 90 μg/ml) and/or thiamin (final concn. 1.0 μg/ml).

Protein expression and purification

The DNA fragments encoding IlvD [36], ThiC [37], aconitase B [38], endonuclease III [39], SoxR [40], ferredoxin [41] and FhuF [42] were amplified from wild-type E. coli genomic DNA using PCR. The primers used for PCR amplifications are listed in Table 1. The PCR products were digested with restriction enzymes and ligated into the plasmid pBAD (Invitrogen). The cloned plasmid was introduced into the iscA/sufA double mutant strain and its parental wild-type strain (MC4100) using electroporation. The DNA sequence of each cloned gene was confirmed by direct sequencing at the Genomic Facility at Louisiana State University (Baton Rouge, LA, U.S.A.). Typically, E. coli cells hosting the expression plasmid were grown to a D600 of 0.6 before the protein expression was induced by adding L-arabinose (final concn. 0.02%) for an additional 3 h under aerobic growth conditions. For expression of ThiC, the final concentration of L-arabinose used was 0.005% (to avoid aggregation of the expressed ThiC). The histidine-tagged proteins were purified using an Ni2+-nitrilotriacetate–agarose column (Qiagen), followed by passage through a High-Trap desalting column as described previously [43]. All solutions used for protein purification were purged with pure argon gas. The concentration of purified proteins was calculated from the absorption peak at 280 nm using an absorption coefficient of 35.2 mM−1·cm−1 for IlvD, 77.9 mM−1·cm−1 for ThiC, 73.6 mM−1·cm−1 for aconitase B, 18.6 mM−1·cm−1 for endonuclease III, 15.7 mM−1·cm−1 for SoxR, 7.8 mM−1·cm−1 for ferredoxin and 56.5 mM−1·cm−1 for FhuF. The amounts of the acid-labile iron and sulfide contents in protein samples were analysed according to the methods of Fischer [44] and Siegel [45] respectively.

Table 1
PCR primers used for the gene cloning
Gene Primer-1 Primer-2 
ilvD 5′-atagagctcatgcctaagtaccgttccgccacc-3′ 5′-cgcgaattcttaaccccccagtttcgatttatc-3′ 
thiC 5′-ataccatggctgcaacaaaactgacccg-3′ 5′-tctaagcttcgcttcctccttacgcaggtag-3′ 
acnB 5′-gaaccgccatggtagaagaatacc-3′ 5′-tgactttttaaagcttagtctgga-3′ 
nth 5′-aatgtcccatggataaagcaaaac-3′ 5′-ttcttcaaagcttaactttctctt-3′ 
fdx 5′-aggtttaccatggcaaagattgtt-3′ 5′-cctctgttaaagcttacgcgcatg-3′ 
soxR 5′-atagagctcatggaaaagaaattacccc-3′ 5′-cgcgaattcttagttttgttcatcttcc-3′ 
fhuF 5′-tatccatggcctatcgttccgcaccgctctatg-3′ 5′-gcgaagctttttcagcgtacaatcgccacattg-3′ 
Gene Primer-1 Primer-2 
ilvD 5′-atagagctcatgcctaagtaccgttccgccacc-3′ 5′-cgcgaattcttaaccccccagtttcgatttatc-3′ 
thiC 5′-ataccatggctgcaacaaaactgacccg-3′ 5′-tctaagcttcgcttcctccttacgcaggtag-3′ 
acnB 5′-gaaccgccatggtagaagaatacc-3′ 5′-tgactttttaaagcttagtctgga-3′ 
nth 5′-aatgtcccatggataaagcaaaac-3′ 5′-ttcttcaaagcttaactttctctt-3′ 
fdx 5′-aggtttaccatggcaaagattgtt-3′ 5′-cctctgttaaagcttacgcgcatg-3′ 
soxR 5′-atagagctcatggaaaagaaattacccc-3′ 5′-cgcgaattcttagttttgttcatcttcc-3′ 
fhuF 5′-tatccatggcctatcgttccgcaccgctctatg-3′ 5′-gcgaagctttttcagcgtacaatcgccacattg-3′ 

Iron-sulfur-cluster assembly in IlvD in vitro

For the iron-sulfur cluster assembly in IlvD, apo-IlvD (10 μM) was incubated with the iron-bound IscA or SufA (containing 75 μM iron) and IscS (0.5 μM) in the presence of dithiothreitol (2 mM), NaCl (200 mM) and Tris (20 mM, pH 8.0) in an open-to-air microcentrifuge tube. The iron-sulfur-cluster assembly reaction was initiated by adding L-cysteine (0.5 mM) to the incubation solution at 37 °C. The [4Fe-4S] cluster assembly in IlvD was monitored by the UV–visible absorption measurements and by an enzyme activity assay (see below). IlvD was then re-purified from the incubation solutions using a Mono-Q column attached to the FPLC system.

Activity assay for IlvD

The enzyme activity of IlvD was measured using the substrate DL-2,3-dihydroxyisovalerate, which was synthesized as described by Cioffi et al. [46]. All of the chemical reagents used for this synthesis were obtained from Sigma–Aldrich. Aliquots of the cell extracts prepared from E. coli cells containing recombinant IlvD were immediately transferred to pre-incubated solutions containing Tris (50 mM, pH 8.0), MgCl2 (10 mM) and DL-2,3-dihydroxyisovalerate (10 mM) at 37 °C as described in [47]. The reaction product (oxo acids) was monitored at 240 nm using an absorption coefficient of 0.19 mM−1·cm−1 [36].

Activity assay for SoxR In vivo

A plasmid (pTN1530) containing the reporter gene soxS::lacZ [48] was introduced into the iscA/sufA double mutant and its parental wild-type (MC4100) cells using electroporation. Overnight-cultured E. coli cells containing pTN1530 were diluted to a D600 of 0.01 in fresh LB medium. After incubation at 37 °C with aeration (agitation at 250 rev./min) to a D600 of 0.1, Methyl Viologen (paraquat; 100 μM) was added to the cell cultures. Aliquots (25 μl) were taken from cell cultures after continuous incubation with aeration (agitation at 250 rev./min) at 37 °C for 0, 20, 40 and 60 min. The β-galactosidase activity in cells was measured as described in [48].

RESULTS

Cell growth of the iscA/sufA double mutant can be partially restored by supplementing with BCAAs and thiamin in growth medium

We reported previously [28] that in-frame deletion of either IscA or SufA in E. coli had only a mild effect on cell growth and that deletion of both IscA and SufA paralogues resulted in a null-growth phenotype in minimal medium under aerobic growth conditions (Figure 1A). Cell growth of the iscA/sufA double mutant was fully restored when either IscA or SufA was re-introduced into the iscA/sufA double mutant [28], demonstrating that IscA and SufA have complementary roles for the biogenesis of iron-sulfur clusters in E. coli cells.

Deletion of IscA/SufA paralogues in E. coli results in deficiency of BCAAs and thiamin under aerobic growth conditions

Figure 1
Deletion of IscA/SufA paralogues in E. coli results in deficiency of BCAAs and thiamin under aerobic growth conditions

E. coli cells (∼107) of the wild-type (1), the sufA mutant (2), the iscA mutant (3) and the iscA/sufA double mutant (4) were inoculated either on a minimal-medium plate containing 0.2% glucose (A) or on an LB plate (B). The photographs were taken after incubation at 37 °C overnight under aerobic growth conditions. (C) Growth curves of the wild-type (▲) and the iscA/sufA mutant (◃) in minimal medium containing 0.2% glucose under aerobic growth conditions. □, iscA/sufA double mutant in minimal medium supplemented with thiamin (1.0 μg/ml); ●, iscA/sufA double mutant in minimal medium supplemented with three BCAAs (90 μg/ml); ■, cell growth of the iscA/sufA double mutant in minimal medium supplemented with both BCAAs (90 μg/ml) and thiamin (1.0 μg/ml). The results are representative of three independent experiments. Abbreviation: O.D., attenuance (D).

Figure 1
Deletion of IscA/SufA paralogues in E. coli results in deficiency of BCAAs and thiamin under aerobic growth conditions

E. coli cells (∼107) of the wild-type (1), the sufA mutant (2), the iscA mutant (3) and the iscA/sufA double mutant (4) were inoculated either on a minimal-medium plate containing 0.2% glucose (A) or on an LB plate (B). The photographs were taken after incubation at 37 °C overnight under aerobic growth conditions. (C) Growth curves of the wild-type (▲) and the iscA/sufA mutant (◃) in minimal medium containing 0.2% glucose under aerobic growth conditions. □, iscA/sufA double mutant in minimal medium supplemented with thiamin (1.0 μg/ml); ●, iscA/sufA double mutant in minimal medium supplemented with three BCAAs (90 μg/ml); ■, cell growth of the iscA/sufA double mutant in minimal medium supplemented with both BCAAs (90 μg/ml) and thiamin (1.0 μg/ml). The results are representative of three independent experiments. Abbreviation: O.D., attenuance (D).

Whereas the iscA/sufA double mutant had no growth on minimal-medium plates under aerobic growth conditions (Figure 1A), it grew slowly on rich-medium LB plates (Figure 1B), indicating that deletion of IscA/SufA paralogues may result in paucity of some essential metabolites for cell growth. Since deficiency of BCAAs [49] and thiamin [50] has been considered a hallmark of oxidative inactivation of iron-sulfur enzymes in bacteria, we postulated that deletion of both IscA and SufA may prevent iron-sulfur-cluster assembly in key enzymes, thus blocking the biosyntheses of BCAAs and thiamin in E. coli cells. Figure 1(C) shows that addition of the three BCAAs leucine, isoleucine and valine indeed partially restored cell growth of the E. coli iscA/sufA double mutant in minimal medium under aerobic growth conditions. Whereas addition of thiamin alone did not result in the recovery of any noticeable cell growth of the iscA/sufA double mutant, addition of both thiamin and BCAAs significantly increased cell growth under aerobic growth conditions. Thus deletion of IscA/SufA paralogues seems to result in BCAAs and thiamin auxotrophy in E. coli cells under aerobic growth conditions.

The iscA/sufA double mutant fails to assemble the [4Fe-4S] cluster in IlvD under aerobic growth conditions

The BCAA-biosynthetic pathway in E. coli contains two ironsulfur enzymes, namely IlvD [36] and LeuCD [49]. IlvD converts 2,3-dihydroxyisovalerate to 2-oxoisovalerate [36], whereas LeuCD converts 2-isopropylmalate to 3-isopropylmalate [49]. Both IlvD and LeuCD require an intact [4Fe-4S] cluster for their catalytic activity. Because LeuCD is a two-subunit enzyme that dissociates during purification [49], we chose IlvD for further investigation.

To explore the function of IscA/SufA paralogues in the [4Fe-4S] cluster assembly in IlvD, we expressed IlvD in the wild-type, the iscA mutant, the sufA mutant and the iscA/sufA-double-mutant cells grown in LB medium under aerobic growth conditions. SDS/PAGE analysis showed that the amounts of IlvD expressed in the wild-type and the IscA/SufA deletion mutants were very similar to each other (Figure 2B). However, unlike IlvD expressed in the wild-type or the iscA and sufA single mutant cells, IlvD expressed in the iscA/sufA-double-mutant cells had little or no enzyme activity (Figure 2A). Recombinant IlvD was then purified from the wild-type and the iscA/sufA-double-mutant cells as described in the Experimental section. As shown in Figure 2(C), whereas IlvD purified from the wild-type E. coli cells had a typical absorption peak at 415 nm of the [4Fe-4S] cluster and was active [36], IlvD purified from the iscA/sufA double mutant had no absorption features of any iron-sulfur clusters and no enzyme activity. The acid-labile iron and sulfide content analyses further revealed that IlvD purified from the wild-type cells contained about 1.6±0.4 mol of iron and 1.4±0.3 mol of sulfide per mol of protein (n=3), indicating that on average about 40% of purified IlvD contained an intact [4Fe-4S] cluster. In contrast, IlvD purified from the iscA/sufA-double-mutant cells had no detectable amounts of the acid-labile iron and sulfide.

IscA/SufA paralogues are required for the [4Fe-4S] cluster assembly in IlvD in E. coli under aerobic growth conditions

Figure 2
IscA/SufA paralogues are required for the [4Fe-4S] cluster assembly in IlvD in E. coli under aerobic growth conditions

(A) Specific activity of IlvD in the cell extracts prepared from the wild-type (●), the sufA mutant (▲), the iscA mutant (◃), and the iscA/sufA double mutant (○) cells grown in LB medium under aerobic growth conditions. The enzyme activity of IlvD was monitored at 240 nm as described in the Experimental section. (B) SDS/PAGE analysis of the cell extracts prepared from the wild-type (1), the sufA mutant (2), the iscA mutant (3) and the iscA/sufA double mutant (4) cells containing recombinant IlvD. The molecular-mass markers are indicated by M (kd=kDa). (C) UV–visible absorption spectra of recombinant IlvD purified from the wild-type (spectrum 1) and the iscA/sufA double mutant (spectrum 2). The concentrations of IlvD were ∼20 μM. The inset shows a photograph of the SDS/PAGE gel of IlvD purified from the wild-type (lane 1) and the iscA/sufA double mutant (lane 2) cells. Abbreviation: O.D., absorbance (A).

Figure 2
IscA/SufA paralogues are required for the [4Fe-4S] cluster assembly in IlvD in E. coli under aerobic growth conditions

(A) Specific activity of IlvD in the cell extracts prepared from the wild-type (●), the sufA mutant (▲), the iscA mutant (◃), and the iscA/sufA double mutant (○) cells grown in LB medium under aerobic growth conditions. The enzyme activity of IlvD was monitored at 240 nm as described in the Experimental section. (B) SDS/PAGE analysis of the cell extracts prepared from the wild-type (1), the sufA mutant (2), the iscA mutant (3) and the iscA/sufA double mutant (4) cells containing recombinant IlvD. The molecular-mass markers are indicated by M (kd=kDa). (C) UV–visible absorption spectra of recombinant IlvD purified from the wild-type (spectrum 1) and the iscA/sufA double mutant (spectrum 2). The concentrations of IlvD were ∼20 μM. The inset shows a photograph of the SDS/PAGE gel of IlvD purified from the wild-type (lane 1) and the iscA/sufA double mutant (lane 2) cells. Abbreviation: O.D., absorbance (A).

The iron-bound IscA/SufA can efficiently provide iron for the [4Fe-4S] assembly in IlvD under aerobic conditions

As reported previously, both IscA and SufA purified from E. coli can bind ferrous iron and act as an iron donor for the iron-sulfur cluster assembly in IscU in vitro [2729]. To test whether IscA/SufA paralogues can provide iron for the [4Fe-4S] cluster assembly in IlvD, we prepared the iron-bound IscA as described in [51] and incubated it with apo-IlvD (purified from the iscA/sufA-double-mutant cells), IscS and L-cysteine in the presence of dithiothreitol at 37 °C under aerobic conditions. Figure 3(A) shows that the enzyme activity of IlvD was quickly restored during the incubation with the iron-bound IscA, IscS and L-cysteine. In a control where the iron-bound IscA was replaced with apo-IscA, no IlvD enzyme activity was recovered after incubation. IlvD was then re-purified from the incubation solution using an anion-exchange Mono-Q column. The UV–visible absorption measurements of re-purified IlvD confirmed that the [4Fe-4S] cluster was assembled in IlvD after incubation with the iron-bound IscA, IscS and L-cysteine (Figure 3B). Similarly, when the iron-bound SufA was included in the incubation solution instead of IscA, the enzyme activity of IlvD was also restored and the [4Fe-4S] cluster assembled in the protein (results not shown). Thus IscA/SufA paralogues may act as iron donors for the [4Fe-4S] cluster assembly in IlvD under aerobic conditions.

Iron-bound IscA can efficiently provide iron for the [4Fe-4S] cluster assembly in apo-IlvD under aerobic conditions

Figure 3
Iron-bound IscA can efficiently provide iron for the [4Fe-4S] cluster assembly in apo-IlvD under aerobic conditions

(A) Re-activation of apo-IlvD by the iron-bound IscA. Apo-IlvD (10 μM) prepared from the iscA/sufA double mutant was incubated with the iron-bound IscA (containing 75 μM iron), L-cysteine (0.5 mM), IscS (0.5 μM) and dithiothreitol (2 mM) (■) at 37 °C aerobically for the indicated times. Apo-IlvD (10 μM) incubated with apo-IscA, L-cysteine (1 mM), IscS (0.5 μM) and dithiothreitol (2 mM) at 37 °C was used as a control (●). The enzyme activity of IlvD was measured as described in the Experimental section. (B) UV–visible absorption spectra of IlvD before (a) and after (b) incubation with the iron-bound IscA, L-cysteine, IscS and dithiothreitol at 37 °C for 30 min under aerobic conditions. IlvD was re-purified from the incubation solutions using a Mono-Q column. The concentrations of IlvD were ∼ 10 μM. Abbreviation: O.D., absorbance (A).

Figure 3
Iron-bound IscA can efficiently provide iron for the [4Fe-4S] cluster assembly in apo-IlvD under aerobic conditions

(A) Re-activation of apo-IlvD by the iron-bound IscA. Apo-IlvD (10 μM) prepared from the iscA/sufA double mutant was incubated with the iron-bound IscA (containing 75 μM iron), L-cysteine (0.5 mM), IscS (0.5 μM) and dithiothreitol (2 mM) (■) at 37 °C aerobically for the indicated times. Apo-IlvD (10 μM) incubated with apo-IscA, L-cysteine (1 mM), IscS (0.5 μM) and dithiothreitol (2 mM) at 37 °C was used as a control (●). The enzyme activity of IlvD was measured as described in the Experimental section. (B) UV–visible absorption spectra of IlvD before (a) and after (b) incubation with the iron-bound IscA, L-cysteine, IscS and dithiothreitol at 37 °C for 30 min under aerobic conditions. IlvD was re-purified from the incubation solutions using a Mono-Q column. The concentrations of IlvD were ∼ 10 μM. Abbreviation: O.D., absorbance (A).

Because IscA/SufA paralogues have also been characterized as alternative scaffolds for the iron-sulfur cluster assembly [2023], it is imperative to examine whether IscA/SufA can directly transfer the assembled iron-sulfur clusters to apo-IlvD. To address this, IscA with a pre-assembled iron-sulfur cluster was prepared as described in [27] and incubated with apo-IlvD in the presence of dithiothreitol at 37 °C under aerobic conditions. Although the enzyme activity of apo-IlvD could indeed be restored by incubation with the iron-sulfur-cluster-bound IscA, pre-incubation of the iron-sulfur-cluster-bound IscA rapidly diminished its ability to re-activate apo-IlvD under aerobic conditions (results not shown). This is probably because the iron-sulfur cluster pre-assembled in IscA is oxygen labile [2022], which results in a failure to re-activate apo-IlvD under aerobic conditions (see the Discussion).

IscA/SufA paralogues are required for the [4Fe-4S] assembly in other enzymes in E. coli under aerobic growth conditions

Because supplementation of the growth medium containing BCAAs with thiamin can increase further the cell growth of the iscA/sufA double mutant under aerobic growth conditions (Figure 1C), we speculated that deletion of IscA/SufA paralogues may also lead to deficiency of thiamin in E. coli cells. The thiamin-biosynthetic pathway in E. coli contains at least two iron-sulfur enzymes, namely ThiC [37] and ThiH [52]. ThiH catalyses the synthesis of the 4-methyl-5β-hydroxyethylthiazole monophosphate moiety of thiamin pyrophosphate [52], whereas ThiC catalyses the formation of 4-amino-5-hydroxymethyl-2-methylpyrimidine [37]. Both ThiC and ThiH require an intact [4Fe-4S] cluster for their catalytic activity [50]. To explore the role of IscA/SufA paralogues in [4Fe-4S] cluster assembly in the enzymes of the thiamin-biosynthetic pathway, we expressed recombinant ThiC in the wild-type and the iscA/sufA-double-mutant cells in LB medium under aerobic growth conditions. SDS/PAGE analysis showed that deletion of IscA/SufA paralogues in E. coli cells did not significantly affect the expression of ThiC and subsequent protein purification. Figure 4(A) shows that ThiC purified from the wild-type cells had a clear absorption peak at 410 nm of the [4Fe-4S] cluster, as reported previously [37], and contained about 1.1±0.2 mol of iron and 1.2±0.2 mol of sulfide per mol of protein (n=3). In contrast, ThiC purified from the iscA/sufA double mutant did not have any absorption peaks of iron-sulfur clusters and no detectable amounts of the acid-labile iron and sulfide. Since the [4Fe-4S] cluster is required for the activity of ThiC [37], lack of the [4Fe-4S] cluster in ThiC would block thiamin biosynthesis in the iscA/sufA double mutant under aerobic growth conditions.

IscA/SufA paralogues are required for the [4Fe-4S] cluster assembly in ThiC, aconitase B and endonuclease III in E. coli under aerobic growth conditions

Figure 4
IscA/SufA paralogues are required for the [4Fe-4S] cluster assembly in ThiC, aconitase B and endonuclease III in E. coli under aerobic growth conditions

(A) UV–visible absorption spectra of ThiC purified from the wild-type (1) and the iscA/sufA double mutant (2) grown in LB medium under aerobic growth conditions. The concentrations of purified ThiC were ∼11 μM. (B) UV–visible absorption spectra of aconitase B (AcnB) purified from the wild-type (1) and the iscA/sufA double mutant (2) grown in LB medium under aerobic growth conditions. The concentrations of purified aconitase B were ∼12 μM. (C) UV–visible absorption spectra of endonuclease III (Nth) purified from the wild-type (1) and the iscA/sufA double mutant (2) grown in LB medium under aerobic growth conditions. The concentrations of purified endonuclease III were ∼15 μM. The inset in each panel is a photograph of the SDS/PAGE gel of purified protein from the wild-type (1) and the iscA/sufA double mutant (2) respectively. Abbreviation: O.D., absorbance (A).

Figure 4
IscA/SufA paralogues are required for the [4Fe-4S] cluster assembly in ThiC, aconitase B and endonuclease III in E. coli under aerobic growth conditions

(A) UV–visible absorption spectra of ThiC purified from the wild-type (1) and the iscA/sufA double mutant (2) grown in LB medium under aerobic growth conditions. The concentrations of purified ThiC were ∼11 μM. (B) UV–visible absorption spectra of aconitase B (AcnB) purified from the wild-type (1) and the iscA/sufA double mutant (2) grown in LB medium under aerobic growth conditions. The concentrations of purified aconitase B were ∼12 μM. (C) UV–visible absorption spectra of endonuclease III (Nth) purified from the wild-type (1) and the iscA/sufA double mutant (2) grown in LB medium under aerobic growth conditions. The concentrations of purified endonuclease III were ∼15 μM. The inset in each panel is a photograph of the SDS/PAGE gel of purified protein from the wild-type (1) and the iscA/sufA double mutant (2) respectively. Abbreviation: O.D., absorbance (A).

Figure 1(C) also showed that supplementation with both BCAAs and thiamin did not fully restore cell growth in the iscA/sufA double mutant, suggesting that deletion of IscA/SufA paralogues may have a much broader effect on iron-sulfur-cluster assembly in E. coli cells. Because the aconitase B [4Fe-4S] cluster represents the major aconitase activity in E. coli cells [38], we chose to examine the [4Fe-4S] cluster assembly in aconitase B in the wild-type and the iscA/sufA-double-mutant cells in LB medium under aerobic growth conditions. SDS/PAGE analysis showed that the amounts of aconitase B expressed in the wild-type and the iscA/sufA-double-mutant cells were similar to each other. However, unlike the aconitase B expressed in the wild-type cells, the aconitase B expressed in the iscA/sufA-double-mutant cells was completely inactive (results not shown). Recombinant aconitase B was then purified from the wild-type and the iscA/sufA-double-mutant cells. As shown in Figure 4(B), aconitase B purified from the wild-type cells had a typical absorption peak at 413 nm of the [4Fe-4S] cluster and contained about 1.4±0.2 mol of iron and 1.0±0.2 mol of sulfide per mol of protein (n=3). In contrast, aconitase B purified from the iscA/sufA-double-mutant cells had no absorption peaks of any iron-sulfur clusters and no detectable amounts of the acidlabile iron and sulfide.

Finally, we explored the [4Fe-4S] cluster assembly in endonuclease III of the DNA-base-excision-repair pathway [39] in the iscA/sufA double mutant cells grown in LB medium under aerobic growth conditions. Unlike IlvD, ThiC or aconitase B, endonuclease III contains an oxygen-resistant [4Fe-4S] cluster [39]. Figure 4(C) shows that, whereas endonuclease III purified from wild-type cells had an absorption peak at 419 nm of the [4Fe-4S] cluster and contained about 2.6±0.2 mol of iron and 2.1±0.3 mol of sulfide per mol of protein (n=3), endonuclease III purified from the iscA/sufA double mutant had no absorption peaks of iron-sulfur clusters and no detectable amounts of the acid-labile iron and sulfide. Thus, IscA/SufA paralogues are required for the [4Fe-4S] cluster assembly in IlvD of the BCAA-biosynthetic pathway, ThiC of the thiamin-biosynthetic pathway, aconitase B of the citrate acid cycle and endonuclease III of the DNA-base-excision-repair pathway in E. coli cells under aerobic growth conditions.

Deletion of IscA/SufA paralogues does not significantly affect [2Fe-2S] cluster assembly in SoxR in E. coli under aerobic growth conditions

Another type of iron-sulfur cluster found in cells is the [2Fe-2S] cluster [13]. It would be pertinent to examine the role of IscA/SufA paralogues in the [2Fe-2S] cluster assembly. We utilized SoxR as an example, since it requires an intact [2Fe-2S] cluster for its redox activation in response to oxidative stress in E. coli cells [40]. Under normal physiological conditions, the SoxR [2Fe-2S] cluster is in a reduced state and SoxR remains inactive [53]. When E. coli cells are subjected to oxidative stress, the reduced SoxR [2Fe-2S] cluster becomes oxidized and SoxR is switched on to stimulate expression of its target gene soxS [53]. To monitor SoxR activation, we introduced a plasmid containing a reporter gene, soxS::lacZ [48], into the wild-type and the iscA/sufA-double-mutant E. coli cells.

Figure 5(A) shows that, for the wild-type E. coli cells, SoxR quickly became activated upon addition of the redox-cycling reagent Methyl Viologen, as reported previously [48]. For the iscA/sufA double mutant, we noticed that the basal level of the SoxR activation was considerably higher than that of the wild-type cells (Figure 5B), indicating that deletion of IscA/SufA paralogues may cause an elevated basal oxidative stress. Nevertheless, when the iscA/sufA double mutant was exposed to Methyl Viologen, the SoxR activation was further increased to a level that is comparable with that of the Methyl Viologen-treated wild-type cells (Figure 5B). The results suggested that the SoxR [2Fe-2S] cluster is fully functional in response to oxidative stress in the iscA/sufA double mutant under aerobic growth conditions.

Redox activation of the transcription factor SoxR by Methyl Viologen in the wild-type and the iscA/sufA-double-mutant E. coli cells

Figure 5
Redox activation of the transcription factor SoxR by Methyl Viologen in the wild-type and the iscA/sufA-double-mutant E. coli cells

(A) Activation of SoxR by Methyl Viologen in the wild-type E. coli cells. Cells hosting a plasmid pTN1530 (containing a reporter gene soxS::lacZ) were incubated in the absence (○) or presence (●) of Methyl Viologen (+PQ) (100 μM) with vigorous aeration (agitation at 250 rev./min) in LB medium. Aliquots (20 μl) were taken from cell cultures at 20 min intervals for assay of β-galactosidase as described in the Experimental section. (B) Activation of SoxR by Methyl Viologen in the iscA/sufA double mutant under aerobic growth conditions. The iscA/sufA-double-mutant cells hosting a plasmid pTN1530 (containing a reporter gene soxS::lacZ) were incubated in the absence (□) or presence (■) of Methyl Viologen (100 μM) with vigorous aeration (agitation at 250 rev./min) in LB medium. Aliquots (20 μl) were taken from cell cultures at 20 min intervals for the assay of β-galactosidase as described in the Experimental section. (C) UV–visible absorption spectrum of recombinant SoxR purified from the wild-type E. coli cells grown in LB medium under aerobic conditions. (D) UV–visible absorption spectrum of recombinant SoxR purified from the iscA/sufA-double-mutant E. coli cells grown in LB medium under aerobic conditions. The protein concentrations of SoxR in (C) and (D) were ∼60 μM. Abbreviation: O.D., absorbance (A).

Figure 5
Redox activation of the transcription factor SoxR by Methyl Viologen in the wild-type and the iscA/sufA-double-mutant E. coli cells

(A) Activation of SoxR by Methyl Viologen in the wild-type E. coli cells. Cells hosting a plasmid pTN1530 (containing a reporter gene soxS::lacZ) were incubated in the absence (○) or presence (●) of Methyl Viologen (+PQ) (100 μM) with vigorous aeration (agitation at 250 rev./min) in LB medium. Aliquots (20 μl) were taken from cell cultures at 20 min intervals for assay of β-galactosidase as described in the Experimental section. (B) Activation of SoxR by Methyl Viologen in the iscA/sufA double mutant under aerobic growth conditions. The iscA/sufA-double-mutant cells hosting a plasmid pTN1530 (containing a reporter gene soxS::lacZ) were incubated in the absence (□) or presence (■) of Methyl Viologen (100 μM) with vigorous aeration (agitation at 250 rev./min) in LB medium. Aliquots (20 μl) were taken from cell cultures at 20 min intervals for the assay of β-galactosidase as described in the Experimental section. (C) UV–visible absorption spectrum of recombinant SoxR purified from the wild-type E. coli cells grown in LB medium under aerobic conditions. (D) UV–visible absorption spectrum of recombinant SoxR purified from the iscA/sufA-double-mutant E. coli cells grown in LB medium under aerobic conditions. The protein concentrations of SoxR in (C) and (D) were ∼60 μM. Abbreviation: O.D., absorbance (A).

We then expressed recombinant SoxR in the wild-type and the iscA/sufA-double-mutant cells in LB medium under aerobic growth conditions. The UV–visible absorption measurements showed that SoxR purified from the wild-type (Figure 5C) and from the iscA/sufA-double-mutant cells (Figure 5D) had nearly identical spectra, further suggesting that IscA/SufA paralogues are not essential for the [2Fe-2S] cluster assembly in SoxR in E. coli cells under aerobic conditions.

IscA/SufA paralogues are dispensable for the [2Fe-2S] cluster assembly in other proteins in E. coli under aerobic growth conditions

To test whether IscA/SufA paralogues are required for the [2Fe-2S] cluster assembly in other proteins, we expressed the recombinant ferredoxin [2Fe-2S] cluster [41] in the wild-type and the iscA/sufA-double-mutant cells. Figure 6(A) shows that ferredoxin purified from the wild-type and the iscA/sufA-double-mutant cells also had a similar UV–visible absorption spectrum, indicating the presence of the [2Fe-2S] cluster. The iron and sulfide content analyses showed that ferredoxin purified from the wild-type [0.6±0.1 mol of iron and 0.5±0.2 mol of sulfide per mol of protein (n=3)] and from the iscA/sufA double mutant [0.5±0.1 mol of iron and 0.4±0.2 mol of sulfide per mol of protein (n=3)] were very similar to each other.

IscA/SufA paralogues are dispensable for the [2Fe-2S] cluster assembly in ferredoxin and FhuF in E. coli under aerobic growth conditions

Figure 6
IscA/SufA paralogues are dispensable for the [2Fe-2S] cluster assembly in ferredoxin and FhuF in E. coli under aerobic growth conditions

Ferredoxin (Fdx) (A) or FhuF (B) was expressed in the wild-type and the iscA/sufA-double-mutant cells grown in LB medium under aerobic growth conditions. Proteins were purified as described in the Experimental section. (A) UV–visible absorption spectra of ferredoxin purified from the wild-type (1) and the iscA/sufA double mutant (2). The concentrations of purified ferredoxin were ∼50 μM. Spectrum 1 was up-shifted by 0.05 A (O.D.). The insets are photographs of the SDS/PAGE gel of purified ferredoxin from the wild-type (1) and the iscA/sufA double mutant (2). (B) UV–visible spectra of FhuF purified from the wild-type (1) and the iscA/sufA double mutant (2). The concentrations of purified FhuF were ∼13 μM. Spectrum 1 was up-shifted by 0.2 A. The insets are photographs of the SDS/PAGE gel of purified FhuF from the wild-type (1) and the iscA/sufA double mutant (2).

Figure 6
IscA/SufA paralogues are dispensable for the [2Fe-2S] cluster assembly in ferredoxin and FhuF in E. coli under aerobic growth conditions

Ferredoxin (Fdx) (A) or FhuF (B) was expressed in the wild-type and the iscA/sufA-double-mutant cells grown in LB medium under aerobic growth conditions. Proteins were purified as described in the Experimental section. (A) UV–visible absorption spectra of ferredoxin purified from the wild-type (1) and the iscA/sufA double mutant (2). The concentrations of purified ferredoxin were ∼50 μM. Spectrum 1 was up-shifted by 0.05 A (O.D.). The insets are photographs of the SDS/PAGE gel of purified ferredoxin from the wild-type (1) and the iscA/sufA double mutant (2). (B) UV–visible spectra of FhuF purified from the wild-type (1) and the iscA/sufA double mutant (2). The concentrations of purified FhuF were ∼13 μM. Spectrum 1 was up-shifted by 0.2 A. The insets are photographs of the SDS/PAGE gel of purified FhuF from the wild-type (1) and the iscA/sufA double mutant (2).

We also examined the [2Fe-2S] cluster assembly in FhuF [42] in the wild-type and the iscA/sufA-double-mutant cells in LB medium under aerobic growth conditions. Figure 6(B) shows that FhuF purified from the wild-type and the iscA/sufA-double-mutant cells had a typical UV–visible absorption spectrum, indicating the presence of the [2Fe-2S] cluster. The iron and sulfide content analyses further revealed that FhuF purified from the wild-type [1.1±0.1 mol of iron and 0.7±0.1 mol of sulfide per mol of protein (n=3)] and from the iscA/sufA double mutant [1.2±0.1 mol of iron and 0.9±0.2 mol of sulfide per mol of protein (n=3)] were essentially the same. Thus deletion of IscA/SufA paralogues does not significantly affect the [2Fe-2S] cluster assembly in SoxR, ferredoxin and FhuF in E. coli cells under aerobic growth conditions.

DISCUSSION

Iron-sulfur-cluster assembly requires a co-ordinated delivery of iron and sulfur in cells. Whereas sulfide in iron-sulfur clusters is derived from L-cysteine via cysteine desulfurases [4], the iron donor for the iron-sulfur cluster assembly still remains elusive. It has been postulated that mitochondrial protein frataxin [54] and its bacterial homologue, CyaY [55], may act as an iron donor for the iron-sulfur-cluster assembly. However, deletion of frataxin/CyaY has little or no effect on iron-sulfur proteins in E. coli [56] and S. cerevisiae [57]. On the other hand, depletion of frataxin has been linked to the human neurodegenerative disease Friedreich's ataxia [58], indicating that frataxin/CyaY may have yet unknown and critical cellular functions. The other proposed iron donors for the iron-sulfur-cluster assembly are IscA/SufA paralogues [27]. in vitro studies have shown that IscA/SufA purified from E. coli can bind ferrous iron and deliver iron for the iron-sulfur-cluster assembly in IscU [2629]. Whereas deletion of either IscA or SufA had only a mild effect on cell growth of E. coli, deletion of both IscA and SufA resulted in a null-growth phenotype in minimal medium under aerobic growth conditions [28]. These results led us to propose that IscA/SufA paralogues have an indispensable role in the biogenesis of iron-sulfur clusters in E. coli cells under aerobic conditions. Here we report that cell growth of the iscA/sufA double mutant can be partially restored by supplementing with BCAAs and thiamin in growth medium under aerobic conditions. Furthermore, we found that the iscA/sufA double mutant fails to assemble the [4Fe-4S] cluster in IlvD of the BCAA-biosynthetic pathway, ThiC of the thiamin-biosynthetic pathway, aconitase B of the citrate acid cycle and endonuclease III of the DNA-base-excision-repair pathway under aerobic growth conditions, suggesting that IscA/SufA paralogues are essential for the [4Fe-4S] cluster assembly in E. coli under aerobic growth conditions. On the other hand, deletion of IscA/SufA paralogues has little or no effect on the [2Fe-2S] cluster assembly in SoxR, ferredoxin and FhuF in E. coli cells under aerobic growth conditions, indicating that IscA/SufA paralogues may be dispensable for the [2Fe-2S] cluster assembly in cells.

The specific function of IscA/SufA paralogues for the [4Fe-4S] cluster assembly in proteins can only be speculated upon. In the present study we found that the iron-bound IscA/SufA can efficiently provide iron for the [4Fe-4S] cluster assembly in IlvD and restore the enzyme activity under aerobic conditions (Figure 3), suggesting that IscA/SufA paralogues may directly provide iron for the [4Fe-4S] cluster assembly in apo-IlvD. However, we could not exclude the possibility that IscA/SufA paralogues may also act as scaffolds for iron-sulfur-cluster assembly as proposed by others [2023]. Indeed, IscA/SufA with pre-assembled iron-sulfur clusters can also restore the enzyme activity of IlvD in the presence of dithiothreitol (results not shown). Nevertheless, the iron-sulfur cluster pre-assembled in IscA/SufA paralogues is oxygen labile [2023], thus limiting its ability to re-activate apo-IlvD under aerobic conditions. On the other hand, the iron centre in IscA is stable under aerobic conditions [51] and can be readily mobilized by L-cysteine [29] for iron-sulfur-cluster assembly in proteins. Although the mechanism underlying the [4Fe-4S] cluster assembly could still not be ascertained, we propose that, under aerobic conditions, IscA/SufA paralogues may act as iron chaperones to make the iron accessible for [4Fe-4S] cluster assembly in proteins such as IlvD, ThiC, aconitase B and endonuclease III in E. coli cells.

A salient finding from the present study is that the iscA/sufA double mutant has an elevated level of basal oxidative stress (Figure 5B). Because IscA or SufA per se does not have anti-(oxidative stress) activity [26], it is possible that deficiency of the [4Fe-4S] cluster assembly in the iscA/sufA double mutant may result in accumulation of intracellular ‘free’ iron and promote cellular oxidative stress. This would be consistent with the previous studies showing that deletion of the IscA homologues in S. cerevisiae led to the accumulation of ‘free’ iron in mitochondria [25]. Alternatively, the iscA/sufA double mutant may fail to assemble the [4Fe-4S] cluster in 6-phosphogluconate dehydratase [14], thus limiting cellular NADPH production and increasing basal oxidative stress. Additional experiments are required to illustrate the mechanism of the elevated basal oxidative stress in the iscA/sufA double mutant E. coli cells under aerobic growth conditions.

There are over 200 distinctive iron-sulfur proteins identified so far [13]. The [2Fe-2S] clusters and the [4Fe-4S] clusters represent two major types of iron-sulfur clusters found in cells. The present model suggests that iron-sulfur cluster assembly scaffolds such as IscU [12] and NfuA [5961] may host both the [2Fe-2S] clusters and the [4Fe-4S] clusters and transfer the ‘correct’ clusters to target proteins. It has also been reported that the [2Fe-2S] clusters in IscU can be reductively converted into the [4Fe-4S] clusters in vitro [19]. Nevertheless, conversion between the [2Fe-2S] clusters and the [4Fe-4S] clusters has yet to be demonstrated In vivo [12], and little is known about the regulation of any scaffold proteins binding the [4Fe-4S] clusters or the [2Fe-2S] clusters. Structurally the [2Fe-2S] clusters and the [4Fe-4S] clusters are very different in their ligand arrangements within proteins. Whereas there are two ligands from protein that bind each iron atom of the [2Fe-2S] cluster, there is only one ligand from protein that binds each iron atom of the [4Fe-4S] cluster. It is plausible that proteins that host the [2Fe-2S] cluster or the [4Fe-4S] cluster may have different binding affinities for ‘free’ iron in cells because of their ligand arrangements for the iron-sulfur clusters. Preliminary studies indicated that, at limited iron concentrations, the [2Fe-2S] clusters are assembled in ferredoxin in preference to [4Fe-4S] clusters in IlvD under aerobic conditions (G. Tan, J. Lu and H. Ding, unpublished work). In summary, the present findings have led us to propose that, under aerobic conditions, (1) IscA/SufA paralogues may act as iron chaperones to provide iron for the [4Fe-4S] cluster assembly, but are dispensable for the [2Fe-2S] cluster assembly in E. coli cells, and (2) that the [2Fe-2S] cluster and [4Fe-4S] cluster may have distinct assembly pathways.

We thank Professor Bruce Demple (Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA, U.S.A.) for generously supplying plasmid pTN1530.

Abbreviations

     
  • BCAA

    branched-chain amino acid

  •  
  • CyaY

    bacterial homologue of the mitochondrial protein frataxin

  •  
  • FhuF

    siderophore-iron reductase

  •  
  • Hsc

    heat-shock cognate protein

  •  
  • IlvD

    dihydroxyacid dehydratase

  •  
  • iscA/sufA double mutant

    Escherichia coli strain in which both iscA and sufA have been in-frame-deleted

  •  
  • IscS

    cysteine desulfurase

  •  
  • LB

    Luria–Bertani

  •  
  • LeuCD

    isopropylmalate isomerase

  •  
  • SoxR

    a redox transcription factor

  •  
  • ThiC

    an enzyme of the thiamin-biosynthetic pathway

FUNDING

This work was supported in part by the Science and Technology Key Program of Zhejiang Province, China [grant number 2006C14025]; the Natural Science Foundation of Zhejiang Province, China [grant numbers Y2081075, Y507233 (to J. L.)]; the United States National Sciences Foundation [grant number MCB-0416537]; and the National Institutes of Health [grant number CA107494 (to H. D.)].

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