The l-Cysteine/l-Cystine Shuttle System Provides Reducing Equivalents to the Periplasm in Escherichia coli

Abstract

Intracellular thiols like l-cysteine and glutathione play a critical role in the regulation of cellular processes. Escherichia coli has multiple l-cysteine transporters, which export l-cysteine from the cytoplasm into the periplasm. However, the role of l-cysteine in the periplasm remains unknown. Here we show that an l-cysteine transporter, YdeD, is required for the tolerance of E. coli cells to hydrogen peroxide. We also present evidence that l-cystine, a product from the oxidation of l-cysteine by hydrogen peroxide, is imported back into the cytoplasm in a manner dependent on FliY, the periplasmic l-cystine-binding protein. Remarkably, this protein, which is involved in the recycling of the oxidized l-cysteine, is also found to be important for the hydrogen peroxide resistance of this organism. Furthermore, our analysis of the transcription of relevant genes revealed that the transcription of genes encoding FliY and YdeD is highly induced by hydrogen peroxide rather than by l-cysteine. These findings led us to propose that the inducible l-cysteine/l-cystine shuttle system plays an important role in oxidative stress tolerance through providing a reducing equivalent to the periplasm in E. coli.

Introduction

A key building block of proteins, l-cysteine is an amino acid with a thiol side chain. Because of its high reactivity, l-cysteine is an important structural and functional component of many proteins. Although l-cysteine is pivotal for various protein functions, the molecule itself is toxic to cells even at low concentrations in both prokaryotes and eukaryotes (1–3). It has been reported that threonine deaminase, an enzyme in l-isoleucine biosynthesis, is inhibited by l-cysteine (4), which could be part of the reason for the cytotoxicity of l-cysteine in this organism. To maintain the l-cysteine concentrations below the threshold of cytotoxicity, the intracellular l-cysteine level is strictly controlled.

Serine acetyltransferase, a key enzyme in the l-cysteine synthesis pathway of Escherichia coli, is under the control of feedback inhibition by l-cysteine. In addition, E. coli has five or more enzymes having l-cysteine desulfhydrase activity (TnaA, CysK, CysM, MalY, and MetC). These systems may prevent the accumulation of excess l-cysteine in cells.

E. coli has l-cysteine transporters in the inner membrane (YdeD, YfiK, and Bcr) (5–7), and in the outer membrane (TolC) (8). It is known that TolC associates with the inner membrane and accessories, e.g. AcrAB or AcrEF, forming tripartite efflux pumps which export toxic compounds directly from the cytoplasm to the outside of the cells. However, in the l-cysteine export system, TolC does not associate with the l-cysteine transporters in the inner membrane (YdeD, YfiK, and Bcr) (8). These findings suggest that l-cysteine, transported from the cytoplasm, is first pooled in the periplasm, and then exported through TolC in the outer membrane. Despite this knowledge, the role of the periplasmic l-cysteine remained elusive.

The electron transport chain in the inner membrane of E. coli is thought to generate reactive oxygen species (ROS),³ such as superoxide and hydrogen peroxide (H₂O₂), due primarily to the leakage of electrons (9). H₂O₂ in the cytoplasm is eliminated by two catalases (KatE and KatG) and a peroxidase (AhpCF). An Hpx⁻ mutant lacking all of these three major enzymes accumulates H₂O₂ in cells (10). However, these enzymes do not exist in the periplasm, but superoxide dismutase (SodC), which generates the H₂O₂, localizes in this compartment. This fact raises a question concerning how H₂O₂ generated in the periplasm is eliminated.

In addition, E. coli is exposed to H₂O₂, which is produced by phagocytes, in the environment. If the cells could detoxify H₂O₂ in the periplasm before its penetration into the cytoplasm, it would diminish its toxicity. Thus, the possession of such H₂O₂ removal ability in the periplasm may be beneficial for the cells. It is known that the sulfhydryl group of l-cysteine can react with H₂O₂ to yield H₂O and l-cystine (11) as in Equation 1.

Thus, we speculated that an l-cysteine transporter such as YdeD exports l-cysteine as a scavenger of H₂O₂ into the periplasm. These considerations have led us to study the role of l-cysteine transporters in E. coli. In this report, we show evidence that the l-cysteine transporter YdeD indeed functions against H₂O₂ stress in E. coli. We also provide evidence that the periplasmic l-cystine-binding protein FliY is involved not only in the uptake of l-cystine, a product of oxidation of l-cysteine, but also in the H₂O₂ tolerance of this organism. Further, our data show that H₂O₂ stress highly induces the expression of the genes encoding YdeD and FliY. From these findings, we propose that the inducible l-cysteine/l-cystine shuttle system plays an important role for the resistance of cells to H₂O₂ in the periplasm.

EXPERIMENTAL PROCEDURES

Bacterial Strains, Plasmids, and Oligonucleotides

E. coli strains and plasmids used in this work are listed in Table 1, and oligonucleotides used are listed in supplemental Table S1. Gene cloning and DNA manipulation and the transformation of E. coli strains were performed according to standard methods (12). E. coli wild-type strain, BW25113, their derivatives (deletion mutants), and plasmids, pCA24N and pDsbA were supplied by the National BioResource Project (NBRP). A Hpx⁻ mutant strain lacking two catalases (KatE and KatG) and a peroxidase (AhpCF)(10) was kindly provided by James A. Imlay. High l-cysteine-producing plasmid pDES (supplied by Ajinomoto) is a derivative of pACYC184 containing the altered cysE gene encoding the l-cysteine feedback inhibition-insensitive mutant SAT (T167A), the wild-type ydeD gene encoding inner membrane l-cysteine transporter (5), and the altered serA gene encoding the l-serine feedback inhibition-insensitive mutant of d-3-phosphoglycerate dehydrogenase (T410stop). Each gene fragment is under the control of the constitutive promoter of the E. coli ompA gene encoding outer membrane protein A precursor (13). The medium copy number vector pSTV29 was purchased from Takara Bio Co. (Kyoto, Japan). The construction of pYdeD has already been described (8).

TABLE 1.

Bacterial strains and plasmids used

E. coli strain or plasmid	Genotype		Ref. or source
Strains
BW25113	lacIq, rrnBT14, lacZWJ16, hsdR514, araBADAH33, rhaBADLD78		(35)
BW25113	pYdeD	(Enhancing periplasmic l-cystine excreation)	(35)
BW25113	pDE, pCA24N	(l-Cystine overproducer)	(35)
JW3832	BW25113 dsbA::KmR		(35)
JW5182	BW25113 dsbB::KmR		(35)
JW5250	BW25113 ydeD::KmR		(35)
JW2663	BW25113 gshA::KmR		(35)
JW2562	BW25113 yfiK::KmR		(35)
JW5363	BW25113 bcr::KmR		(35)
JW1905	BW25113 fliY::KmR	(Weakening l-cystine uptake)	(35)
JW1905	fliY::KmR, pDES, pCA24N	(Weakening l-cystine uptake)	(35)
pCA24N	T5-lacpromoter, pQB2-based, CmR		(36)
pFliY	pCA24N, fliYgene on 0.8 kb DNA fragment		(36)
pDsbA	pCA24N, dsbAgene on 0.6 kb DNA fragment		(36)
pSTV29	lacpromoter, pACYC184-based, CmR		(8)
pYdeD	pSTV29, ydeDgene on 1.5 kb DNA fragment		(8)

Media and Growth Conditions

Luria-Bertani (LB) complete medium or SM1 medium that is supplemented with LB broth, l-methionine, and thiosulfate (8) was used for the general cultivations of E. coli. When appropriate, antibiotics were added at 50 μg/ml (for kanamycin), 30 μg/ml (for chloramphenicol), and 10 μg/ml (for tetracycline). Growth of cultures was monitored by measuring of the optical density at 660 nm (OD₆₆₀). H₂O₂ was added to the medium at the indicated concentration. For solid medium, 1.5% agar was added.

l-Cystine Uptake Assays

Cells grown to mid-exponential phase were harvested by centrifugation, washed twice with cold KPM solution (10 mm MgSO₄, 0.1 m K₂HPO₄; pH was adjusted to 6.5 with H₃PO₄), and suspended in cold KPM solution to a density of 10⁸ cells per ml. Portions of the cell suspension (6 ml each) were energized, by the addition of 0.57 ml of 40% d-glucose, followed by incubation for 10 min at 37 °C. The l-cysteine uptake assay was initiated by the addition of 2.5 μl of l-[¹⁴C]cystine (291.3 mCi/mmol). Following the incubation of the cells at room temperature for the indicated time, the cells were collected by filtration through a GF/C filter (Whatman), and the cells collected on the filter were washed three times with KPM solution. Then, the radioactivity derived from ¹⁴C incorporated into the cells on the filter was determined by liquid scintillation counter LS6500 (Beckman).

Preparation of Intracellular l-Cysteine

After E. coli cells were grown to stationary phase at 30 °C in LB medium or SM1 medium that was supplemented with L broth, l-methionine, and thiosulfate, 1 ml of the cell culture was harvested, washed with distilled water, and suspended in 0.2 ml of distilled water. The intracellular amino acids were then extracted from the cells by boiling the cell suspension for 10 min using a heat block. After centrifugation (1 min at 15,000 × g) of the heated sample, the supernatant was used as an intracellular amino acid extract (7).

Quantification of l-Cysteine

The amount of l-cysteine in culture supernatants was determined according to the method of Gaitonde (14). 100 μl of the sample was incubated with 200 μl of Gaitonde reagent (250 mg ninhydrin dissolved in a mixture of 4 ml of concentrated HCl and 16 ml of glacial acetic acid) at 100 °C for 15 min. Under the strongly acidic conditions, ninhydrin reacts specifically with l-cysteine even in the presence of other thiols, forming a pink-colored product (E_max 560 nm). The reaction product was immediately cooled on ice and diluted to 1.8 ml with 99.5% (v/v) ethanol. The concentration of l-cysteine in the original sample was then determined by measuring the absorbance at 560 nm of the diluted sample.

Quantification of Total Free l-Cysteine (l-Cysteine Plus l-Cystine)

To determine the amount of the total l-cysteine, l-cystine in the sample was reduced by incubation with 5 mm dithiothreitol in 200 mm Tris-HCl buffer (pH 8.6) for 10 min. Then the amount of l-cysteine in the reduced sample was determined using the method by Gaitonde.

Determination of l-Cystine

The amount of l-cystine was calculated by subtracting the amount of l-cysteine from that of total free l-cysteine.

Real-time Quantitative Polymerase Chain Reaction Analysis

Primers used in this study were designed using the Primer Express (Applied Biosystems, Foster City, CA; supplemental Table S1). E. coli cells were lysed with 1 mg/ml lysozyme in 10 mm Tris-HCl (pH 8.0) containing 1 mm EDTA buffer. Total RNA was prepared using the RNeasy Mini Kit (Qiagen, Valencia, CA) and the RNase-Free DNase Set (Qiagen). Complementary DNA was synthesized from 1 μg of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For real-time quantitative polymerase chain reaction (qPCR), cDNA was amplified with oligonucleotide primers specific to each target gene using the 7300 Real-Time PCR System (Applied Biosystems). Reactions contained Power SYBR PCR Master Mix (Applied Biosystems), forward and reverse primers (0.1 μm each), and a cDNA template (20 ng). For the dissociation curve analysis, the following conditions were used: initial steps at 50 °C for 2 min, 95 °C for 10 min; 40 cycles of PCR at 95 °C for 15 s, 60 °C for 1 min; and final steps at 95 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s (15). The melting curve for each PCR product was determined according to the supplier's guidelines, ensuring specific amplification of the target gene. Quantitative values were obtained as the threshold PCR cycle number (Ct) when the increase in the fluorescent signal of the PCR product showed exponential amplification. The mRNA level of each gene was normalized to that of rrsH in the same sample. The cycle threshold (Ct) value for each reaction was determined using the 7300 Real-time PCR System software package (Applied Biosystems). The Ct values were used to calculate the mean-fold change of the reactions via the 2^−ΔΔCt method for each sample in triplicate, in which 1 indicates no change in abundance (16).

Hydrogen Peroxide Measurements

The concentration of H₂O₂ in the sample was measured using coloring reagent mixture containing peroxidase, 4-aminoantipyrine, and phenol as a specific pink product forms from oxidative condensation of phenol, 4-aminoantipyrine, and H₂O₂. 5 μl of sample was incubated with 1 ml of the coloring reagent mixture (Wako) at room temperature for 10 min. Then, the amount of the pink product derived from H₂O₂ in the original sample was determined by measuring absorbance at 505 nm (A₅₀₅).

Redox State Analysis on DsbA

To determine the in vivo redox states of DsbA, the free cysteine residues of the protein were acid trapped and alkylated with the high molecular mass reagent AMS (Invitrogen) as described (17). Alkylated samples were separated by SDS-PAGE, and detected by Western blot analysis with anti-DsbA that has been described (18).

Microscopic Analysis

Cultures, grown in LB complete medium at 37 °C overnight, were diluted 1:100-fold into the same medium with or without 100 μm H2O2. After growth was continued for 8 h, cells were harvested for microscopic analyses. The pictures of the cells were taken with the Axiovert 200 m microscope (ZEISS, Osaka, Japan). Images were collected and processed using the AxioVision 4.5 software (ZEISS, Osaka, Japan).

RESULTS

The l-Cysteine Transporter YdeD Contributes to Hydrogen Peroxide Tolerance in E. coli

To address our hypothesis concerning the role of l-cysteine transporters in the detoxification of H₂O₂, we first investigated whether YdeD is involved in H₂O₂ resistance in E. coli. For this purpose, ΔydeD mutant and wild-type cells were transformed with either pYdeD, a middle copy plasmid carrying the ydeD gene with the original promoter, or the empty vector pSTV29. Serial dilutions of the overnight culture in LB medium were spotted onto the surface of a H₂O₂-containing LB agar plate. The cells were then grown at 30 °C for 24 h. The ydeD deletion mutant cells displayed increased H₂O₂ sensitivity (Fig. 1A). In contrast, mutants missing either one of the other l-cysteine transporter genes, yfiK or bcr, did not show an increased H₂O₂ sensitivity (supplemental Fig. S1). Remarkably, both wild-type cells and ΔydeD cells overexpressing YdeD showed higher levels of H₂O₂ tolerance than that of wild-type cells (Fig. 1A). These results demonstrate that YdeD contributes to H₂O₂ resistance in E. coli.

FIGURE 1.

l-Cysteine transporter, YdeD, is important for the tolerance of E. coli to oxidative stress. A, serial dilutions of the overnight cultures were spotted on a LB solid medium plate containing 0 10 mm or 0.5 mm H₂O₂. The sensitivity of each strain to H₂O₂ was then examined by incubating the plates at 30 °C for 1 day. The strains used are: the wild-type strain BW25113 (WT) harboring an empty vector (pSTV29) or a plasmid encoding YdeD (pYdeD), and the ΔydeD mutant JW5250 (ΔydeD) harboring pSTV29 or pYdeD. B, growth of the wild-type strain expressing YdeD from plasmid (pYdeD) on LB solid medium containing 0.5 mm H₂O₂. Cells were treated with 0.88 mm H₂O₂ for 30 min prior to spotting. C, relative transcription levels of the ydeD gene after addition of 0.88 mm H2O2 (filled bar) compared with those without stress treatment (empty bar). D, growth of the wild-type strain (BW25113), or a ΔgshA mutant (JW2663) carrying pSTV29 or pYdeD on LB solid medium containing 0.5 mm H2O2.

The levels of H₂O₂ tolerance were clearly elevated by H₂O₂ pretreatment (Fig. 1B), suggesting that E. coli has an inducible H₂O₂ tolerance system. Interestingly, qPCR showed an 11.2-fold increase in the ydeD gene expression after treatment with 0.88 mm H₂O₂ for 30 min (Fig. 1C).

Glutathione is a redox-active tripeptide (l-γ-glutamyl-l-cysteinylglycine; GSH) that is present in the cytoplasm of many organisms. It is reported that GSH also exists in the periplasm of E. coli (19). To examine whether GSH plays a role in the H₂O₂ resistance of E. coli cells, we tested the effects of the deletion of the gshA gene on the H₂O₂ resistance of E. coli. This gene encodes γ-glutamylcysteine synthetase, a key enzyme in GSH synthesis. We found that H₂O₂ tolerance was increased by YdeD overexpression even in the ΔgshA mutant (Fig. 1D). Importantly, no significant difference in H₂O₂ tolerance was observed between the wild-type cells and the ΔgshA mutant. These findings suggest that, in contrast to l-cysteine, GSH may not contribute to the H₂O₂ resistance of E. coli cells.

l-Cysteine Is Oxidized to l-Cystine in the Periplasm

As the periplasm is under oxidative conditions, it is inferred that l-cysteine is oxidized into l-cystine in this oxidative cellular compartment. Thus, we investigated whether l-cystine is, in fact, produced in the periplasm. Because E. coli cells maintain an extremely low cellular l-cysteine level, we could not determine the cellular contents of l-cysteine and l-cystine. Therefore, we used cells transformed with pDES, which allows the cells to produce a large amount of l-cysteine and effectively export it into the periplasm. The pDES plasmid encodes serine acetyltransferase (T167A) and d-3-phosphoglycerate dehydrogenase (T410stop), which are released from feedback inhibition by l-cysteine and l-serine, respectively, and YdeD (8).

As shown in Fig. 2A, l-cystine accounted for about 30% of the total free cysteine (l-cysteine plus l-cystine) in E. coli cells carrying the pDES plasmid. The further overexpression of DsbA, which oxidizes peptidyl-l-cysteine residues into a disulfide bridge in the periplasm (20), facilitated l-cystine formation: the ratio of l-cystine to the total cysteine reached about 50% (Fig. 2A). These findings suggest that a significant fraction of endogenously produced l-cysteine is in fact exported into the periplasm, where it is oxidized into l-cystine even in the absence of a specific exogenous oxidative stressor. These observations are consistent with the proposed role of l-cysteine as a reducing equivalent in the periplasm.

FIGURE 2.

l-Cysteine is oxidized to l-cystine in the periplasm. A, ratio of the oxidized l-cysteine (l-cystine) to the total intracellular l-cysteine. The wild-type strain (BW25113) and its ΔfliY-derivative (JW1905) were transformed with plasmid pDES for the hyperproduction of l-cysteine. Where indicated, pDsbA plasmid was co-transformed to express DsbA. The pCA24N plasmid is an empty vector. Cell extract was prepared by incubating the cell suspension in hot water for 10 min. To determine the total l-cysteine, l-cystine in the sample was once reduced with DTT to l-cysteine. The l-cysteine content was then determined using the method by Gaitonde (14). B, wild-type strain (BW25113) was transformed with pDES plasmid. The transformant was grown to stationary phase in LB supplemented with 3% glucose and 10 mm sodium thiosulfate. The culture was then incubated with 0.88 mm H₂O₂ for 10 min and the cells were harvested. The ratio of the l-cystine to the total intracellular l-cysteine was determined as described under “Experimental Procedures.” C, l-[¹⁴C]cystine uptake by the wild-type strain (closed circle), and the ΔfliY mutant (open circle).

The Intracellular l-Cystine/l-Cysteine Ratio Increases Upon Treatment of the Cells with Hydrogen Peroxide

If l-cysteine is used as a reducing equivalent to detoxify H₂O₂ in vivo, a portion of l-cysteine will be oxidized after treatment of the cells with H₂O₂. To test this, we also determined the intracellular contents of l-cysteine and l-cystine after treatment of cells with H₂O₂. To detect the changes in the intracellular l-cystine/l-cysteine ratio before and after H₂O₂ treatment, we used E. coli cells carrying the pDES plasmid. As shown in Fig. 2B, the ratio of l-cystine to the total l-cysteine of the cells after treatment with 0.88 mm H₂O₂ (52%) was higher than that of the cells before treatment with H₂O₂ (41%). This ratio reached 73% after treatment with 8.8 mm H₂O₂ (data not shown). These results are consistent with the finding that endogenous l-cysteine can detoxify exogenous H₂O₂ in the periplasm.

Overexpression of the l-Cysteine Transporter YdeD Can Affect the Redox Environment of the Periplasm

We showed evidence that the overexpression of the l-cysteine transporter, YdeD, confers H₂O₂ resistance to E. coli (see above). We next wanted to examine the influence of the l-cysteine transporter overexpression on the redox environment of the periplasm. To this end, we investigated the redox state of DsbA after the overexpression of the l-cysteine transporter from the pYdeD plasmid because DsbA is a periplasmic protein with a pair of redox-active l-cysteines that can alternate between the oxidized and the reduced states. Importantly, these two l-cysteines are normally maintained in the fully oxidized state in vivo by the membrane protein DsbB, which passes electrons from DsbA to quinones in the respiratory chain (17). To study the oxidative state of DsbA in vivo, we used an alkylating reagent, AMS, which modifies free l-cysteines. The modification of free l-cysteines with AMS allows the separation of the oxidized and the reduced form of DsbA on a gel (17). Upon overexpression of the l-cysteine transporter, a fraction of DsbA was indeed reduced in the cells (supplemental Fig. S2). This observation is consistent with the hypothesis that l-cysteine exported into the periplasm by YdeD can function as a reducing equivalent in the periplasm

l-Cystine Is Imported Dependently of the Periplasmic l-Cystine-binding Protein, FliY

In Lactobacillus fermentum BR11, the l-cystine binding protein BspA is responsible for l-cystine uptake. FliY of E. coli was identified as a BspA ortholog and has been shown to bind l-cystine (21). However, the mechanism of the l-cystine import remained unclear in this organism. To examine whether the uptake of l-cystine is dependent on FliY, the rate of uptake of l-[¹⁴C]cystine in a ΔfliY strain was compared with that of E. coli wild-type strain. It was found that the intracellular uptake of l-[¹⁴C]cystine was significantly impaired in the ΔfliY mutant, only 37% of that in wild-type cells (Fig. 2C), indicating that FliY is required for the efficient uptake of l-cystine by E. coli.

Consistent with the role of FliY in the uptake of l-cystine, the disruption of the fliY gene increased the ratio of intracellular l-cystine to l-cysteine plus l-cystine by about 50% (Fig. 2A). Remarkably, the ratio reached 96% in the ΔfliY cells that overexpress DsbA from the pDsbA plasmid. It should be noted that, in these experiments, we used an E. coli strain that exports a large amount of l-cysteine so that we could detect l-cysteine and l-cystine in the periplasm (see above). These results led us to draw a model in which endogenous l-cysteine is exported to the periplasm by YdeD and oxidized to l-cystine, which is then imported back into the cytoplasm in a FliY-dependent manner.

FliY Is Also Involved in the H₂O₂ Resistance of E. coli

We envisaged that, in addition to YdeD, FliY may also contribute to H₂O₂ tolerance as it can be expected that l-cystine uptake will promote the recycling of the l-cystine into l-cysteine (22). To test this possibility, serial dilutions of ΔfliY or ΔydeD mutant cells were spotted onto the surface of an H2O2-containing LB agar plate. The cells were then grown at 30 °C for 24 h. The ΔfliY and ΔydeD mutants showed a higher sensitivity to H₂O₂ than wild-type cells (Fig. 3A). Moreover, in liquid medium, the growth of the ΔfliY cells was completely inhibited in the presence of 0.5 mm H₂O₂, though the mutant would normally grow in the same medium without H₂O₂ (Fig. 3B). The growth defect of ΔfliY was partially restored by the transformation of the mutant cells with the pFliY plasmid. These results demonstrate that FliY contributes to growth in liquid medium with H₂O₂.

FIGURE 3.

The roles of FliY in E. coli resistance to H₂O₂. A, serial dilutions of the overnight cultures of the wild-type strain BW25113 (WT), a ΔfliY mutant JW1905 (ΔfliY), and a ΔydeD mutant JW5250 (ΔydeD) were spotted on a LB solid medium plate containing 0 mm or 0.75 mm H₂O₂. The sensitivity of each mutant to H₂O₂ was then examined by incubating the plates at 30 °C for 1 day. B, overnight cultures of various E. coli strains were inoculated in LB liquid medium and grown in the presence (closed) or the absence (open) of 0.5 mm H₂O₂ at 30 °C. Growth was monitored by measuring the optical density at 660 nm (OD₆₆₀). The strains used are: the wild-type strain BW25113 carrying an empty vector pCA24N (circles), and the ΔfliY mutant JW1905 carrying pCA24N (triangles) or pFliY encoding FliY (diamonds).

It should be noted that, as we showed before, overexpression of YdeD conferred an increased H₂O₂ resistance to the wild-type cells. However, such an increased resistance was not observed when FliY was overproduced in the wild-type cells (not shown and Fig. 3D). The difference in the effect of protein overexpression between YdeD and FliY may reflect the fact that, while YdeD directly promotes the export of l-cysteine into the periplasm, FliY is involved in the uptake of l-cystine that has been exported in the form of l-cysteine and is then oxidized in the periplasm. Nevertheless, the increased sensitivity of the ΔfliY mutant to H₂O₂ indicates the importance of the l-cystine uptake system in the H₂O₂ defense mechanism of E. coli. Taken together, we conclude that YdeD and FliY are the key components in the l-cysteine/l-cystine shuttle system, which confers H₂O₂ resistance to E. coli cells.

The l-Cysteine/l-Cystine Shuttle System Is Induced by H₂O₂

Next, we elucidated whether this system is inducible under oxidative stress. For this purpose, we quantified the expression level of related genes using real-time qPCR. As positive controls for the H₂O₂ treatment of the cells, we also examined the expression of the katG and ahpC genes, which are known to be up-regulated in response to H₂O₂ in an oxyR-dependent manner. The levels of katG and ahpC expression increased significantly under the stress conditions (Fig. 4), consistent with the observation by Strorz et al. (23). Among the genes encoding l-cysteine/l-cystine transporters, ydeD, yfiK, and fliY were highly induced, at 11.2-, 4.0-, and 10.2-fold, respectively, after treatment of the cells with 0.88 mm H₂O₂ for 20 min (Fig. 4). Both YdeD and FliY are key components involved in H₂O₂ resistance of E. coli. The dramatic increases in the expression of the genes encoding these proteins are consistent with our observation in Fig. 1B that the levels of tolerance of E. coli to H₂O₂ were elevated by pretreatment of the cells with this reagent.

FIGURE 4.

Induction of the ydeD and fliY gene expression by H₂O₂. Induction of the transcription of relevant genes was analyzed after treatment of the cells with 1 mm H₂O₂ for 20 min. Relative expression (y-axis) represents fold change of each mRNA level after treatment of the cells with H₂O₂ as compared with that of the untreated cells (dotted line).

Curiously, in addition to H₂O₂, exogenous l-cysteine also induced the expression of ydeD, yfiK, and fliY genes (Fig. 5). However, the induction of these genes by exogenous l-cysteine was not as significant as that by H₂O₂ (Fig. 4). Further, in contrast to the katG and ahpC genes whose H₂O₂-dependent induction was abolished in an ΔoxyR mutant, expression of the ydeD and fliY genes was still highly induced by H₂O₂ even in the same ΔoxyR mutant.⁴ Thus, the induction of the ydeD and fliY genes by H₂O₂ may not depend on oxyR, which is a regulator of oxidative stress response.

FIGURE 5.

Induction of the ydeD and fliY gene expression by l-cysteine. Induction of the transcription of relevant genes in cells 20 min after addition of 10 mm l-cysteine to the culture. Relative expression represents the fold change of each mRNA level in cells 20 min after incubation with l-cysteine as compared with that of the cells without stress treatment (dotted line).

Our analysis of the expression of the other relevant genes during H₂O₂ stress also revealed their unique regulatory features. First, the expression of the cysE gene encoding serine acetyltransferase, a key enzyme in l-cysteine biosynthesis and the tnaA gene encoding a major l-cysteine desulfhydrase, was not induced, suggesting that H₂O₂ treatment does not promote l-cysteine production. Second, the system to uptake periplasmic GSH is composed of l-glutamyl transferase (Ggt), which degrades GSH into l-glutamate and l-cysteinylglycine in the periplasm; the machinery importing l-cysteinylglycine encoded by yliA, B and C; and cytosolic aminopeptidase, pepA, B, D, and N (24). Among them, H₂O₂ treatment induced the expression of ggt (12.7-fold), yliA (4.0-fold), yliB (5.4-fold), yliC (4.8-fold), pepB (4.2-fold), and pepN (7.4-fold). However, no expression of gshA, a gene encoding a key enzyme of GSH synthesis, or cydD (25), encoding a GSH exporter, was induced. In addition, cellular l-cysteine was undetectable even under oxidative stress conditions. These findings indicate that, under oxidative stress conditions, E. coli up-regulates the expression of the genes involved in the export of l-cysteine or the uptake of l-cystine but not the expression of the genes involved in the synthesis of l-cysteine. Thus, to resist oxidative stress, E. coli appears to promote the l-cysteine/l-cystine shuttle system rather than enhancing the synthesis of l-cysteine.

The Growth Defect of Hpx⁻ (katG katE ahpCF) Mutants under the Presence of 100 μm H₂O₂ Is Suppressed by Overexpression of the l-Cysteine Transporter YdeD

It has been reported that an Hpx⁻ mutant, which lacks alkyl hydroperoxide (Ahp) and two catalases (KatE and KatG), has very little H₂O₂-scavenging activity (10). Consistent with its decreased H₂O₂-scavenging ability, the growth of this Hpx⁻ mutant was impaired for 12 h in LB liquid medium containing 100 μm H₂O₂ at 37 °C (Fig. 6). It should be noted here that the same Hpx⁻ mutant grew rather normally with 50 μm H₂O₂ (not shown). This finding suggests that the presence of 100 μm or higher concentrations of H₂O₂ is required even for the H₂O₂-sensitive mutant to exhibit a clear growth defect.

FIGURE 6.

The growth defect of Hpx⁻ mutants is suppressed by overexpression of l-cysteine transporter, YdeD. The cellular morphology of Hpx⁻ cells carrying (top left) pSTV29 or (top right) pYdeD cultures was analyzed by microscopy, and samples were taken at the 8-h point. Bottom, overnight cultures of various E. coli strains were inoculated in LB liquid medium and grown in 11 the presence (circles) or the absence (diamonds or triangle) of 100 μm H₂O₂ at 37 °C. Growth was monitored by measuring the optical density at 660 nm (OD₆₆₀). The strains used are: the Hpx⁻ mutants carrying an empty vector pSTV29 (open diamonds, triangle, or circles), and pYdeD (filled diamonds, triangle, or circles).

We showed that overexpression of YdeD but not of FliY conferred increased H₂O₂ resistance to the wild-type cells. We envisaged that, if overexpression of YdeD leads to the enhanced detoxication of H₂O₂, the overexpression may also suppress the H₂O₂-hypersensitive growth phenotype of the Hpx⁻ mutant grown in the presence of 100 μm H₂O₂. To test this, we compared the growth of the Hpx⁻ mutant with that of the same mutant overexpressing YdeD in LB liquid medium containing 100 μm H₂O₂. As shown in Fig. 6, the growth of the Hpx⁻ mutant was partially but clearly restored by the transformation of the mutant cells with the pYdeD plasmid. In addition, overexpression of YdeD in the Hpx⁻ mutant decreased the number of the cells forming filaments. These finding further supports the protective role of YdeD against H₂O₂.

DISCUSSION

Mutants missing the l-cysteine transporter, YdeD, or the l-cystine-binding protein, FliY, exhibited an increased sensitivity to H₂O₂. Further, our data indicated that E. coli FliY is involved in the uptake of l-cystine from the periplasm to the cytoplasm. Interestingly, the expressions of the genes encoding these proteins were dramatically increased upon treatment of the cells with H₂O₂. These findings led us to propose that E. coli removes periplasmic H2O2 using l-cysteine supplied to the periplasm from the cytoplasm by a l-cysteine/l-cystine shuttle system (Fig. 7).

FIGURE 7.

A model for the inducible l-cysteine/l-cystine shuttle system. A model for the l-cysteine/l-cystine shuttle system responsible for defense to oxidative stress in E. coli. Superoxide (O₂^˙̄) generated during respiratory electron transfer is converted to H₂O₂ by superoxide dismutase SodA in the cytoplasm or superoxide dismutase SodC in the periplasm. Cytoplasmic H₂O₂ is scavenged by catalases, while periplasmic H₂O₂ is detoxified by l-cysteine exported from cytoplasm. l-Cystine, the oxidized form of l-cysteine, is then uptaken by the l-cystine transporter into the cytoplasm to regenerate l-cysteine. I, II, and IV represent NADH dehydrogenase, succinic acid dehydrogenase, and terminal oxidase, respectively.

The question then is why does E. coli require such a l-cysteine/l-cystine shuttle system? The inner membrane is the place where E. coli produces ATP via the respiratory chain. The process often generates superoxide (O₂^˙̄) due primarily to the leakage of electrons (Fig. 7) (9). The respiratory electron transport chain-linked ROS generation accounts for as much as 87% of the total H₂O₂ production in aerobic growth (26). Superoxide dismutases (SODs) convert O₂^˙̄ to H₂O₂. SODs exist in the periplasm (SodC) as well as the cytoplasm (SodA and SodB). Thus, as a result of the reaction to destroy the superoxide, H₂O₂ would be generated. In addition, E. coli cells are exposed to H₂O₂, which is produced by phagocytes, in the environment. Local H₂O₂ concentrations may rise up to 100 μm inside phagocytes and even more than 1 mm near H₂O₂-generating lactic acid bacteria (10). As H₂O₂ also can damage molecules in the cells, it needs to be removed. However, no H₂O₂ scavenger has been identified in the periplasm, in contrast to the cytoplasm, where multiple H₂O₂ scavengers (KatE, KatG, AhpC) exist. It should be noted that thiol peroxidase (Tpx), which was initially characterized as a periplasmic H₂O₂ scavenger, actually localizes in the cytoplasm (27). We suggest that the absence of an efficient enzyme catalyst to remove H₂O₂ from the periplasm may be one of the reasons why l-cysteine is exported to the periplasm.

Interestingly, H₂O₂ but not exogenous l-cysteine (Fig. 5) significantly induced the expression of the ydeD gene (Figs. 1C, 4, and 5). Thus, YdeD seems to be intrinsically used to protect cells from H₂O₂. However, other l-cysteine transporters, such as Yfik and Bcr, might not contribute much to this defense mechanism because the mutants that are missing one of the latter genes displayed no increase in H₂O₂ sensitivity (supplemental Fig. S1)

GSH is a predominant low molecular weight thiol in E. coli, as in the case of many other organisms, and it has been proposed to protect cells from oxidative damages such as those caused by H₂O₂ and ionizing radiations (28). However, it has been reported that a ΔgshA mutation does not cause increased sensitivities to H₂O₂ or cumene hydroperoxide (29). In addition, we, ourselves, observed here that there was no detectable change in the levels of resistance to H₂O₂ between the wild-type cells and the ΔgshA mutant whether or not YdeD is overproduced (Fig. 1D). Thus, we propose that E. coli uses l-cysteine to protect cells from periplasmic H₂O₂ even though GSH exists in the periplasm as well as in the cytoplasm (17). Interestingly, under H₂O₂ stress, genes involved in the utilization of periplasmic GSH as a source of l-cysteine were induced (Fig. 4). This finding may imply that the periplasmic GSH is also used as a source of l-cysteine that is to be provided to the periplasm to reduce oxidative stress.

Furthermore, we showed evidence that FliY is involved in l-cystine uptake in E. coli (Fig. 2C). FliY is a homologue of BspA, an L. fermentum BR11 l-cysteine binding protein. BspA is a component of the l-cysteine transport system in this organism. Here we showed that FliY is an important component of l-cystine import and that this protein is necessary for resistance of E. coli to oxidative stress (Fig. 3, A and B). The latter finding is consistent with the previous observation in L. fermentum (30). It should be noted that, as FliY is a periplasmic solute-binding protein, FliY probably cooperates with a certain transporter for the uptake of l-cystine (Fig. 7).

Interestingly, H₂O₂ induced the fliY gene as well as the ydeD gene. These findings led us to propose that the l-cysteine exporter, YdeD, and the l-cystine-binding protein, FliY, are the components of the l-cysteine/l-cystine shuttle system in the inner membrane that contribute to H₂O₂ scavenging.

As shown in Fig. 4, H₂O₂ stress did not cause any detectable increase in the expression of genes involved in the production of intracellular thiols. This finding is consistent with the finding that the intracellular l-cysteine level is strictly controlled even under oxidative stress conditions (31). If H₂O₂ is detoxified by de novo synthesized l-cysteine, cells will again face growth inhibition by the increase in l-cysteine toxicity. Thus, the l-cysteine recycle system may be beneficial for the growth of E. coli cells, because the system allows cells to scavenge H₂O₂ without growth inhibition from the presence of a large amount of de novo synthesized l-cysteine.

An intermembrane ROS defense system that is analogous to the E. coli YdeD/FliY system may also exist in the cellular compartments of eukaryotes. In fact, in lung cancer cells, an xc- l-cystine transporter (xCT) and Multi-drug Resistant Proteins (MRP) are the components of the l-cysteine/l-cystine cycle system and are involved in selenite reduction in the extracellular space (supplemental Fig. S3A) (32). In addition, Burkitt's Lymphoma cells overexpressing xCT were reported to become highly resistant to oxidative stress. Importantly, the cells exhibited H₂O₂ resistance even in the absence of GSH synthesis (33), implying the importance of l-cysteine in the resistance to H₂O₂.

Protein-disulfide isomerase, PDI, catalyzes the formation of protein disulfide bonds in the ER of eukaryotes. After the oxidation of its substrates, PDI is reduced. The reduced PDI, in turn, is oxidized by Ero1p, which then passes electrons to molecular oxygen. The latter reaction results in the generation of H₂O₂, which probably damages cells (34). However, it is not understood how cells are protected from the damage caused by the generation of ROS in the ER (supplemental Fig. S3B). An l-cysteine/l-cystine shuttle system that scavenges H₂O₂ instead of catalase might also exist in the ER lumen. Indeed, Yct1p, an l-cysteine-specific transporter from the Saccharomyces cerevisiae, was found to transport l-cysteine into the ER (35). Thus, it may be possible that, in addition to the plasma membrane, the the ER membrane might also utilize a l-cysteine/l-cystine shuttle system to supply l-cysteine as a reducing equivalent to the ER lumen. Finally, these findings including our results suggest that an analogous l-cysteine/l-cystine shuttle system may be conserved from bacteria to mammals and may play important roles by supplying a reducing equivalent to the oxidative cellular compartments.