The L-cysteine/L-cystine shuttle system provides reducing equivalents to the periplasm in Escherichia coli

Article abstract of DOI:10.1074/jbc.M109.081356

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.

Full text of DOI:10.1074/jbc.M109.081356

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 23, pp. 17479–17487, June 4, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
The L-Cysteine/L-Cystine Shuttle System Provides Reducing
□
S
Equivalents to the Periplasm in Escherichia coli*
Received for publication, November 2, 2009, and in revised form, March 26, 2010 Published, JBC Papers in Press, March 29, 2010, DOI 10.1074/jbc.M109.081356
Iwao Ohtsu1,2, Natthawut Wiriyathanawudhiwong1, Susumu Morigasaki, Takeshi Nakatani, Hiroshi Kadokura,
and Hiroshi Takagi
From the Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho,
Ikoma, Nara 630-0192, Japan
Intracellular thiols like L-cysteine and glutathione play a crit-
ical 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 toler-
ance of E. coli cells to hydrogen peroxide. We also present evi-
dence 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 recy-
cling of the oxidized L-cysteine, is also found to be important for
the hydrogen peroxide resistance of this organism. Further-
more, our analysis of the transcription of relevant genes re-
vealed 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.
threshold of cytotoxicity, the intracellular L-cysteine level is
strictly controlled.
Serine acetyltransferase, a key enzyme in the L-cysteine syn-
thesis pathway of Escherichia coli, is under the control of feed-
back 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 cyto-
plasm 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
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 con-
centrations in both prokaryotes and eukaryotes (1–3). It has
been reported that threonine deaminase, an enzyme in L-iso-
leucine 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
Ϫ
Hpx mutant lacking all of these three major enzymes accumu-
lates H2O2 in cells (10). However, these enzymes do not exist in
the periplasm, but superoxide dismutase (SodC), which gener-
ates 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.
* This work was supported by KAKENHI (Grant-in-Aid for Young Scientists on
priority areas (B) and funding from the NAIST Global COE Program) from
the Ministry of Education, Culture, Sports, Science and Technology (MEXT)
of Japan (to I. O.). This work was also supported in part by KAKENHI (Grant-
in-Aid for Scientific Research) on Priority Areas “Applied Genomics” from
MEXT of Japan and by a grant from Ajinomoto Co., Inc. (to I. O. and H. T.).
This work was further supported in part by an international research fel-
lowship from the Global COE Program in NAIST from MEXT of Japan and a
Grant-in-Aid for Scientific Research (C) (21580092) from JSPS (to H. K.).
H2O2 ϩ 2 L-cysteine 3 2H2O ϩ L-cystine
(Eq. 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
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Table S1 and Figs. S1–S3.
1 Both authors contributed equally to this work.
2 To whom correspondence should be addressed: Graduate School of Biolog-
ical Sciences, Nara Institute of Science and Technology, 8916-5 Takayama,
Ikoma, Nara 630-0192, Japan. Tel.: 81-743-72-5422; Fax: 81-743-72-5429; 3 The abbreviations used are: ROS, reactive oxygen species; ER, endoplasmic
E-mail: iohtsu@bs.naist.jp.
reticulum; SOD, superoxide dismutase.
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Cysteine/Cystine Shuttle Provides Reducing Force
TABLE 1
Bacterial strains and plasmids used
E. coli strain or plasmid
Genotype
Ref. or source
Strains
BW25113
BW25113
BW25113
JW3832
JW5182
JW5250
JW2663
JW2562
JW5363
JW1905
JW1905
pCA24N
pFliY
lacIq, rrnBT14, lacZWJ16, hsdR514, araBADAH33, rhaBADLD78
(35)
(35)
(35)
(35)
(35)
(35)
(35)
(35)
(35)
(35)
(35)
(36)
(36)
(36)
(8)
pYdeD
(Enhancing periplasmic L-cystine excreation)
pDE, pCA24N
(L-Cystine overproducer)
BW25113 dsbA::KmR
BW25113 dsbB::KmR
BW25113 ydeD::KmR
BW25113 gshA::KmR
BW25113 yfiK::KmR
BW25113 bcr::KmR
BW25113 fliY::KmR
(Weakening L-cystine uptake)
(Weakening L-cystine uptake)
fliY::KmR, pDES, pCA24N
T5-lacpromoter, pQB2-based, CmR
pCA24N, fliYgene on 0.8 kb DNA fragment
pCA24N, dsbAgene on 0.6 kb DNA fragment
lacpromoter, pACYC184-based, CmR
pSTV29, ydeDgene on 1.5 kb DNA fragment
pDsbA
pSTV29
pYdeD
(8)
L-cysteine transporters in E. coli. In this report, we show evi- (OD660). H2O2 was added to the medium at the indicated con-
dence 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 pro-
pose that the inducible L-cysteine/L-cystine shuttle system
plays an important role for the resistance of cells to H2O2 in the
periplasm.
centration. 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 ␮lofL-[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 incorpo-
rated into the cells on the filter was determined by liquid
scintillation counter LS6500 (Beckman).
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
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.
Theintracellularamino acidswerethenextractedfrom the cells by
boiling the cell suspension for 10 min using a heat block. After
centrifugation (1 min at 15,000 ϫ 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 ␮l of the sample was incubated with 200 ␮l 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 (Emax 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.
Ϫ
supplied by the National BioResource Project (NBRP). A Hpx
mutant strain lacking two catalases (KatE and KatG) and a per-
oxidase (AhpCF)(10) was kindly provided by James A. Imlay.
High L-cysteine-producing plasmid pDES (supplied by Ajino-
moto) 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 pre-
cursor (13). The medium copy number vector pSTV29 was pur-
chased from Takara Bio Co. (Kyoto, Japan). The construction of
pYdeD has already been described (8).
Media and Growth Conditions—Luria-Bertani (LB) com-
plete medium or SM1 medium that is supplemented with LB
broth, L-methionine, and thiosulfate (8) was used for the gen-
eral cultivations of E. coli. When appropriate, antibiotics were
added at 50 ␮g/ml (for kanamycin), 30 ␮g/ml (for chloram-
phenicol), and 10 ␮g/ml (for tetracycline). Growth of cultures
Quantification of Total Free L-Cysteine (L-Cysteine Plus
L-Cystine)—To determine the amount of the total L-cysteine,
was monitored by measuring of the optical density at 660 nm L-cystine in the sample was reduced by incubation with 5 mM
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Cysteine/Cystine Shuttle Provides Reducing Force
each gene was normalized to that of
rrsH in the same sample. The cycle
threshold (Ct) value for each reac-
tion 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
Ϫ⌬⌬Ct
via the 2
method for each
sample in triplicate, in which 1 indi-
cates no change in abundance (16).
Hydrogen Peroxide Measure-
ments—The concentration of H2O2
in the sample was measured using
coloring reagent mixture contain-
ing peroxidase, 4-aminoantipyrine,
and phenol as a specific pink prod-
uct forms from oxidative condensa-
tion of phenol, 4-aminoantipyrine,
and H2O2. 5 ␮l of sample was incu-
bated with 1 ml of the coloring rea-
gent mixture (Wako) at room tem-
perature for 10 min. Then, the
amount of the pink product derived
from H2O2 in the original sample
was determined by measuring ab-
sorbance at 505 nm (A505).
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 ⌬ydeD mutant JW5250 (⌬ydeD) 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
afteradditionof0.88mM H2O2(filledbar)comparedwiththosewithoutstresstreatment(emptybar). D, growth
of the wild-type strain (BW25113), or a ⌬gshA mutant (JW2663) carrying pSTV29 or pYdeD on LB solid medium
containing 0.5 mM H2O2.
Redox State Analysis on DsbA—
dithiothreitol in 200 mM Tris-HCl buffer (pH 8.6) for 10 min. To determine the in vivo redox states of DsbA, the free cysteine
Then the amount of L-cysteine in the reduced sample was residues of the protein were acid trapped and alkylated with the
determined using the method by Gaitonde.
high molecular mass reagent AMS (Invitrogen) as described
Determination of L-Cystine—The amount of L-cystine was (17). Alkylated samples were separated by SDS-PAGE, and
calculated by subtracting the amount of L-cysteine from that of detected by Western blot analysis with anti-DsbA that has been
total free L-cysteine.
described (18).
Real-time Quantitative Polymerase Chain Reaction Analysis—
Microscopic Analysis—Cultures, grown in LB complete
Primers used in this study were designed using the Primer medium at 37 °C overnight, were diluted 1:100-fold into the
Express (Applied Biosystems, Foster City, CA; supplemental same medium with or without 100 ␮M H2O2. After growth was
Table S1). E. coli cells were lysed with 1 mg/ml lysozyme in 10 continued for 8 h, cells were harvested for microscopic analy-
mM Tris-HCl (pH 8.0) containing 1 mM EDTA buffer. Total ses. The pictures of the cells were taken with the Axiovert 200 M
RNA was prepared using the RNeasy Mini Kit (Qiagen, Valen- microscope (ZEISS, Osaka, Japan). Images were collected and
cia, CA) and the RNase-Free DNase Set (Qiagen). Complemen- processed using the AxioVision 4.5 software (ZEISS, Osaka,
tary DNA was synthesized from 1 ␮g of total RNA using the Japan).
High Capacity cDNA Reverse Transcription Kit (Applied Bio-
RESULTS
systems). For real-time quantitative polymerase chain reaction
(qPCR), cDNA was amplified with oligonucleotide primers spe-
The L-Cysteine Transporter YdeD Contributes to Hydrogen
cific to each target gene using the 7300 Real-Time PCR System Peroxide Tolerance in E. coli—To address our hypothesis con-
(Applied Biosystems). Reactions contained Power SYBR PCR cerning the role of L-cysteine transporters in the detoxification
Master Mix (Applied Biosystems), forward and reverse primers of H2O2, we first investigated whether YdeD is involved in
(0.1 ␮M each), and a cDNA template (20 ng). For the dissocia- H2O2 resistance in E. coli. For this purpose, ⌬ydeD mutant and
tion curve analysis, the following conditions were used: initial wild-type cells were transformed with either pYdeD, a middle
steps at 50 °C for 2 min, 95 °C for 10 min; 40 cycles of PCR at copy plasmid carrying the ydeD gene with the original pro-
95 °C for 15 s, 60 °C for 1 min; and final steps at 95 °C for 15 s, moter, or the empty vector pSTV29. Serial dilutions of the over-
60 °C for 30 s, and 95 °C for 15 s (15). The melting curve for each night culture in LB medium were spotted onto the surface of
PCR product was determined according to the supplier’s guide- a H2O2-containing LB agar plate. The cells were then grown
lines, ensuring specific amplification of the target gene. Quan- at 30 °C for 24 h. The ydeD deletion mutant cells displayed
titative values were obtained as the threshold PCR cycle num- increased H2O2 sensitivity (Fig. 1A). In contrast, mutants
ber (Ct) when the increase in the fluorescent signal of the PCR missing either one of the other L-cysteine transporter genes,
product showed exponential amplification. The mRNA level of yfiK or bcr, did not show an increased H2O2 sensitivity
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Cysteine/Cystine Shuttle Provides Reducing Force
(supplemental Fig. S1). Remarkably, both wild-type cells and
⌬ydeD 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 resist-
ance in E. coli.
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-␥-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 resis-
tance of E. coli. This gene encodes ␥-glutamylcysteine syn-
thetase, a key enzyme in GSH synthesis. We found that H2O2
tolerance was increased by YdeD overexpression even in the
⌬gshA mutant (Fig. 1D). Importantly, no significant differ-
ence in H2O2 tolerance was observed between the wild-type
cells and the ⌬gshA mutant. These findings suggest that, in
contrast to L-cysteine, GSH may not contribute to the H2O2
resistance of E. coli cells.
FIGURE2. L-CysteineisoxidizedtoL-cystineintheperiplasm. A, ratioofthe
oxidized L-cysteine (L-cystine) to the total intracellular L-cysteine. The wild-
type strain (BW25113) and its ⌬fliY-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)wastransformedwithpDESplasmid. Thetransformantwasgrown
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 ⌬fliY
mutant (open circle).
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 main-
tain 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-phospho-
glycerate dehydrogenase (T410stop), which are released cells carrying the pDES plasmid. As shown in Fig. 2B, the ratio
from feedback inhibition by L-cysteine and L-serine, respec- of L-cystine to the total L-cysteine of the cells after treatment
tively, and YdeD (8).
with 0.88 mM H2O2 (52%) was higher than that of the cells
As shown in Fig. 2A, L-cystine accounted for about 30% of the before treatment with H2O2 (41%). This ratio reached 73% after
total free cysteine (L-cysteine plus L-cystine) in E. coli cells car- treatment with 8.8 mM H2O2 (data not shown). These results
rying the pDES plasmid. The further overexpression of DsbA, are consistent with the finding that endogenous L-cysteine can
which oxidizes peptidyl-L-cysteine residues into a disulfide detoxify exogenous H2O2 in the periplasm.
bridge in the periplasm (20), facilitated L-cystine formation: the
Overexpression of the L-Cysteine Transporter YdeD Can
ratio of L-cystine to the total cysteine reached about 50% (Fig. Affect the Redox Environment of the Periplasm—We showed
2A). These findings suggest that a significant fraction of endo- evidence that the overexpression of the L-cysteine transporter,
genously produced L-cysteine is in fact exported into the YdeD, confers H2O2 resistance to E. coli (see above). We next
periplasm, where it is oxidized into L-cystine even in the wanted to examine the influence of the L-cysteine transporter
absence of a specific exogenous oxidative stressor. These obser- overexpression on the redox environment of the periplasm. To
vations are consistent with the proposed role of L-cysteine as a this end, we investigated the redox state of DsbA after the over-
reducing equivalent in the periplasm.
expression of the L-cysteine transporter from the pYdeD plas-
The Intracellular L-Cystine/L-Cysteine Ratio Increases Upon mid because DsbA is a periplasmic protein with a pair of redox-
Treatment of the Cells with Hydrogen Peroxide—If L-cysteine is active L-cysteines that can alternate between the oxidized and
used as a reducing equivalent to detoxify H2O2 in vivo, a portion the reduced states. Importantly, these two L-cysteines are
of L-cysteine will be oxidized after treatment of the cells with normally maintained in the fully oxidized state in vivo by the
H2O2. To test this, we also determined the intracellular con- membrane protein DsbB, which passes electrons from DsbA
tents of L-cysteine and L-cystine after treatment of cells with to quinones in the respiratory chain (17). To study the oxi-
H2O2. To detect the changes in the intracellular L-cystine/L- dative state of DsbA in vivo, we used an alkylating reagent,
cysteine ratio before and after H2O2 treatment, we used E. coli AMS, which modifies free L-cysteines. The modification of
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