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Journal of Bacteriology, December 2000, p. 6842-6844, Vol. 182, No. 23
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Hydrogen Peroxide Activates the SoxRS Regulon
In Vivo
Manuel
Manchado,
Carmen
Michán, and
Carmen
Pueyo*
Departamento de Bioquímica y
Biología Molecular, Universidad de Córdoba, 14071 Córdoba, Spain
Received 12 July 2000/Accepted 12 September 2000
 |
ABSTRACT |
By multiplex reverse transcription-PCR, we demonstrate that the
SoxRS response, which protects cells against superoxide toxicity, is
triggered also by hydrogen peroxide. SoxR-dependent inductions of 7.3-, 7.6-, 4.6-, 2.2-, and 2.6-fold were quantified for soxS, micF, sodA, inaA, and
fpr transcripts, respectively. This finding suggests an
extensive and tight connectivity between different regulatory pathways
in the Escherichia coli response to oxidative stress.
| |
TEXT |
Key regulators of adaptive responses
to hydrogen peroxide (H2O2) and superoxide
anion (O2·
) are OxyR and SoxR
together with SoxS, respectively (recently reviewed in reference
11). H2O2 oxidizes the
transcription factor OxyR, leading to the formation of an
intramolecular disulfide bond (17). Oxidized OxyR then
induces the transcription of a set of genes, including katG
(hydroperoxidase I), dps (a nonspecific DNA binding
protein), ahpCF (alkyl hydroperoxide reductase) and gorA (glutathione reductase). Superoxide-generating
compounds, such as paraquat, activate the transcription factor
SoxR by the univalent oxidation of the 2Fe-2S clusters of the protein
through an unknown mechanism (11). Oxidized SoxR then
induces the expression of a second transcription factor, SoxS, which in
turn activates the transcription of a set of genes. Among the
SoxRS-regulated genes are micF (a regulatory RNA),
sodA (manganese superoxide dismutase), inaA
(unknown function), and fpr (NADPH- ferredoxin reductase) (10, 11). Redox-cycling agents also activate the syntheses of proteins that are induced by H2O2
and controlled by OxyR, due to the spontaneous and superoxide
dismutase-mediated conversion of
O2· to H2O2
(4, 15). In contrast, it is commonly believed that H2O2 is unable to switch on the SoxRS regulon
expression (4, 8, 14, 15).
We have devised a multiplex reverse transcription-PCR for the
simultaneous quantitation of the in vivo transcription of more than 10 different target genes (2) (M. Manchado,
C. Michán, M. Cousinou, G. Dorado and C. Pueyo,
unpublished data). In this protocol, all target genes, a housekeeping
gene, and one or two external standards are amplified in the same
reaction tube. Specific fluorescent primers are used, and amplification
products are analyzed with a DNA sequencer. Putative variations in the
expression of the housekeeping gene are controlled by the external
standards. Expressions of the targets relative to the reference (or to
one of the external standards) are measured. Recently, we have
experimentally demonstrated that our methodology fulfills all
theoretical requirements for precise quantification of both induction
and repression of gene transcription (M. Manchado, C. Michán, M. Cousinou, G. Dorado and C. Pueyo,
unpublished data). Because of the PCR amplification step, our method
displays a much higher sensitivity than those of current techniques for
mRNA quantitation, such as Northern blotting or primer extension
analyses. Here, we used this sensitive experimental approach to
investigate if H2O2 is able to trigger the
expression of the SoxRS regulon, in order to better define the
coordination between the OxyR and SoxRS regulatory networks of Escherichia coli.
H2O2 induces sodA
transcription.
The putative activation of the SoxRS regulon by
H2O2 was first investigated by examining the
expression of the sodA gene in wild-type cells exposed to a
wide range of H2O2 concentrations (varying from
0.25 to 1,000 µM) (Fig. 1). While the
lower-dose treatments (from 1 to 100 µM H2O2)
induced specifically the transcription of selected OxyR-regulated genes
(katG, dps, ahpCF, gorA),
the higher-dose treatments (
500 µM H2O2)
resulted in the activation of sodA. In contrast with
previous data (14), inductions of 2.1- and 4.7-fold were
readily seen for sodA mRNA in response to 500 and 1,000 µM
H2O2, respectively, immediately after the addition of the oxidant (Fig. 1).
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FIG. 1.
H2O2 induces sodA
transcription. Wild-type cells grown in M9 minimal medium (optical
density at 600 nm of 0.2) were treated with the
H2O2 concentration (µM) indicated in the
abscissa. Samples were collected immediately (<1 min) after the
addition of the oxidant. RNA purification, cDNA synthesis, and
multiplex PCRs were carried out as described previously (7).
An exogenous fragment of the gene (CYP1A) coding for
cytochrome P4501A from Liza aurata (9) was
coamplified with the target genes and the reference gapA
gene. The fluorescence signal of each PCR product was compared to that
of CYP1A (noncompetitor heterologous standard). Data were
from an average of eight multiplexed PCR amplifications. Values from
treated samples were divided by those from the corresponding control
(unexposed bacteria). Statistical comparisons were done by an analysis
of variance test. Significant increments are indicated by filled-in
symbols.
|
|
In previous studies (
2,
7,
9), the amounts of the mRNAs of
interest were determined with reference to the mRNA level
of the
gapA gene (used as an internal standard), which codes for
a
key enzyme of the glycolytic and gluconeogenesis pathways
(
D-glyceraldehyde-3-phosphate
dehydrogenase). As discussed
elsewhere (M. Manchado, C. Michán,
M. Cousinou, G. Dorado and C. Pueyo, unpublished data), it cannot
be taken for
granted that the so-called housekeeping gene will
maintain a steady
level of expression under all circumstances.
Indeed, we observed a
small (
twofold), though statistically significant,
increase in
gapA transcription in response to
500 µM
H
2O
2. Thus,
to circumvent possible
underestimations of transcriptional inductions,
all data in this
work were compared to an external standard. A
noncompetitor
heterologous standard was used for Fig.
1, and a
competitor
homologous standard was used for Fig.
2 and
3. As described
elsewhere
(M. Manchado, C. Michán, M. Cousinou, G. Dorado and
C. Pueyo,
unpublished data), both types of external standards
are equally useful
for monitoring changes in the expression levels
of the genes, but the
competitor has the additional advantage
of using the same pair of
primers that amplifies the reference
gene.
SoxRS regulon activation by H2O2 depends on
SoxR.
The results above and the particularly complex
multiregulated transcription of sodA (13)
prompted us to investigate the effect of H2O2
treatment on the expression of other SoxRS-regulated genes, such as
soxS, micF, inaA, and fpr
(Fig. 2 and
3).
H2O2 at a 100 µM concentration increased
instantaneously the transcript levels of soxS and
micF (Fig. 2), whereas 5- to 10-fold-higher concentrations
were required to induce significantly the sodA expression
(Fig. 1 and 2). Nonetheless, significant increments in sodA
transcription and also in inaA and fpr
transcription were observed when bacteria were exposed to 100 µM
H2O2 for 10 min (Fig. 3). These results clearly
demonstrate that H2O2 stress conditions that
are regularly used to induce the OxyR response (e.g., references 1, 12 and 17) strongly activate
the in vivo transcription of the SoxRS regulon. Therefore, induction
levels of 7.3- and 7.6-fold (<1 min of treatment) and of 4.6-, 2.2-, and 2.6-fold (10 min of treatment) were quantified for soxS,
micF, sodA, inaA, and fpr,
respectively, in response to 100 µM H2O2
(Fig. 3). Interestingly, the activation of the SoxRS-regulated genes by
H2O2 was seen concomitantly with a decline in
the amounts of the OxyR-regulated transcripts (e.g., the
katG transcript levels shown in Fig. 2).
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FIG. 2.
H2O2 induces SoxRS regulon
transcription. Treatments and analyses were as described in the legend
to Fig. 1. A laboratory-engineered fragment (gapA*) of the
housekeeping gene was used as an external standard (M. Manchado, C. Michán, M. Cousinou, G. Dorado, and C. Pueyo, unpublished data).
The fluorescence signal of each PCR product was compared to that of
gapA* (competitor homologous standard). Error bars were
estimated from the corresponding standard error of the mean values.
Significant increments are marked with an asterisk.
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FIG. 3.
SoxR regulates the induction of SoxRS regulon
transcription by H2O2. Wild-type,
 oxyR::kan, and
soxR::cat bacteria were treated with
100 µM H2O2. Samples were collected
immediately (<1 min) or at 10 min after the addition of
H2O2. The fluorescence signal of each PCR
product was compared to that of a competitor homologous standard
(gapA*). See the legends to Fig. 1 and 2 for more details.
|
|
Figure
3 provides further evidence that the induction of
soxS,
micF,
sodA,
inaA, and
fpr expression by H
2O
2 was abolished
by the introduction of the
soxR9::
cat mutation (
16),
indicating
a strict dependence on a functional SoxR regulator. In
contrast,
this transcriptional up-regulation was preserved in the
strain
with the
oxyR::
kan mutation
(
6), indicating that OxyR is not
involved in the
SoxR-mediated response to H
2O
2. In fact,
generally,
the induction ratios of the SoxRS-regulated genes by
H
2O
2 were
somewhat higher in the
oxyR::
kan mutant than in the
wild-type
strain, which might be attributed to the inability of
OxyR-defective
bacteria to induce
katG transcription
(
7), i.e., the H
2O
2 breakdown
catalyzed by hydroperoxidase
I.
The activation of the SoxRS regulon by H
2O
2
stress in a SoxR-dependent manner might be an indirect result,
depending on the
formation of an
H
2O
2-generated signal that is sensed by SoxR
rather
than on H
2O
2 itself. In fact, a crucial
unanswered question concerns
the nature of the signal(s) sensed by
SoxR. Another possibility
is that H
2O
2
somehow could interfere with the unknown system that
keeps SoxR
in its reduced, inactive form in vivo, e.g., by depleting
the electron
source for the recently discovered SoxR reductase
(
5).
Indeed, we have demonstrated (
9) that both glutaredoxin
and
thioredoxin pathways, which consume NADPH, are triggered in
the
OxyR-mediated response to H
2O
2. Whatever the
mechanism, the
H
2O
2-mediated induction of the
SoxRS response may reflect the
existence of multiple pathways for SoxR
activation and deactivation,
as discussed by others (
3).
This versatility would let the
SoxR system respond to a wide range of
environmental and intracellular
changes indicative of possible
oxidative stress. The multiplex
reverse transcription-PCR
methodology used in this work for quantification
of transcription
will be of relevance in further experiments.
Nevertheless,
quantifications at the protein level will also be
necessary in order to
unravel the relationships between mRNA production
and protein
synthesis.
| |
ACKNOWLEDGMENTS |
M.M. and C.M. contributed equally to this paper, and both should be
considered first authors.
This work was supported by grant PB98-1627 (DGES) and by Junta de
Andalucía (group CVI 0187). M.M. was the recipient of a predoctoral fellowship from the Spanish Ministry of Education and
Culture (MEC).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Bioquímica y Biología Molecular, Campus de Rabanales
edificio C-6, Carretera Madrid-Cádiz Km 396-a, Universidad de
Córdoba, 14071 Córdoba, Spain. Phone: 34 957 218695. Fax:
34 957 218688. E-mail: bb1pucuc{at}uco.es.
| |
REFERENCES |
| 1.
|
Altuvia, S.,
M. Almiron,
G. Huisman,
R. Kolter, and G. Storz.
1994.
The dps promoter is activated by OxyR during growth and by IHF and  S in stationary phase.
Mol. Microbiol.
13:265-272[Medline].
|
| 2.
|
Gallardo-Madueño, R.,
J. F. Leal,
G. Dorado,
A. Holmgren,
J. Lopez-Barea, and C. Pueyo.
1998.
In vivo transcription of nrdAB operon and of grxA and fpg genes is triggered in Escherichia coli lacking both thioredoxin and glutaredoxin 1 or thioredoxin and glutathione, respectively.
J. Biol. Chem.
273:18382-18388[Abstract/Free Full Text].
|
| 3.
|
Gaudu, P.,
S. Dubrac, and D. Touati.
2000.
Activation of SoxR by overproduction of desulfoferrodoxin: multiple ways to induce the soxRS regulon.
J. Bacteriol.
182:1761-1763[Abstract/Free Full Text].
|
| 4.
|
Greenberg, J. T., and B. Demple.
1989.
A global response induced in Escherichia coli by redox-cycling agents overlaps with that induced by peroxide stress.
J. Bacteriol.
171:3933-3939[Abstract/Free Full Text].
|
| 5.
|
Kobayashi, K., and S. Tagawa.
1999.
Isolation of reductase for SoxR that governs an oxidative response regulon from Escherichia coli.
FEBS Lett.
451:227-230[CrossRef][Medline].
|
| 6.
|
Kullik, I.,
J. Stevens,
M. B. Toledano, and G. Storz.
1995.
Mutational analysis of the redox-sensitive transcriptional regulator OxyR: regions important for DNA binding and multimerization.
J. Bacteriol.
177:1285-1291[Abstract/Free Full Text].
|
| 7.
|
Michán, C.,
M. Manchado,
G. Dorado, and C. Pueyo.
1999.
In vivo transcription of the Escherichia coli oxyR regulon as a function of growth phase and in response to oxidative stress.
J. Bacteriol.
181:2759-2764[Abstract/Free Full Text].
|
| 8.
|
Nunoshiba, T.,
E. Hidalgo,
C. F. Amabile Cuevas, and B. Demple.
1992.
Two-stage control of an oxidative stress regulon: the Escherichia coli SoxR protein triggers redox-inducible expression of the soxS regulatory gene.
J. Bacteriol.
174:6054-6060[Abstract/Free Full Text].
|
| 9.
|
Prieto-Álamo, M. J.,
J. Jurado,
R. Gallardo-Madueño,
F. Monje-Casas,
A. Holmgren, and C. Pueyo.
2000.
Transcriptional regulation of glutaredoxin and thioredoxin pathways and related enzymes in response to oxidative stress.
J. Biol. Chem.
275:13398-13405[Abstract/Free Full Text].
|
| 10.
|
Rosner, J. L., and J. L. Slonczewski.
1994.
Dual regulation of inaA by the multiple antibiotic resistance (Mar) and superoxide (SoxRS) stress response systems of Escherichia coli.
J. Bacteriol.
176:6262-6269[Abstract/Free Full Text].
|
| 11.
|
Storz, G., and J. A. Imlay.
1999.
Oxidative stress.
Curr. Opin. Microbiol.
2:188-194[CrossRef][Medline].
|
| 12.
|
Tao, K.,
K. Makino,
S. Yonei,
A. Nakata, and H. Shinagawa.
1989.
Molecular cloning and nucleotide sequencing of oxyR, the positive regulatory gene of a regulon for an adaptive response to oxidative stress in Escherichia coli: homologies between OxyR protein and a family of bacterial activator proteins.
Mol. Gen. Genet.
218:371-376[CrossRef][Medline].
|
| 13.
|
Touati, D.
1997.
Superoxide dismutases in bacteria and pathogen protists, p. 447-493.
In
J. G. Scandalios (ed.), Oxidative stress and the molecular biology of antioxidant defenses. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 14.
|
Touati, D.
1988.
Transcriptional and posttranscriptional regulation of manganese superoxide dismutase biosynthesis in Escherichia coli, studied with operon and protein fusions.
J. Bacteriol.
170:2511-2520[Abstract/Free Full Text].
|
| 15.
|
Walkup, L. K., and T. Kogoma.
1989.
Escherichia coli proteins inducible by oxidative stress mediated by the superoxide radical.
J. Bacteriol.
171:1476-1484[Abstract/Free Full Text].
|
| 16.
|
Wu, J., and B. Weiss.
1992.
Two-stage induction of the soxRS (superoxide response) regulon of Escherichia coli.
J. Bacteriol.
174:3915-3920[Abstract/Free Full Text].
|
| 17.
|
Zheng, M.,
F. Aslund, and G. Storz.
1998.
Activation of the OxyR transcription factor by reversible disulfide bond formation.
Science
279:1718-1721[Abstract/Free Full Text].
|
Journal of Bacteriology, December 2000, p. 6842-6844, Vol. 182, No. 23
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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