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J Biol Chem, Vol. 275, Issue 18, 13398-13405, May 5, 2000
From the We examined the in vivo expression of
up to 16 genes encoding for components of both glutaredoxin and
thioredoxin systems and for members of the OxyR and SoxRS regulons. We
demonstrated that grxA (Grx1) transcription is triggered in
bacteria lacking Trx1 (trxA) and GSH (gshA) in
an OxyR-dependent manner. We also indicated that, unlike
OxyR, SoxR is not constitutively activated in the oxidizing environment
of trxA gshA mutants. We discovered that the lack of Trx1
plus GSH increases the steady-state levels of Trx reductase
(trxB) and Trx2 (trxC) transcripts. This
increase and the trxB and trxC up-regulation
caused by the constitutive oxyR2 allele indicate that OxyR
also plays a role in the regulation of the thioredoxin pathway. On the
contrary, no change in the expression of genes for Trx1, Grx2, and Grx3
was observed. Transcription of nrdAB (RRase) was not
induced by oxidative stress yet was induced by hydroxyurea (RRase
inhibitor). Induction level was as the enhanced nrdAB basal
expression of trxA grxA mutants, indicating that RRase operation without Trx1 and Grx1 must lead to disturbances sensed as
those caused by hydroxyurea. We also demonstrated an inverse relation
between nrdAB expression and that of genes coding for components of both glutaredoxin (grxA, gorA)
and thioredoxin (trxB, trxC) systems.
Thioredoxins (Trxs)1 and
glutaredoxins (Grxs) are known hydrogen donors for ribonucleotide
reductase (RRase), the essential key enzyme for deoxyribonucleotide and
DNA biosynthesis. In the reduced form, both Trxs and Grxs contain two
redox-active cysteine thiols, which by dithiol-disulfide interchange
reduce an acceptor disulfide in the active center of RRase. Trxs and
Grxs differ in the manner they are reduced in the cell, although
ultimately reducing equivalents come from NADPH. Thus, the thioredoxin
system is composed of NADPH, the flavoprotein thioredoxin reductase, and Trx; and the glutaredoxin system of NADPH consists of the flavoprotein glutathione reductase, the ubiquitous tripeptide glutathione (GSH), and Grx (1) (Fig.
1).
Escherichia coli contains genetic information for three
different RRases (2). The NrdAB (encoded by the nrdAB
operon) is active during aerobiosis, NrdDG is strictly anaerobic and
uses formate as electron donor, and NrdEF is a cryptic enzyme of
unknown physiological role. At present, two thioredoxins (Trx1 and
Trx2) and three glutaredoxins (Grx1, Grx2, and Grx3) have been
discovered in the bacterial cytoplasm (1, 3, 4). Grx1 (grxA)
and Trx1 (trxA) are the two main hydrogen donors of E. coli RRase (5, 6). In comparison, Grx3 (grxC) is a very
inefficient reductant (about 5% of the catalytic activity of Grx1),
while Grx2 (grxB) lacks activity as hydrogen donor for RRase
(3). Similar to Trx1, Trx2 (trxC) is a functional electron
donor for NrdAB. However, compared with Trx1, Trx2 is a 5-fold less
abundant protein and a 2.5-fold less efficient enzyme (4). A fifth
potential reductant is the NrdH-redoxin, which has thioredoxin-like
activity but a glutaredoxin-like amino acid sequence (7). NrdH is a functional hydrogen donor for RRase with higher specificity for the
NrdEF than for the NrdAB enzyme. However, the physiological function of
NrdH in E. coli is not well understood, since the nrdH gene is located in the same poorly transcribed operon
as nrdEF (8).
Different inducible responses are critical in protecting E. coli from the damage caused by oxidative stress (9). Key
regulators of adaptive responses to hydrogen peroxide
(H2O2) and superoxide anion (O The OxyR protein is directly sensitive to oxidation, and only the
oxidized OxyR is capable of activating transcription (11). Recent
studies have revealed that oxidation of OxyR leads to the formation of
an intramolecular disulfide bond and that OxyR is reduced and
deactivated by enzymatic reduction with Grx1 at the expense of
glutathione (12). SoxR is a homodimer containing two [2Fe-2S] centers
essential for its transcriptional activity (13). The univalent
oxidation of the iron-sulfur clusters appears to be the mechanism for
activating SoxR as a transcriptional factor (14, 15). The mechanism of
SoxR reduction/deactivation has not been elucidated, except for an
NADPH-dependent SoxR reductase that has recently been
purified from E. coli (16). Likewise, it has recently been
suggested that cellular monothiols, like GSH, and low molecular weight
dithiols and dithiols proteins, such as Trx1, may contribute to SoxR
regulation by promoting the disassembly (14) and reassembly (17) of the
[2Fe-2S] clusters.
Recently, we reported that the in vivo transcription of the
nrdAB operon and of the grxA and fpg
(coding for formamidopyrimidine-DNA glycosylase; Ref. 18) genes is triggered in E. coli lacking both Trx1 and Grx1 (trxA grxA mutant) or
Trx1 and GSH (trxA gshA mutant) (19). The present study
aimed to examine the role of OxyR and SoxRS in determining such
increased basal levels of expression. To this end, trxA grxA
and trxA gshA defective bacteria, additionally mutated in
the oxyR or soxRS regulatory genes, have been
isolated, and the in vivo expression of up to 16 different
genes has been examined by reverse transcription/multiplex polymerase
chain reaction (RT/MPCR). The genes studied include most components of
both thioredoxin and glutaredoxin systems as well as known members of
the OxyR and SoxRS regulons.
Materials--
Media and chemical reagents were prepared or
purchased as described previously (19). Hydrogen peroxide, paraquat,
and hydroxyurea were purchased from Sigma.
Media--
Bacteria were grown in Luria-Bertani (LB) nutrient
broth or M9 minimal medium (20). The media were supplemented (when
necessary) with chloramphenicol (34 µg/ml), kanamycin (50 µg/ml),
or tetracycline (20 µg/ml). The minimal medium contained arginine (40 µg/ml), D-biotin (5 µg/ml), thiamine (5 µg/ml),
glucose (2 g/liter), and casamino acids (2 g/liter).
Bacterial Strains--
Strains used are listed in Table
I. All strains are E. coli
K-12, UC5710 (21) being considered the parental wild type. UC844,
UC827, and UC859 have been previously described (22, 23). Strains were
constructed by P1 transduction (24). The mutant alleles of the genes
used to construct the strains are as follows:
Treatments--
E. coli cells from an overnight
culture in LB broth were diluted 100-fold into 50 ml of M9 minimal
medium and incubated at 37 °C and 150 rpm to reach an
A600 of 0.2. At this stage, the bacteria were
further grown in the absence or the presence of hydrogen peroxide,
paraquat, hydroxyurea, or diethylmaleate for a fixed time period. The
cells were then rapidly cooled to 0 °C for total RNA purification.
RNA Purification--
Total RNA was extracted using the hot
phenol method previously described (30). The quality of the samples was
checked electrophoretically, and quantification was done
spectrophotometrically. At least two independent RNA preparations were
isolated for each experimental condition.
Primers--
Primers used are listed in Table
II. Set A has been previously described
(19). Set B and set C were designed with the Primer Select 3.03/96 (DNA
Star, Madison, WI) and Oligo 5.0/96 (National Biosciences, Plymouth,
MN) programs, in order to obtain the highest specificity and
performance in multiplexed PCRs. Target genes code for the two subunits
of NrdAB (nrdA, nrdB); Trx1 and Trx2 (trxA, trxC); Trx reductase (trxB);
Grx1, Grx2, and Grx3 (grxA, grxB,
grxC); glutathione reductase (gorA);
Reverse Transcription/Multiplex PCR--
Synthesis of cDNA
was carried out with the GeneAmp RNA PCR kit, as described previously
(19). In short, 1 µg of bacterial RNA (plus 30 pg of external
standard RNA, in the case of set C) was retrotranscribed for 15 min at
42 °C with 2.5 units of murine leukemia virus reverse transcriptase,
using random hexamers. The enzyme was inactivated by heating for 5 min
at 99 °C. Each RNA sample was retrotranscribed on three separated
occasions. PCR amplification of cDNA was carried out using the
primer pair sets listed in Table II. As detailed by
Gallardo-Madueño et al. (19) for set A, PCR conditions
were optimized to produce fluorescence intensities only of the desired
products and in the range of linearity. The primer concentrations were
titrated so that the genes yielded quantifiable amounts of PCR products
over a wide range of different expression levels when co-amplified. The
multiplex PCR amplification was performed in a mixture (25 µl final
volume) containing 1.5 units of DNA polymerase, 2.5 µl of MPCR buffer
3, a 1 mM concentration of each dNTP, and primers at the
following amounts: (i) set A, 2.75 pmol (nrdA), 3 pmol
(nrdB), 1.25 pmol (trxA), 1.25 pmol
(grxA), 3 pmol (gorA), 2.5 pmol (fpg),
and 2 pmol (gapA); (ii) set B, 0.8 pmol (nrdA),
3.8 pmol (trxA), 2.8 pmol (grxA), 0.76 pmol
(grxB), 1.4 pmol (grxC), 0.36 pmol
(katG) or 2.4 pmol (oxyS), 1.15 pmol (soxS), and 0.74 pmol (gapA); (iii) set C, 2.75 pmol (trxB), 3.25 pmol (trxC), 2.75 pmol
(gorA), 3.0 pmol (gshA), 2.5 pmol
(tpx), 3.5 pmol (zwf), 1.25 pmol
(gapA), and 3.5 pmol (external standard). Twenty-seven
cycles of PCR were performed with set A, 24 cycles with set B, and 25 cycles with set C. Each cycle consisted of 1 min of denaturation at
94 °C, 15 s of annealing at 70 °C, and 30 s for
enzymatic primer extension at 72 °C. Multiplex PCR products were
quantified as described previously (19). Differences among PCR outcomes
were normalized by comparing the fluorescence intensity of each band to
that resulting from gapA amplification (internal standard).
Samples for comparison of different experimental conditions or
different bacterial strains were handled in parallel. Data are the
means ± S.E. from n independent multiplexed PCR
amplifications. Comparison between groups was done by Student's
t test.
We have recently devised a sensitive, rapid, nonradioactive, and
semiquantitative procedure (RT/MPCR) for comparing the in vivo expression of metabolically related genes (19, 31). In this
technique, the target genes and a constitutively expressed reporter
gene are coamplified in the same reaction vessel. Specific fluorophor-labeled primers are used, and amplification products are
subsequently analyzed with a DNA sequencer (GeneScan). The levels of
expression of the genes of interest are presented in reference to the
internal standard. Here we have used three sets of primers to examine
the expression of genes coding for most components of both thioredoxin
and glutaredoxin systems as well as pivotal members of the OxyR and
SoxRS regulons. One of the sets includes an external standard to
control the potential variability of the reporter gene.
Gene Expression Induction by Oxidative Stress--
Hydrogen
peroxide activates the transcription factor OxyR through the oxidation
of two cysteines and formation of an intramolecular disulfide bond
(12). Superoxide-generating compounds, such as paraquat, activate the
transcription factor SoxR by oxidizing the [2Fe-2S] clusters in the
protein through an unknown mechanism (14-15, 33). By using the set A
of primers (19), we examined the expression of genes coding for the
NrdAB ribonucleotide reductase (nrdA and nrdB),
its two main hydrogen donors Trx1 (trxA) and Grx1
(grxA), the glutaredoxin pathway enzyme glutathione
reductase (gorA), and the DNA repair glycosylase Fpg
(fpg), in response to increasing concentrations of
H2O2 and paraquat (Fig.
2). Treatment with
H2O2 stimulated the expression of the two genes
(gorA and grxA), identified as being under the
oxyR control (11, 31, 34), as well as that of the
fpg gene. Maximal induction levels of ~3.5-, ~1.6-, and
~13.0-fold were observed for fpg, gorA, and grxA, respectively, after 10 min of treatment with
concentrations of 100 µM H2O2.
Paraquat also activated the expression of these three genes but at
higher concentrations than did H2O2. On the contrary, expression of nrdA, nrdB, and
trxA genes was not induced after oxidative stress by either
H2O2 or paraquat.
To examine the role of OxyR and SoxRS regulators in fpg,
gorA, and grxA expression induction by
H2O2 and paraquat, strains carrying the
Construction of Mutants Lacking Components of the Thioredoxin and
Glutaredoxin Systems and Additionally Mutated in the soxRS or oxyR
Regulatory Genes--
We have recently demonstrated that untreated
trxA grxA and trxA gshA mutant bacteria show
increased basal levels of nrdAB and of grxA and
fpg expression (19). Likewise, it has been recently reported
that both trxA gorA and trxA gshA double mutants
display partial induction of the OxyR-regulated oxyS gene
(36) and that trxA gorA bacteria exhibit also slightly
elevated basal expression of the SoxR-regulated soxS gene
(17). To explore the role of OxyR and SoxRS regulators on basal levels
of gene expression in bacteria lacking components of both thioredoxin
and glutaredoxin systems, soxRS and oxyR mutant
derivatives were isolated from trxA grxA and trxA
gshA double mutant strains. Strains UC1337 ( Effects of Mutations in soxRS or oxyR on Basal Levels of Gene
Expression of Mutants Lacking Components of the Thioredoxin and
Glutaredoxin Systems--
The lack of SoxRS did not change
significantly the steady-state levels of fpg,
gorA, grxA, nrdA, and nrdB
transcripts in both trxA grxA and trxA gshA
double mutant strains (Fig. 4). Thus, UC1337 and UC1336, which lack the functional soxRS genes,
did not appear to be hindered in their abilities to display large increments in nrdAB and grxA and fpg
expression, respectively, as compared with the parental wild type. As
indicated above, we failed in the isolation of an OxyR
Transcription of grxA in the
As shown in Fig. 5, the expression of katG (OxyR-regulated)
and that of trxB and trxC genes followed
basically the same pattern as the expression of grxA; the
exception was that basal expression levels were not significantly
altered upon the introduction of the
Taking advantage of the RT/MPCR technique, we have monitored for
the first time the simultaneous in vivo transcription of up
to 16 different genes in response to oxidative stress by either exposure to chemical oxidants (H2O2 or
paraquat) or by a decrease in the cellular thiol-disulfide ratio (Trx1
and Grx1 or Trx1 and GSH deficient bacteria). Genes under study code
for most components of both glutaredoxin and thioredoxin pathways, some
related enzymes, and several pivotal members of the OxyR and SoxRS regulons.
We have been able to show dramatic increases in the yield of
grxA transcript (maximal induction ratio of about 50-fold)
upon exposure to 100 µM H2O2 in
an oxyR-dependent and
soxRS-independent manner. The OxyR-mediated response to 100 µM H2O2 was previously reported
(34), yet much lower induction ratios (from 1.3- to 3.0-fold) were
quantified for It has been demonstrated that both the thioredoxin and glutaredoxin
systems play a role in determining the thiol-disulfide balance in the
E. coli cytoplasm (40). Therefore, disulfide bond formation
was found to be 8.4 times more efficient in bacteria simultaneously
lacking Trx1 and GSH (the physiological reductant of all Grxs) than in
wild type. Likewise, we have shown recently that the in vivo
transcription of grxA is triggered in trxA gshA double mutant cells (19). Since OxyR becomes active through the
oxidative formation of a disulfide bond between two of its cysteine
residues (12), it is feasible that trxA gshA mutant cells
provide enough titer of oxidized OxyR to activate grxA
expression. Here we proved this possibility by demonstrating that a
triple trxA gshA oxyR mutant does not show increased basal
levels of grxA mRNA and that a double trxA
oxyR mutant fails to induce grxA transcription after
depletion of GSH with diethylmaleate. In addition, we established that
trxA gshA mutants show increased basal expression of other
OxyR-regulated genes, like gorA and katG, and we
confirmed (data not shown) the induction of OxyS RNA in these mutants
in the absence of oxidative stress by H2O2
(36). Interestingly, the extent of activation of grxA
transcription by either H2O2 or deficiencies in
the cellular disulfide-reducing systems (trxA gshA double
mutants) was much greater (by a factor of 10) than the extent of
induction observed for gorA. This suggests the need for
higher levels of Grx1 than for glutathione reductase (the enzyme that
regenerates its reduced form) in the OxyR-mediated cellular defense
against oxidative stress. The extent of katG induction was
in between those of grxA and gorA, thus following the same order that the information content previously calculated for
their respective OxyR binding sites (i.e.
grxA > katG > gorA) (39).
The results presented here demonstrate that different trxA
gshA double mutant strains can display different degree of OxyR activation, which seems to indicate differences in their respective ability to form cytoplasmic disulfide bonds. Interestingly, the expression level of the OxyR-regulated genes in these strains correlates with their growth rate (higher for the fastest-growing strain). Suppressor mutations, arising at relatively high frequency, that improve the growth of mutant strains defective in different components of both the thioredoxin and glutaredoxin systems, like trxA grxA, trxB gshA, or trxB gorA,
have been previously reported (40, 22, 38). Suppressor mutations
unwittingly selected during the manipulation of mutants missing parts
of both systems might explain why the ability of proteins to form
disulfide bonds in the cytoplasm of these strains is not always altered
in the expected ways (38).
Trx2 accounts for the viability of trxA gshA bacteria, since
the triple trxA trxC gshA mutant is nonviable in the absence of the disulfide reductant dithiothreitol (41). Our data demonstrate for the first time that the lack of Trx1 and the simultaneous blockage
of the GSH/glutaredoxin pathway stimulate the thioredoxin pathway by
increasing the steady-state level of trxB and
trxC transcripts. Like in the case of the OxyR-regulated
genes, the fastest growing trxA gshA bacteria displayed the
highest induction ratios. In addition, we showed that transcription of
trxB and trxC is up-regulated by the constitutive
oxyR2 mutant allele. Taken together, these results indicate
that OxyR, either directly or indirectly, play also a role in the
regulation of the thioredoxin pathway by controlling Trx2 and the
enzyme that regenerates its reduced form. In contrast, we did not
observe any significant change in the expression of the gene
(trxA) coding for Trx1 either in the expression of
grxB or grxC coding for the two new glutaredoxins.
With regard to the nrdAB operon, the data presented show
that its transcription is not induced in response to
H2O2 or paraquat, indicating that
nrdAB is not a member of the OxyR or SoxRS regulons. In
contrast, nrdAB transcription was readily induced by the
RRase inhibitor hydroxyurea (data not shown), at concentrations that block DNA synthesis without effect on other metabolic processes (42).
Interestingly, maximal induction ratios by hydroxyurea (7.5- and
5.2-fold for nrdA and nrdB genes, respectively)
were similar to the increments in basal level of nrdAB
expression displayed by trxA grxA mutant bacteria.
Therefore, operation of RRase in the absence of its two main reductans
Trx1 and Grx1 must lead to disturbances in deoxyribonucleotide
production equivalent to those caused by hydroxyurea treatment (42).
Moreover, we demonstrated a tight and inverse relation between the
level of nrdAB expression and those of genes coding for some
components of both the glutaredoxin (grxA and
gorA) and the thioredoxin (trxB and
trxC) systems. This compensation between RRase and its
reductants might explain why, ironically, the fastest growing
trxA gshA mutant bacteria showed the lowest increments in
nrdAB mRNA. In the absence of Trx1 and the physiological
reductant of all Grxs, Trx2 is the alternate hydrogen donor for RRase
in ribonucleotide reduction (38), but a relatively low level of Trx2
expression has been reported (4). If the Trx2 level is limiting, the
induction of high levels of trxC message concomitantly to
induction of trxB transcription would be clearly beneficial
for trxA gshA mutants, thus explaining that the highest
increments in trxC and trxB expression, compared with wild type, were observed in the trxA gshA mutant
(UC859) that grows faster in both rich and minimal media.
Interestingly, OxyR was found to be essential for the viability of UC859.
Like OxyR, the transcription factor SoxR has the unusual property of
being inactive under reducing conditions but is activated when the
redox potential of the environment becomes more oxidizing (43). Cells
mutated in genes for both Trx1 and GSH provide a highly oxidizing
cytoplasmic environment by negatively affecting the two major
redox-balancing systems in the E. coli cytoplasm (38). The
experiments presented here demonstrated that in vivo transcription of the SoxR-dependent soxS and
zwf genes is not elevated in trxA gshA mutant
cells, indicating that, unlike OxyR, SoxR is not constitutively
activated in such a deficient genetic background. These in
vivo results are not easily interpreted because while reduced Trx1
promotes in vitro the aerobic assembly of SoxR [2Fe-2S]
clusters (17), GSH has an opposite disrupting effect (44). Considerable
additional investigation will be required to unravel the in
vivo role of thioredoxins and glutaredoxins on SoxR regulation. To
this end, some of the mutant strains isolated in this work in
combination with the RT/MPCR technique for in vivo analysis
of gene expression can be of great utility.
Finally, data on fpg expression deserve some particular
comments. Here we demonstrated that fpg transcription is
induced in response to oxidative stress by H2O2
in an OxyR-dependent manner, therefore suggesting the
possibility of the fpg gene being considered a new member of
the oxyR regulon. A previous report (45) has excluded such a
possibility based on the finding that H2O2
(even at concentrations of up to 1000 µM) failed to
induce the 8-hydroxyguanine endonuclease activity of the Fpg protein.
This apparent discrepancy may be explained if the putative increment in
8-hydroxyguanine endonuclease upon H2O2
exposure had fallen back to basal levels after 1 h of treatment
(45), in agreement with the observation that OxyR-dependent
genes exhibit a remarkably rapid and reversible induction in response
to H2O2 (31). Besides, our data confirm at the
transcriptional level that the induction of 8-hydroxyguanine endonuclease by paraquat is independent of SoxRS (45), but they further
attribute such an induction to the conversion of O We are indebted to members of the laboratory
for discussion and technical advice; to Dr. J. Alhama for GSH
determination; and to Dr. B. Weiss, Dr. G. Storz, and Dr. M. Blanco for
providing E. coli strains.
*
This work was supported by Junta de Andalucía (group
CVI 0187) Grants PB95-0557-CO2-01 and PB98-1627 (to D. G. E. S.) and by the Swedish Cancer Society.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Recipient of a postdoctoral contract from the Ministerio de
Educación y Cultura.
¶
Recipient of a predoctoral fellowship from Junta de
Andalucía.
**
To whom correspondence and reprint requests should be addressed: C. Pueyo, Departamento de Bioquímica y Biología Molecular, Avda. de Medina Azahara s/n, Universidad de Córdoba, 14071 Córdoba, España. Tel.: 34-957-218695; Fax: 34-957-218688;
E-mail: bb1pucuc@uco.es.
The abbreviations used are:
Trx, thioredoxin;
Grx, glutaredoxin;
RRase, ribonucleotide reductase;
RT/MPCR, reverse
transcription/multiplex polymerase chain reaction;
Tc, tetracycline;
Kan, kanamycin;
Cam, chloramphenicol;
Tpx, thiol peroxidase;
Fpg, formamidopyrimidine-DNA glycosylase;
DEM, diethylmaleate;
PCR, polymerase chain reaction.
Transcriptional Regulation of Glutaredoxin and Thioredoxin
Pathways and Related Enzymes in Response to Oxidative Stress*
,§,,¶,
, and**
Departamento de Bioquímica y
Biología Molecular, Universidad de Córdoba,
14071-Córdoba, España and the Medical Nobel
Institute for Biochemistry, Department of Medical Biochemistry and
Biophysics, Karolinska Institute, S-171 77, Stockholm, Sweden
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
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Fig. 1.
Known components of the thioredoxin and
glutaredoxin systems and related enzymes. Gene names are given in
parentheses. OxyR* and SoxR* denote
the oxidized states of these two regulatory proteins.
OxyR-dependent regulation of grxA,
trxB, and trxC is shown in the present
study.
2) are
the OxyR and SoxRS transcription factors, respectively (recently
reviewed in Ref. 10). Among the OxyR-regulated genes are those encoding
for hydroperoxidase I (catalase, katG), glutathione reductase (gorA), Grx1 (grxA), and OxyS RNA
(oxyS). Regulation of the soxRS regulon occurs by
a two-stage process; the constitutively expressed SoxR protein is first
converted to an active form, which stimulates soxS
transcription, and the increased levels of SoxS in turn activate
expression of the regulon. Among the SoxRS-regulated genes are those
encoding for manganese superoxide dismutase (sodA) and
glucose-6-phosphate dehydrogenase (zwf).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
sox-8::cat (25),
oxyR::kan (26),
btuB::Tn10 (obtained from M. Blanco),
oxyR2 (27) linked to
btuB::Tn10, and gshA linked
to srl::Tn10 (28). The
sox-8::cat deletion covers the
region encoding both the SoxR and SoxS proteins (25). Strains with the
sox-8::cat or
oxyR::kan allele did not induce glucose-6-phosphate dehydrogenase (coded for by the zwf
gene) or hydroperoxidase I catalase (coded for by the katG
gene) activity upon exposure to 100 µM paraquat or 500 µM H2O2, respectively. Strains
carrying the oxyR2 allele exhibited constitutive high levels
of catalase activity. UC1351 was made because the generation of a
tetracycline-sensitive clone leaves a genetic lesion at the original
site of the insertion. TcS bacteria were selected as
described (29). The genetic lesion carried by UC1351 extended outward
from the Tn10 insertion site into the grxA locus,
making the bacteria additionally sensitive to kanamycin
(KanS). UC1351 and its parental UC827 showed identical
growth rates in LB broth and the same basal levels of gene expression,
as measured by the set A of primers. Strains with either the
gshA::Tn10kan (UC859, UC1336, and
UC614) or the gshA (UC1358, UC1369, UC1363, and UC1395)
mutant allele exhibited identical sensitive to diamide and undetectable
levels of glutathione, as determined by high pressure liquid
chromatography (23). The second trxA gshA double mutant
(UC1358) displayed a greater growth defect than the first construction
(UC859) (doubling time in LB of 60 ± 1 and 37 ± 2 min,
respectively). Nonetheless, suppressor mutations (no reversions of the
gshA mutant allele) that allow UC1358 to grow faster arose at a high frequency (approximately 10
4). UC1369 was
constructed by selecting for spontaneous reversion to utilization of
sorbitol (Srl+). This precise eductant became sensitive to
tetracycline.
Bacterial strains
-glutamylcysteine synthetase (gshA); thioredoxin-linked
Tpx (tpx); formamidopyrimidine-DNA glycosylase
(fpg); hydroperoxidase I catalase (katG); OxyS
RNA (oxyS); SoxS transcription factor (soxS); and
glucose-6-phosphate dehydrogenase (zwf). As described
previously (19, 31), the gapA gene, which codes for
D-glyceraldehyde-3-phosphate dehydrogenase, was used as
internal standard. An exogenous fragment (referred as external
standard) of the gene coding for cytochrome P4501A1 from Liza
aurata (32) was coamplified with the target genes and the internal
standard of set C. Primers for katG and oxyS were
alternately included in set B, since both were labeled with the same
fluorophor and gave PCR products of identical length. As control,
primers of new sets amplify some of the genes of set A. Thus, set A
shares the nrdA, trxA, and grxA genes
with set B and the gorA gene with set C. For those genes in
common, data obtained with different primers were essentially
identical; accordingly, only the results obtained with one of the sets
are presented.
PCR primer characteristics
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
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Fig. 2.
Gene expression induction by hydrogen
peroxide and paraquat. Wild-type cells were treated for 10 min
with the hydrogen peroxide (H2O2) or paraquat
concentration (in µM) indicated on the
abscissa. cDNAs were amplified by using the set A
primers. The fluorescence signal of each PCR product was referred to
that of gapA (internal standard). Data are from an average
of six independent multiplexed PCR amplifications. Values from treated
samples were divided by those from the corresponding control and
plotted as a function of H2O2 or paraquat
concentration. All genes of set A were analyzed, but only those genes
for which highly statistically significant (p 
0.001)
increases were observed at a given H2O2 or
paraquat concentration are represented. Error
bars were estimated from the corresponding S.E.
oxyR::kan (OxyR) or
sox-8::cat (SoxRS)
mutant allele were constructed and subjected to oxidative stress conditions in conjunction with wild-type bacteria (Fig.
3). H2O2 was used
at a concentration of 100 µM, and paraquat was used at a
concentration of 500 µM; gene expression was examined at
5 min upon exposure. The induction of fpg, gorA,
and grxA expression by H2O2 or
paraquat was abolished by the introduction of the
oxyR::kan mutation, indicating that
activation by both oxidants is OxyR-dependent. In contrast,
this transcriptional up-regulation was preserved in the strain with the
sox-8::cat mutation. It should be
noted that induction factors for fpg (~3.0-fold),
gorA (~3.5-fold), and particularly grxA
(~45.5-fold) genes were higher at 5 min (Fig. 3) than at 10 min (Fig.
2) after the addition of H2O2 or paraquat, in
agreement with previous observations showing that oxyR-regulated genes are most strongly induced within the
first 5 min of H2O2 exposure (31). The paraquat
activation of genes that are induced by H2O2
and controlled by OxyR can be explained by the spontaneous and
superoxide dismutase-mediated conversion of O2 to
H2O2 (35). In agreement with this, the lower
levels of paraquat induction in the SoxRS deletion mutant (Fig. 3), as compared with wild type, are expected for the inability of
SoxRS bacteria to induce sodA (Mn-superoxide
dismutase) transcription upon paraquat exposure (25).
View larger version (20K):
[in a new window]
Fig. 3.
OxyR and SoxRS regulation of gene
expression induced by hydrogen peroxide and paraquat. UC5710 (wild
type), UC1342 ( oxyR::kan), and
UC1333 (sox-8::cat) cells were
treated for 5 min with 100 µM hydrogen peroxide
(H2O2) or 500 µM paraquat. The
fluorescence signal of each PCR product was referred to that of
gapA (internal standard). Data were from an average of six
independent multiplexed PCR amplifications. Values from treated samples
were divided by those from the corresponding control. All genes of set
A were analyzed, but only those genes for which highly statistically
significant (p 0.001) increases were observed with a
given bacterial strain are represented. Error
bars were estimated from the corresponding S.E.
trxA
grxA::kan zbi::Tn10
sox-8::cat), UC1336 (trxA
gshA::Tn10kan sox-8::cat), and UC1356
(trxA grxA oxyR::kan)
were readily constructed by transducing to CamR or
KanR the corresponding isogenic parental strain (UC827,
UC859, or UC1351). We were unable to construct an
oxyR::kan derivative from UC859 (P1
(UC1381) × UC859; selection for TcR and screening for
hydroperoxidase I catalase induction). However, the
oxyR::kan allele was successfully
moved into a second trxA gshA
srl::Tn10 double mutant (UC1358). The
oxyR2 mutation causes overexpression of
oxyR-regulated proteins in the absence of oxidative stress
(27). Unlike the oxyR::kan insertion
mutation, oxyR2 was moved with similar efficiency into both
UC859 and UC1358 genetic backgrounds.
derivative from UC859. Unexpectedly, however, the
oxyR::kan allele was readily
transferred to the new trxA gshA double mutant (UC1358)
isolated in this work. By using the set A of primers, we detected
substantial differences in the basal levels of nrdAB and
grxA expression between the two trxA gshA mutants
UC859 and UC1358; of note was the additional observation that the
transcriptional up-regulation of grxA in UC1358 was
completely absent from its OxyR derivative. Encouraged by
these results, we proceeded to investigate in more detail the effect of
OxyR on the steady-state levels of gene expression exhibited by
bacteria simultaneously defective in Trx1 and GSH and the reasons for
differences between mutant strains. To this end, constitutive
OxyRc derivatives were constructed, and new sets of primers
were designed in order to examine by RT/MPCR the expression of 10 additional genes. These genes code for the two new Grx2
(grxB) and Grx3 (grxC) glutaredoxins, the
recently discovered Trx2 (trxC) thioredoxin, the thioredoxin
pathway enzyme Trx reductase (trxB), the OxyR-regulated hydroperoxidase I catalase (katG) and OxyS RNA
(oxyS), the SoxR-regulated SoxS transcriptional factor
(soxS), the SoxRS-regulated NADPH supplier
glucose-6-phosphate dehydrogenase (zwf), the Tpx enzyme linked to the thioredoxin system (tpx), and the GSH
biosynthetic pathway enzyme -glutamylcysteine synthetase
(gshA). The expression levels of 15 genes were examined in
UC5710, UC859, UC1358, and their respective OxyR and/or
OxyRc mutant derivatives. The amounts of the
trxA, grxB, grxC, soxS, zwf, tpx, and gshA transcripts
remained basically unchanged among the strains. Genes trxA
and gshA are not expressed in trxA gshA mutants.
Putative differences in gapA (internal standard) expression among strains were ruled out by expressing the levels of
gapA in reference to the heterologous external standard
included in set C. Those genes for which significant differences with
respect to basal levels of wild-type parent strain (UC5710) were found are shown in Fig. 5.
View larger version (12K):
[in a new window]
Fig. 4.
Basal levels of gene expression in SoxRS
proficient and deficient bacteria. UC5710 (wild type), UC827
( trxA grxA::kan
zbi::Tn10), UC859 (trxA
gshA::Tn10kan), UC1333
(sox-8::cat), UC1337 (trxA
grxA::kan zbi::Tn10
sox-8::cat), and UC1336
(trxA gshA::Tn10kan
sox-8::cat) cells were grown in LB
broth to reach an A600 of 0.7. cDNAs were
amplified by using the set A primers. The fluorescence signal of each
PCR product was referred to that of gapA (internal
standard). Data are from an average of six independent multiplexed PCR
amplifications. Values from UC827 and UC859 were divided by those from
UC5710, and values from UC1337 and UC1336 were divided by those from
UC1333. -Fold increases relative to UC5710 (SoxRS+) or
UC1333 (SoxRS) were plotted for the different genes of
set A. Bacteria carrying the null trxA allele had
undetectable levels of the corresponding mRNA.
View larger version (24K):
[in a new window]
Fig. 5.
OxyR regulation of basal levels of gene
expression. UC5710 (wild type), UC1342
( oxyR::kan), UC1394 (oxyR2
btuB::Tn10), UC859 (trxA
gshA::Tn10kan), UC614 (trxA
gshA::Tn10kan oxyR2
btuB::Tn10), UC1358 (trxA gshA
srl::Tn10), UC1363 (trxA gshA
srl::Tn10
oxyR::kan), and UC1395
(trxA gshA oxyR2 btuB::Tn10) cells
were grown in LB broth to reach an A600 of 0.7. cDNAs were amplified by using the set A, set B (including
katG), and set C primers. The fluorescence signal of each
PCR product was referred to that of gapA (internal
standard). Data were from an average of six independent multiplexed PCR
amplifications. -Fold increases relative to UC5710 were plotted for the
different bacterial strains. All genes were examined, but only those
genes (gorA, fpg, nrdA, and
nrdB from set A; grxA and katG from
set B; trxB and trxC from set C) for which highly
statistically significant (p 0.001) increases were
observed with a given bacterial strain are represented.
Error bars were estimated from the corresponding
S.E.
oxyR::kan mutant (UC1342) was
2.9-fold lower than in UC5710. On the contrary, a dramatic increase of
33-fold in grxA expression was observed in the strain
(UC1394) carrying the oxyR2 missense mutation, which is
considered highly effective causing a constitutively active phenotype
(26). The trxA gshA double mutant UC859 showed a basal level
of grxA message as high as that of the constitutive mutant
UC1394. This level was further elevated in its oxyR2
constitutive derivative (UC614), where a 62-fold increase in the amount
of grxA transcript was observed. The second trxA
gshA double mutant UC1358 showed lower increment in
grxA expression (8.2-fold relative to wild type) than UC859.
This basal level was further modulated by mutations in the
oxyR regulatory gene. Thus, grxA expression was
not elevated in the absence of OxyR (UC1363), while it was induced
45-fold in the isogenic constitutive derivative (UC1395). It should be noted that the three oxyR2 derivatives displayed different
amounts of grxA message, which increased in the same
order that the grxA basal expression of their respective
parental strains (UC5710 < UC1358 < UC859). This finding
seems to indicate that oxyR2 mutants are still
redox-active, as previously demonstrated for other constitutive mutations (26). This was confirmed by demonstrating that UC1394 (oxyR2) displayed a 2-fold induction of grxA
expression (over its high steady-state level) upon treatment with 100 µM H2O2 for 5 min (data not shown).
oxyR::kan mutation in either UC5710
or UC1358 background. The expression pattern of gorA
(OxyR-regulated) showed an additional exception with that of
grxA gene, since both trxA gshA double mutants
(UC859 and UC1358) displayed an identical 2.5-fold increase in the
amount of gorA transcript. Expression of nrdA and
nrdB genes followed a pattern opposite to that of the
OxyR-regulated genes. Therefore, the second trxA gshA double
mutant UC1358 showed a higher (not lower) increment in nrdAB
expression than UC859. Likewise, the oxyR2 constitutive
mutation caused a decrease (not an increase) in the steady-state levels
of UC5710, UC1358, and UC859. The fpg expression pattern was
unique in that basal levels were not apparently altered by mutations in
the oxyR regulatory gene. This last finding was further
substantiated by examining the effect of diethylmaleate (DEM) on gene
expression of bacteria defective in Trx1 (UC844) compared with the
isogenic oxyR::kan derivative
(UC1343). DEM is an electrophilic compound that conjugates with GSH and
thus is considered a very effective agent for in vivo
depletion of glutathione (37). As previously reported (19), GSH
depletion by DEM triggered both the grxA and fpg
expression in UC844 (Fig. 6); induction
ratios similar to the increased basal levels displayed by the second
trxA gshA mutant UC1358 (Fig. 5) were detected after 10 min
of treatment with 30 mM DEM. However, while the
fpg induction ratio was identical in UC844 and UC1343, no
significant increment in grxA expression was observed in the trxA oxyR double mutant strain.
View larger version (27K):
[in a new window]
Fig. 6.
Gene expression induction by
diethylmaleate. UC844 ( trxA) and UC1343
(trxA oxyR::kan)
bacteria were treated for 10 min with 30 mM DEM. cDNAs
were amplified by using the set A primers. The fluorescence signal of
each PCR product was referred to that of gapA (internal
standard). Data were from an average of six independent multiplexed PCR
amplifications. Values from treated samples were divided by those from
the corresponding control. All genes of set A were analyzed, but only
those genes for which highly statistically significant
(p 0.001) increases were observed upon
diethylmaleate exposure of UC844 are represented. Error
bars were estimated from the corresponding S.E.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity by means of two
grxA-lacZ operon fusions. We demonstrated that the
redox-cycling agent paraquat induces also high levels of
grxA expression. However, OxyR, but not SoxRS, was required
for paraquat induction, thus indicating an indirect effect via the
conversion of O2 to H2O2. The OxyR
regulation of grxA expression was further proved by showing that deletion of the oxyR locus from wild-type UC5710
decreases about 3-fold the basal level of grxA transcript,
and that, conversely, the grxA message is overexpressed
greater than 30-fold upon the introduction of the constitutive
oxyR2 mutant allele.
2 into H2O2. Notwithstanding, in contrast to other
OxyR-regulated genes, the fpg mRNA basal level of
wild-type cells and the enhanced transcript level exhibited by
trxA gshA double mutant strains were not increased to a
greater level in a strain constitutive in OxyR. This difference might
be a consequence of the complex regulation that apparently controls
8-hydroxyguanine endonuclease in E. coli, as suggested by a
recent report (45), demonstrating that this DNA repair activity
increases under anaerobic conditions in mutant strains deficient in Fnr
in particular, as well as in Fur, ArcA, and combinations thereof.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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 P. C. Schulze, G. W. De Keulenaer, J. Yoshioka, K. A. Kassik, and R. T. Lee Vitamin D3-Upregulated Protein-1 (VDUP-1) Regulates Redox-Dependent Vascular Smooth Muscle Cell Proliferation Through Interaction With Thioredoxin Circ. Res., October 18, 2002; 91(8): 689 - 695. [Abstract] [Full Text] [PDF]
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 H. K. Hamadeh, K. J. Trouba, R. P. Amin, C. A. Afshari, and D. Germolec Coordination of Altered DNA Repair and Damage Pathways in Arsenite-Exposed Keratinocytes Toxicol. Sci., October 1, 2002; 69(2): 306 - 316. [Abstract] [Full Text] [PDF]
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C. Michan, M. Manchado, and C. Pueyo SoxRS Down-Regulation of rob Transcription J. Bacteriol., September 1, 2002; 184(17): 4733 - 4738. [Abstract] [Full Text] [PDF]
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A. Potamitou, A. Holmgren, and A. Vlamis-Gardikas Protein Levels of Escherichia coli Thioredoxins and Glutaredoxins and Their Relation to Null Mutants, Growth Phase, and Function J. Biol. Chem., May 17, 2002; 277(21): 18561 - 18567. [Abstract] [Full Text] [PDF]
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A. Vlamis-Gardikas, A. Potamitou, R. Zarivach, A. Hochman, and A. Holmgren Characterization of Escherichia coli Null Mutants for Glutaredoxin 2 J. Biol. Chem., March 22, 2002; 277(13): 10861 - 10868. [Abstract] [Full Text] [PDF]
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M. Bebien, G. Lagniel, J. Garin, D. Touati, A. Vermeglio, and J. Labarre Involvement of Superoxide Dismutases in the Response of Escherichia coli to Selenium Oxides J. Bacteriol., March 15, 2002; 184(6): 1556 - 1564. [Abstract] [Full Text] [PDF]
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 R. Harper, K. Wu, M. M. J. Chang, K. Yoneda, R. Pan, S. P.-M Reddy, and R. Wu Activation of Nuclear Factor-kappa B Transcriptional Activity in Airway Epithelial Cells by Thioredoxin but Not by N-Acetyl-Cysteine and Glutathione Am. J. Respir. Cell Mol. Biol., August 1, 2001; 25(2): 178 - 185. [Abstract] [Full Text] [PDF]
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 M. J. Horsburgh, M. O. Clements, H. Crossley, E. Ingham, and S. J. Foster PerR Controls Oxidative Stress Resistance and Iron Storage Proteins and Is Required for Virulence in Staphylococcus aureus Infect. Immun., June 1, 2001; 69(6): 3744 - 3754. [Abstract] [Full Text] [PDF]
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 J. Courcelle, A. Khodursky, B. Peter, P. O. Brown, and P. C. Hanawalt Comparative Gene Expression Profiles Following UV Exposure in Wild-Type and SOS-Deficient Escherichia coli Genetics, May 1, 2001; 158(1): 41 - 64. [Abstract] [Full Text]
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L. M. S. Baker, A. Raudonikiene, P. S. Hoffman, and L. B. Poole Essential Thioredoxin-Dependent Peroxiredoxin System from Helicobacter pylori: Genetic and Kinetic Characterization J. Bacteriol., March 15, 2001; 183(6): 1961 - 1973. [Abstract] [Full Text]
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 K.'i. Ogawa and M. Iwabuchi A Mechanism for Promoting the Germination of Zinnia elegans Seeds by Hydrogen Peroxide Plant Cell Physiol., March 1, 2001; 42(3): 286 - 291. [Abstract] [Full Text] [PDF]
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M. Manchado, C. Michán, and C. Pueyo Hydrogen Peroxide Activates the SoxRS Regulon In Vivo J. Bacteriol., December 1, 2000; 182(23): 6842 - 6844. [Abstract] [Full Text]
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 L. Sahlin, H. Wang, Y. Stjernholm, M. Lundberg, G. Ekman, A. Holmgren, and H. Eriksson The expression of glutaredoxin is increased in the human cervix in term pregnancy and immediately post-partum, particularly after prostaglandin-induced delivery Mol. Hum. Reprod., December 1, 2000; 6(12): 1147 - 1153. [Abstract] [Full Text] [PDF]
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M. Lundberg, C. Johansson, J. Chandra, M. Enoksson, G. Jacobsson, J. Ljung, M. Johansson, and A. Holmgren Cloning and Expression of a Novel Human Glutaredoxin (Grx2) with Mitochondrial and Nuclear Isoforms J. Biol. Chem., July 6, 2001; 276(28): 26269 - 26275. [Abstract] [Full Text] [PDF]
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F. Monje-Casas, J. Jurado, M.-J. Prieto-Alamo, A. Holmgren, and C. Pueyo Expression Analysis of the nrdHIEF Operon from Escherichia coli. CONDITIONS THAT TRIGGER THE TRANSCRIPT LEVEL IN VIVO J. Biol. Chem., May 18, 2001; 276(21): 18031 - 18037. [Abstract] [Full Text] [PDF]
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S. Rahlfs, M. Fischer, and K. Becker Plasmodium falciparum Possesses a Classical Glutaredoxin and a Second, Glutaredoxin-like Protein with a PICOT Homology Domain J. Biol. Chem., September 28, 2001; 276(40): 37133 - 37140. [Abstract] [Full Text] [PDF]
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