Previous Article | Next Article 
Journal of Bacteriology, May 1999, p. 2759-2764, Vol. 181, No. 9
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
In Vivo Transcription of the Escherichia coli oxyR
Regulon as a Function of Growth Phase and in Response to
Oxidative Stress
Carmen
Michán,
Manuel
Manchado,
Gabriel
Dorado, and
Carmen
Pueyo*
Departamento de Bioquímica y
Biología Molecular, Universidad de Córdoba, 14071 Córdoba, Spain
Received 8 September 1998/Accepted 20 February 1999
 |
ABSTRACT |
Simultaneous expression of seven genes in Escherichia
coli was measured by a reverse transcription-multiplex
PCR fluorescence procedure. Genes studied were (i) oxyR
(transcriptional regulator); (ii) katG, dps,
gorA, and ahpCF (controlled by OxyR);
(iii) sodA (controlled by SoxRS); and (iv) trxA
(not related to OxyR or SoxRS). Except for trxA,
transcription of all genes was activated during the course of growth of
wild-type bacteria, though notable variations were observed
with respect to both the time and extent of activation. Whereas
oxyR, katG, dps, and
gorA were activated during exponential growth,
ahpCF and sodA were stimulated in stationary
phase. Maximal induction ranged from 4.6- to 86.5-fold, for
gorA and dps, respectively. Treatment with
H2O2 stimulated expression of the genes
(katG, dps, ahpCF, and
gorA) previously identified as members of the OxyR
regulon, except for oxyR itself. Induction by
H2O2 was a remarkably rapid and reversible
process that took place in an OxyR-dependent and
S-independent manner. NaCl induced expression of the
genes controlled by OxyR, including the oxyR locus. This
transcriptional up-regulation was preserved in a strain with the
oxyR::kan mutation, but
it was abolished (ahpCF) or significantly reduced
(oxyR and dps) in a strain with the
rpoS::Tn10 mutation, potentially
reflecting positive transcriptional regulation of the oxyR
regulon by S. Expression of trxA was not
increased either by H2O2 stress or by a shift
to high-osmolarity conditions.
| |
INTRODUCTION |
Different inducible responses
are critical for survival of Escherichia coli
after oxidative stress (20). Key regulators of adaptive
responses to hydrogen peroxide (6) and superoxide anion
(9) are OxyR (5) and SoxR together with SoxS
(15, 30, 31), respectively.
The oxyR regulon contains at least eight genes,
including those encoding hydroperoxidase I (katG),
glutathione reductase (gorA), alkyl hydroperoxide reductase
(ahpCF), and a nonspecific DNA-binding protein
(dps) (2, 5). OxyR behaves as a transcriptional autorepressor under both inducing and noninducing conditions but activates the different regulon promoters only after
H2O2 treatment (26). Direct
oxidation of OxyR is the mechanism whereby cells sense hydrogen
peroxide and induce the OxyR regulon (28). Recent studies
have revealed that OxyR is activated by the formation of an
intramolecular disulfide bond and is deactivated by enzymatic reduction
with glutaredoxin 1 at the expense of reduced glutathione (32).
The soxRS regulon controls the expression of at least
10 genes, among them the gene encoding manganese-containing
superoxide dismutase (sodA) (15). Regulation of
the soxRS regulon occurs by a two-stage process. The
constitutively expressed SoxR protein is first converted to an
active form, which enhances soxS transcription. The
increased levels of SoxS in turn activate expression of the various regulon genes. The mechanism of SoxR activation and the nature of the signaling molecule are still under debate (17, 18,
20); current mechanisms involve one-electron oxidation and
assembly of the iron-sulfur centers of the molecule (7, 18).
An additional regulator for survival against oxidative stress in
E. coli is the rpoS-encoded S
subunit of RNA polymerase (20). The S regulon
comprises a large number of genes, including katG
(19), gorA (3), and dps
(2), which are also controlled by OxyR. S expression is tightly regulated at the
transcriptional, translational, and posttranslational levels
(21). The cellular concentration of S
increases during entry into stationary phase. Additionally,
S and a rather large subset of
S-controlled genes exhibit hyperosmotic induction
during exponential growth (16).
The in vivo transcriptional activities of E. coli genes are
regularly investigated by assaying for 
-galactosidase activity in
bacterial strains with fusions of the lac operon that
contain the ribosome-binding site for lacZ (22).
Data obtained by this procedure are not always consistent.
Discrepancies are partly due to problems associated with differences
among fusion constructions. We have recently designed and optimized a
semiquantitative reverse transcription-multiplex PCR (RT-MPCR)
procedure to examine simultaneously the in vivo expression of up to
seven different genes (10). The assay is based on
primer extension reactions with specific fluorophor-labeled
primers and subsequent DNA sequencer (GeneScan) analysis of
PCR products. This work investigates the applicability of
the method to the study of the expression of genes involved in
E. coli adaptive responses to oxidative stress.
| |
MATERIALS AND METHODS |
Chemicals.
Saturated phenol II and acrylamide-bis19:1
mixture were from Amresco (Solon, Ohio). The GeneAmp RNA PCR kit, Prism
GeneScan-350 TAMRA
(carboxytetramethylrhodamine-N-hydroxysuccinimide) ladder, loading buffer, fluorescence-labeled primers, deoxynucleoside triphosphates (dNTPs), MgCl2 and AmpliTaq
were from Perkin-Elmer (Norwalk, Conn.). DNase (RNase free) was
from Boehringer (Mannheim, Germany). MPCR buffer 3 was from
Maxim Biotech (San Francisco, Calif.). Hydrogen peroxide
(H2O2) and other chemicals were purchased from
Sigma (St. Louis, Mo.). Reactive protein assay was from Bio-Rad (Hercules, Calif.).
Bacterial strains and growth conditions.
Bacterial strains
used in this work are derived from E. coli K-12. Strain
UC574 (arg56 nad113 araD81) (1) was considered the parental wild type. Strains UC1247
(oxyR::kan) and UC1311 (rpoS::Tn10) were constructed by P1
transduction of the oxyR::kan (obtained
from G. Storz) or rpoS::Tn10
(obtained from P. Loewen) mutant allele into strain UC574.
Transductants were first selected on Luria-Bertani (LB) medium with
kanamycin or tetracycline and then scored for induction of
hydroperoxidase I (HPI) (coded for by the katG gene) or
hydroperoxidase II (coded for by the katE gene) activity
upon exposure to 10 µM H2O2 (UC1247) or 500 mM NaCl (UC1311). Bacteria were grown in LB nutrient broth or M9 minimal medium (11). Minimal medium was supplemented with
2 g of glucose per liter, 40 µg of arginine per ml, 5 µg of
D-biotin per ml, 5 µg of thiamine per ml, and 10 g of Casamino Acids per liter. Growth was monitored by
measuring the optical density at 600 nm (OD600). Bacteria
were inoculated into LB broth or M9 minimal medium and incubated for
15 h at 37°C with shaking at 150 rpm. The overnight cultures
were then diluted into fresh LB or M9 medium (OD600 = 0.03)
and incubated at 37°C and 150 rpm for the times indicated in Table 2
and the figure legends.
RNA purification.
Total RNA was extracted by the hot-phenol
method, as previously described (8). 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.
RT-MPCR.
Synthesis of cDNA was carried out with the GeneAmp
RNA PCR kit, as previously described (10). Each RNA sample
was retrotranscribed at least twice. PCR amplification of cDNA was
carried out with the primer pair sets listed in Table
1. Primers were designed with the Primer
Select 3.03/96 (DNA Star, Madison, Wis.) and Oligo 5.0/96 (Molecular
Biology Insights, Plymouth, Minn.) software programs, in order to
obtain the highest specificity and performance. Conditions for MPCR
were optimized as detailed by Gallardo-Madueño et al.
(10) to produce fluorescence intensities of the desired products in the range of linearity. Thirty-five cycles were performed, each consisting of 1 min of denaturation at 94°C, 15 s of
annealing at 68°C, and 30 s of enzymatic primer extension at
72°C. The PCR amplification mixture contained AmpliTaq
(1.25 U), MPCR buffer 3 (2.5 µl), MgCl2 (25 nmol), dNTP
(at 1 mM each), and primers (3.1 pmol, oxyR; 1.5 pmol,
katG; 1.4 pmol, dps; 1.125 pmol, gorA; 2.7 pmol, ahpCF; 3.1 pmol, sodA; 0.475 pmol,
trxA; 0.7 pmol, gapA) in a final volume of 25 µl. PCR fragments of 129 bp (oxyR), 137 bp
(katG), 142 bp (dps), 125 bp (gor),
128 bp (ahpCF), 121 bp (sodA), 148 bp
(trxA) and 131 bp (gapA) were obtained. At least two MPCRs were performed for each cDNA.
MPCR product quantification.
Following amplification, 0.5 µl of the MPCR product was mixed with 0.2 µl of Prism GeneScan-350
TAMRA ladder, 1.4 µl of deionized formamide, and 0.2 µl of loading
buffer. Samples were denatured at 95°C for 2 min. Samples were run on
a denaturing 4.24% polyacrylamide gel at 750 V in an ABI Prism 377 DNA
sequencer/GeneScan from the Applied Biosystems Division of Perkin-Elmer
(Foster City, Calif.). Data were collected and analyzed with the ABI
Prism 377 Collection 2.1/97 and GeneScan Analysis 2.0.2/95 software
programs, respectively (Perkin-Elmer/Applied Biosystems Division).
Differences in amplification efficiencies among samples were normalized
by comparing the fluorescence intensity of each band to that resulting
from gapA amplification, which was used as reference gene.
Samples for comparison of different experimental conditions were
handled in parallel. Data are presented as means ± standard
errors of the means (SEM) from n MPCR amplifications. Comparison between groups was done by Student's t test.
Significances at a P level of <0.05 are indicated in the text.
Enzymatic assays.
Ten milliliters of bacterial cultures were
centrifuged at 16,000 × g at 4°C for 5 min. The cell
pellet was washed and resuspended in 0.5 ml of 20 mM potassium
phosphate buffer (pH 7.5) with 0.1 mM EDTA. Cells were disrupted at
4°C by ultrasonic disintegration (three 12-s pulses, 25 W). The
peroxidase activity of HPI was assayed in dialyzed extracts by
monitoring H2O2 decomposition at 460 nm with
o-dianisidine as the hydrogen donor (24). One unit of peroxidase activity is defined as the amount of enzyme that
decomposes 1 µmol of H2O2 per min at 25°C.
GAPDH (glyceraldehyde-3-phosphate dehydrogenase) activity was
determined by using a coupled system in a reaction mixture
containing 100 mM triethanolamine buffer (pH 7.6), 0.1 mM EDTA, 0.7 mM
MgSO4, 0.1 mM ATP, 6 mM 3-phosphoglycerate, 1.5 U of
phosphoglycerate kinase from chicken muscle per ml, and 0.2 mM NADH. A
GAPDH unit of activity is defined as the amount of enzyme
catalyzing the utilization of 1 µmol of substrate per minute at
30°C. Protein concentration was estimated by the method of Bradford
(4). Specific activity values were expressed as means ± SEM for at least three independent determinations. Comparison between groups was done by Student's t test. Significances
at a P level of <0.05 are indicated in the text.
| |
RESULTS AND DISCUSSION |
Growth-phase dependent variation in gene expression.
Eight primer pairs were designed (Table 1) to study by RT-MPCR
the simultaneous expression of genes related to E. coli
adaptive responses to oxidative stress. The procedure was
basically as previously described for a different group of genes
(10). The fluorescent PCR products were separated on
acrylamide gels with an ABI Prism 377 DNA sequencer and analyzed
with GeneScan software. For purposes of semiquantitative
analysis, data are expressed as the ratio of the signal obtained for
each individual gene divided by the signal obtained from the reference
mRNA of the corresponding sample.
As described previously (
10), the
gapA gene,
which codes for GAPDH, was used as reference. Bacteria displayed
similar basal
GAPDH enzymatic activities through the
growth curve (average of
790 ± 150 mU/mg of protein from
three independent determinations).
Genes under study were (i)
oxyR (transcriptional regulator); (ii)
katG,
dps,
gorA, and
ahpCF (members of the
H
2O
2-inducible regulon
controlled by OxyR);
(iii)
sodA (member of the
O
2·
-induced regulon controlled by SoxRS);
and (iv)
trxA. The last
gene, coding for thioredoxin,
was included because its expression
has not been linked to OxyR or
SoxRS.
Expression of genes throughout the culture time of wild-type bacteria
in rich LB medium (for which most data are available)
is shown in Table
2. With the possible exception of
trxA, all
genes under study were clearly activated over the
12-h period
of cultivation. In agreement with the oxidant
resistance of stationary-phase
E. coli
(
14), minimal expression levels were observed during
the
first 2 h of growth; then, gene expression increased with
the time
of culture, giving maximal induction levels at the late-exponential
or
stationary growth phase (5 to 12 h). Notable variations with
respect to both the time and the extent of activation, however,
were observed. Thus, whereas
oxyR,
katG,
dps, and
gorA were activated
at exponential
phase,
ahpCF and
sodA were stimulated somewhat
later, at stationary phase. Maximal values ranged from 4.6- to
86.5-fold increases, for
gorA and
dps,
respectively.
González-Flecha and Demple (
13) reported a 3.5-fold
increase in the steady-state level of
oxyR mRNA during
exponential
growth (2 to 4 h) of
E. coli in rich
LB medium, followed by a
rapid decline to yield initial values of
expression. While we
detected a similar rise in
oxyR mRNA
during exponential growth,
we did not observe any decrease at
stationary phase (Table
2).
To the contrary, a maximal induction factor
of 5.6-fold was quantified
at 12 h of
growth.
Activation of
katG expression, however, exhibited a biphasic
profile, with a minimal value at 1.5 h of outgrowth and a
maximal
induction factor of 7.4-fold at 5 h. After
reaching this peak,
the amount of
katG transcript declined
during stationary phase,
and this decrease was statistically
significant with respect to
the 5-h value. This result is basically in
agreement with a previous
study (
12) in which transcription
was monitored by using a
katG::
lacZ operon
fusion. However, the induction and maximum for
katG mRNA
reported in Table
2 preceded by ~2 h that seen for
-galactosidase
expression with the fusion gene. A delay for protein synthesis
and the
effect of the stability of the protein can explain differences
between
the pattern of enzymatic activity and the transcriptional
behavior of a gene. These circumstances are investigated in Fig.
1 with respect to the peroxidase activity
of HPI, coded for by
katG. Low levels of peroxidase activity
were observed during the
first 3 h of outgrowth. Induction
occurred at late exponential
phase and was maintained during stationary
phase. The maximal
increase in peroxidase activity (7.3-fold) was
identical to that
observed in
katG mRNA. As in the case of
peroxidase, glutathione
reductase (GRase) activity presented also an
~2-h delay with respect
to
gorA expression (data not
shown).
View larger version (33K):
[in this window]
[in a new window]
|
FIG. 1.
Peroxidase activity during course of growth of wild-type
bacteria in nutrient LB medium. Cells were grown as described in Table
2, footnote a. Peroxidase activity (mU/mg of protein) was
determined as specified in Materials and Methods. Activity data (darkly
shaded bars) are from an average of four independent determinations.
Expression data (lightly shaded bars) are those given in Table 2. Error
bars represent SEM. Statistical significance (P < 0.05), for comparisons with minimal values at 1.5 h of
outgrowth, is marked with asterisks. Bacterial growth, monitored as
OD600, is indicated by the line.
|
|
Transcriptional regulation as a function of bacterial growth has been
reported for two other genes controlled by OxyR, namely
dps (
2) and
gorA (
3). Our
data agree with both previous
reports in showing maximal
transcriptional levels after the onset
of stationary phase.
Furthermore, they indicate the need for a
much higher level of
induction for
dps than for
gor transcripts
(86.5- versus 4.6-fold increments). The physiological sense of
such a
difference might be in the ability of Dps to directly protect
DNA
from oxidative damage (
23), compared with proteins which
are
either enzymes (HPI, GRase, AHP [alkyl hydroperoxide reductase],
and
Mn-SOD [manganese-containing superoxide dismutase]) or regulators
(OxyR). To our knowledge, we present here the first examples of
variations in the levels of
ahpCF and
sodA
transcripts during
aerobic growth in
E. coli. It is
noteworthy that transcriptional
induction of
ahpCF genes
occurs much later than that of other
OxyR-regulated genes (7 versus 3 h of outgrowth). This observation
seems to be in
disagreement with the idea of an H
2O
2-mediated
change in OxyR regulon expression during growth (
12).
Gene expression induction by hydrogen peroxide.
While the
oxyR locus is central to the adaptive response of
exponentially growing cells to H2O2, the
constitutive increased resistance to oxidants of stationary phase
cells is linked to rpoS (20). To examine the role
of these two regulators on expression of the genes under study, strains
carrying the oxyR::kan or
rpoS::Tn10 mutant allele were constructed and
subjected to different stress conditions in conjunction with wild-type bacteria.
Optimal conditions for induction of gene expression by
H
2O
2 were first established in experiments with
the wild-type strain.
Induction of transcripts was readily seen with 10 to 100 µM H
2O
2 during exponential growth
(data not shown). Treatments in M9 minimal
medium resulted in the
induction of higher levels of gene expression
than those in
nutrient LB (data not shown), in agreement with
previous data on
the induction level of enzymes controlled by
OxyR (
5).
The kinetics of induction following 10 µM
H
2O
2 addition
to wild-type cells at early
exponential growth in M9 is shown
in Fig.
2. The influence of the regulatory
oxyR::
kan and
rpoS::Tn
10 mutations on
H
2O
2 induction is investigated in Fig.
3.
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 2.
Wild-type cells from an overnight culture in M9 minimal
medium were diluted in fresh medium and incubated at 37°C with
shaking at 150 rpm. At an OD600 of 0.2, H2O2 was added to half of the culture, to make
a final 10 µM solution, and the rest was used as a control. Samples
were collected immediately (<1 min) after the addition of
H2O2 and at 5, 10, 15, and 20 min of exposure.
Samples were frozen with liquid nitrogen, and total RNA was purified as
described in Materials and Methods. The fluorescence signal of each PCR
product was compared to that of gapA. Data are from an
average of eight MPCR amplifications. Values from treated samples were
divided by those from the corresponding control. All genes were
analyzed, but only those genes for which statistically significant
(P < 0.05) increases were observed at a given time are
represented. Error bars were estimated from the corresponding SEM.
|
|
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 3.
Wild-type (wt),
oxyR::kan, and
rpoS::Tn10 bacteria from overnight cultures
in M9 minimal medium were diluted in fresh medium and incubated at
37°C with shaking at 150 rpm. At an OD600 of 0.2, H2O2 was added to half of each culture, to make
a final 10 µM solution, and the rest was used as a control. Samples
were collected at 5 min after the addition of
H2O2 and frozen with liquid nitrogen. Total RNA
was purified as described in Materials and Methods. The fluorescence
signal of each PCR product was compared to that of gapA.
Data are from an average of eight MPCR amplifications. Values from
treated samples were divided by those from the corresponding control.
All genes were analyzed, but only those genes for which statistically
significant (P < 0.05) increases were observed are
represented. Error bars were estimated from the corresponding SEM.
|
|
Treatment with H
2O
2 stimulated expression
of the four genes identified as being under
oxyR
control (
2,
5). Therefore,
maximal induction levels of
12.9-, 11.3-, 3.3-, and 2.0-fold were
observed for
katG,
dps,
ahpCF, and
gorA, respectively
(Fig.
2).
Induction of
katG and
dps transcription
in response to H
2O
2 was
previously reported
(
2,
27). However, though
oxyR-regulated
promoters
have been mapped upstream from the
Salmonella typhimurium ahp genes and the
E. coli gorA gene (
27,
28), and a constitutive
oxyR1 mutant of
S. typhimurium contains higher levels of GRase
and AHP activities
than do wild-type extracts (
5), the putative
increments in
ahpCF and
gorA transcription by
H
2O
2 stress have
not been reported so far for
E. coli.
The induction of
katG,
dps,
gorA, and
ahpCF expression produced
by H
2O
2 was abolished by the introduction of
the
oxyR::
kan mutation (Fig.
3),
indicating that the induction is OxyR dependent,
in agreement with
published reports on regulation of
katG and
dps expression (
2,
27). In contrast, this
transcriptional
up-regulation was preserved in the strain with the
rpoS::Tn
10 mutation, indicating that
S is not required in the OxyR-mediated response to
H
2O
2 of exponentially
growing cells. In fact,
the factors of induction by H
2O
2 were
somewhat
higher in the
rpoS::Tn
10 mutant than in the
wild-type
strain, which might suggest that more
70-containing RNA polymerase remains in the
rpoS mutant than in
wild-type cells to support
OxyR-dependent
transcription.
The four OxyR-dependent genes exhibited remarkably rapid
induction in response to 10 µM H
2O
2
(Fig.
2). Therefore, transcription
increased to maximal (or near
maximal) induction levels immediately
after addition of the oxidant and
then fell back to basal levels
within 10 to 20 min of treatment.
Longer periods for this transient
phenomenon were reported for
oxyS expression upon exposure of
wild-type cells to a much
higher (200 µM) H
2O
2 concentration
(
32).
Since expression of
oxyR was not induced
after oxidative stress
with 10 µM H
2O
2, in
agreement with previous results of OxyR protein
synthesis
(
26), our data indicate that activation by oxidation
of the
OxyR transcription factor is an extremely rapid event in
vivo, the
oxidized OxyR being then quickly converted to the reduced
and inactive
form in the presence of cellular reductants such
as glutaredoxins and
thioredoxins (
10,
32).
While expression of the OxyR-dependent genes was readily induced by
H
2O
2, induction (fivefold) of
sodA
transcription (under
the control of
soxRS) was specific to
paraquat (a redox-cycling
compound) treatment (data not shown), in
agreement with previous
results (
29). Expression of
trxA was not induced after oxidative
stress by either
H
2O
2 or
paraquat.
Gene expression induction by sodium chloride.
To obtain
evidence of S-dependent regulation, we exploited the
observation that S expression is induced
posttranscriptionally in response to osmotic upshift in the growth
medium (21). Osmotic shock by increasing the NaCl
concentration to 500 mM resulted in the induction of gene expression in
wild-type bacteria at both the exponential and stationary phases when
grown in either nutrient LB or minimal M9 medium (data not
shown). The kinetics of induction of transcription in response to
increased medium osmolarity and comparisons of the wild-type strain and
isogenic derivatives defective in OxyR or S are
examined in Fig. 4 and
5, respectively.
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 4.
Wild-type cells from an overnight culture in M9 minimal
medium were diluted in fresh medium and incubated at 37°C with
shaking at 150 rpm. At an OD600 of 0.2, NaCl was added to
half of the culture, to make a final 500 mM solution, and the rest was
used as a control. Samples were collected immediately (<1 min) after
the addition of NaCl and at 5, 10, and 15 min of exposure. Samples were
frozen with liquid nitrogen, and total RNA was purified as described in
Materials and Methods. The fluorescence signal of each PCR product was
compared to that of gapA. Data are from an average of eight
MPCR amplifications. Values from treated samples were divided by those
from the corresponding control. All genes were analyzed, but only those
genes for which statistically significant (P < 0.05)
increases were observed at a given time are represented. Error bars
were estimated from the corresponding SEM.
|
|
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 5.
Wild-type (wt),
oxyR::kan, and
rpoS::Tn10 bacteria from overnight cultures
in M9 minimal medium were diluted in fresh medium and incubated at
37°C with shaking at 150 rpm. At an OD600 of 0.2, NaCl
was added to half of each culture, to make a final 500 mM solution, and
the rest was used as a control. Samples were collected at 15 min after
the addition of NaCl and frozen with liquid nitrogen. Total RNA was
purified as described in Materials and Methods. The fluorescence signal
of each PCR product was compared to that of gapA. Data are
from an average of eight MPCR amplifications. Values from treated
samples were divided by those from the corresponding control. All genes
were analyzed, but only those genes for which statistically significant
(P < 0.05) increases were observed are represented.
Error bars were estimated from the corresponding SEM. Bacteria carrying
the oxyR::kan mutation had undetectable
levels of the corresponding mRNA.
|
|
Osmotic induction of transcription was a rapid process (Fig.
4), as
previously reported for several
rpoS-dependent genes
(
16).
Nevertheless, induction by increased osmolarity was
not as fast
as induction in response to H
2O
2
(Fig.
2), considering that substantial
increases in
oxyR,
katG,
dps,
gorA,
ahpCF, and
sodA expression
were not observed until after 10 or 15 min of osmotic upshift.
Osmotic induction of
oxyR
corresponded more or less with the maximal
values found during
the transition into stationary phase (Table
2). In contrast,
stationary-phase induction greatly exceeded
osmotic induction for
dps and
katG. Expression of
gorA,
ahpCF,
and
sodA was only weakly induced
(two- to threefold) by NaCl under
our experimental conditions. These
results agree with the observation
that for a given
rpoS-dependent gene, the extents of osmotic induction
and of
stationary-phase induction do not necessarily correlate
(
16).
Induction by elevated osmolarity was unaffected in the
oxyR::
kan mutant, but it was abolished
(
ahpCF) or significantly reduced
(
oxyR,
dps, and
sodA) in the
rpoS::Tn
10 mutant (Fig.
5), in agreement
with
published data on osmotic regulation of
rpoS-dependent loci
(
16). Only
katG and
gorA expression
did not follow this regulatory
pattern, since neither a mutation in
oxyR nor one in
rpoS were
able to prevent twofold
induction by NaCl, which might correspond
to a mild stimulation of
transcription by
70-containing RNA polymerase as a
consequence of high-osmolarity
stress.
Whereas positive transcriptional regulation by
S has
been previously reported for three of the genes of the
oxyR
regulon
dps (
2),
katG
(
19), and
gorA (
3)
negative
regulation by
S has been described for the
oxyR regulatory locus (
13). Therefore,
the
level of
-galactosidase expression from a single-copy
oxyR'::
lacZ fusion in a
S-defective strain was higher (not lower) than
in its wild-type
parent strain as the cells entered and remained in
stationary
phase. Moreover, elevated expression of
S
prevented (not induced) normal expression of
oxyR
(
13). In
contrast, our data in Fig.
4 and
5 suggest that
S is a direct or indirect positive regulator for
oxyR transcription
as well as for other OxyR-regulated
genes, in accordance with
the increased expression of
oxyR
at the onset of stationary phase
(Table
2). It is not clear at present
if differences in genetic
backgrounds or methods for
measurement of gene expression account
for the apparent
contradiction between our results and those of
González-Flecha and Demple (
13) on the role of
S in transcriptional regulation of the
oxyR gene.
Expression of
trxA was not substantially induced by osmotic
upshift and did not show an evident growth-phase-dependent variation
(Table
2). Little is known about the control of the
trxA
gene
in bacteria except for a recent report suggesting that expression
of
trxA (monitored with a
trxA-lac translational
fusion) in
E. coli is negatively regulated by cyclic
AMP (
25). This regulation
would adjust
trxA
expression to the growth rate of the bacteria,
in accordance with the
role of thioredoxin as a cofactor in the
synthesis of
deoxyribonucleotides. Nevertheless, by RT-MPCR, we
have not found
significant variations in
trxA transcription under
conditions in which a large increment, >30-fold, was observed
in
expression of
grxA (which codes for a second cofactor for
ribonucleotide
reduction) (
10).
In brief, this work monitors for the first time the simultaneous in
vivo expression of multiple genes related to protection
of bacteria
against oxidative stress. The data presented contain
new valuable
information on gene expression during different stages
of growth and in
response to osmotic stress and confirm previous
regulatory
relationships. We propose that the RT-MPCR method applied
in this work
is a powerful tool for monitoring gene expression,
particularly
when the genes under study present a complex pattern
of regulation,
with outstanding advantages over alternative experimental
approaches
such as the use of
lacZ fusions.
| |
ACKNOWLEDGMENTS |
C.M. and M.M. contributed equally to this work, and both should
be considered first authors.
We are grateful to J. F. M. Leal, J. López-Barea, and
R. Gallardo-Madueño for helpful discussions and to N. Abril for
help in UC1247 construction.
M.M. was a recipient of a predoctoral fellowship from the Spanish
Ministry of Education and Culture (MEC). This work was supported by
grant P95-0557-CO2-01 (DGES) and by Junta de Andalucía (group CVI 0187).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Bioquímica y Biología Molecular, Avda. de Medina
Azahara s/n, Universidad de Córdoba, 14071 Córdoba, Spain.
Phone: 34-957-218695. Fax: 34-957-218688. E-mail:
bb1pucuc{at}uco.es.
| |
REFERENCES |
| 1.
|
Abril, N.,
T. Roldán-Arjona,
M. J. Prieto-Álamo,
A. A. van Zeeland, and C. Pueyo.
1992.
Mutagenesis and DNA repair for alkylation damages in Escherichia coli K-12.
Environ. Mol. Mutagen.
19:288-296[Medline].
|
| 2.
|
Altuvia, S.,
M. Almirón,
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].
|
| 3.
|
Becker-Hapak, M., and A. Eisenstark.
1995.
Role of rpoS in the regulation of glutathione oxidoreductase (gor) in Escherichia coli.
FEMS Microbiol. Lett.
134:39-44[Medline].
|
| 4.
|
Bradford, M. M.
1976.
A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:248-254[Medline].
|
| 5.
|
Christman, M. F.,
R. W. Morgan,
F. S. Jacobson, and B. N. Ames.
1985.
Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella typhimurium.
Cell
41:753-762[Medline].
|
| 6.
|
Demple, B., and J. Halbrook.
1983.
Inducible repair of oxidative DNA damage in Escherichia coli.
Nature
304:466-468[Medline].
|
| 7.
|
Ding, H., and B. Demple.
1996.
Glutathione-mediated destabilization in vitro of [2Fe-2S] centers in the SoxR regulatory protein.
Proc. Natl. Acad. Sci. USA
93:9449-9453[Abstract/Free Full Text].
|
| 8.
|
Emory, S. A., and J. G. Belasco.
1990.
The ompA 5' untranslated RNA segment functions in Escherichia coli as a growth-rate-regulated mRNA stabilizer whose activity is unrelated to translational efficiency.
J. Bacteriol.
172:4472-4481[Abstract/Free Full Text].
|
| 9.
|
Farr, S. B.,
D. O. Natvig, and T. Kogoma.
1985.
Toxicity and mutagenicity of plumbagin and the induction of a possible new DNA repair pathway in Escherichia coli.
J. Bacteriol.
164:1309-1316[Abstract/Free Full Text].
|
| 10.
|
Gallardo-Madueño, R.,
J. F. M. Leal,
G. Dorado,
A. Holmgren,
J. López-Barea, and C. Pueyo.
1998.
In vivo transcription of nrdAB operon and of grxA and fpg genes is triggered in E. coli lacking both thioredoxin and glutaredoxin 1, or thioredoxin and GSH, respectively.
J. Biol. Chem.
273:18382-18388[Abstract/Free Full Text].
|
| 11.
|
Gerhardt, P.,
R. G. E. Murray,
W. A. Wood, and N. R. Krieg (ed.).
1994.
Methods for general and molecular bacteriology.
American Society for Microbiology, Washington, D.C.
|
| 12.
|
González-Flecha, B., and B. Demple.
1997.
Homeostatic regulation of intracellular hydrogen peroxide concentration in aerobically growing Escherichia coli.
J. Bacteriol.
179:382-388[Abstract/Free Full Text].
|
| 13.
|
González-Flecha, B., and B. Demple.
1997.
Transcriptional regulation of the Escherichia coli oxyR gene as a function of cell growth.
J. Bacteriol.
179:6181-6186[Abstract/Free Full Text].
|
| 14.
|
Goodrich-Blair, H.,
M. Uría-Nickelsen, and R. Kolter.
1996.
Regulation of gene expression in stationary phase, p. 571-583.
In
E. C. C. Lin, and A. S. Lynch (ed.), Regulation of gene expression in Escherichia coli. Chapman and Hall, New York, N.Y.
|
| 15.
|
Greenberg, J. T.,
P. Monach,
J. H. Chou,
P. D. Josephy, and B. Demple.
1990.
Positive control of a global antioxidant defense regulon activated by superoxide-generating agents in Escherichia coli.
Proc. Natl. Acad. Sci. USA
87:6181-6185[Abstract/Free Full Text].
|
| 16.
|
Hengge-Aronis, R.,
R. Lange,
N. Henneberg, and D. Fisher.
1993.
Osmotic regulation of rpoS-dependent genes in Escherichia coli.
J. Bacteriol.
175:259-265[Abstract/Free Full Text].
|
| 17.
|
Hidalgo, E., and B. Demple.
1996.
Adaptive responses to oxidative stress: the soxRS and oxyR regulons, p. 435-452.
In
E. C. C. Lin, and A. S. Lynch (ed.), Regulation of gene expression in Escherichia coli. Chapman and Hall, New York, N.Y.
|
| 18.
|
Hidalgo, E.,
H. Ding, and B. Demple.
1997.
Redox signal transduction: mutations shifting [2Fe-2S] centers of the SoxR sensor-regulator to the oxidized form.
Cell
88:121-129[Medline].
|
| 19.
|
Ivanova, A.,
C. Miller,
G. Glinsky, and A. Eisenstark.
1994.
Role of rpoS (katF) in oxyR-independent regulation of hydroperoxidase I in Escherichia coli.
Mol. Microbiol.
12:571-578[Medline].
|
| 20.
|
Jamieson, D. J., and G. Storz.
1997.
Transcriptional regulators of oxidative stress response, p. 91-115.
In
J. G. Scandalios (ed.), Oxidative stress and the molecular biology of antioxidant defenses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 21.
|
Lange, R., and R. Hengge-Aronis.
1994.
The cellular concentration of the S subunit of RNA polymerase in Escherichia coli is controlled at the levels of transcription, translation, and protein stability.
Genes Dev.
8:1600-1612[Abstract/Free Full Text].
|
| 22.
|
Lin, E. C. C., and A. S. Lynch (ed.).
1996.
Regulation of gene expression in Escherichia coli.
Chapman and Hall, New York, N.Y.
|
| 23.
|
Martinez, A., and R. Kolter.
1997.
Protection of DNA during oxidative stress by the nonspecific DNA-binding protein Dps.
J. Bacteriol.
179:5188-5194[Abstract/Free Full Text].
|
| 24.
|
Mukhopadhyay, S., and H. Schellhorn.
1994.
Induction of Escherichia coli hydroperoxidase I by acetate and other weak acids.
J. Bacteriol.
176:2300-2307[Abstract/Free Full Text].
|
| 25.
|
Sa, J.-H.,
M. A. Namgung,
C.-J. Lim, and J. A. Fuchs.
1997.
Expression of the Escherichia coli thioredoxin gene is negatively regulated by cyclic AMP.
Biochem. Biophys. Res. Commun.
234:564-567[Medline].
|
| 26.
|
Storz, G.,
L. A. Tartaglia, and B. N. Ames.
1990.
Transcriptional regulator of oxidative stress-inducible genes: direct activation by oxidation.
Science
248:189-248[Abstract/Free Full Text].
|
| 27.
|
Tartaglia, L. A.,
G. Storz, and B. N. Ames.
1989.
Identification and molecular analysis of oxyR-regulated promoters important for the bacterial adaptation to oxidative stress.
J. Mol. Biol.
210:709-719[Medline].
|
| 28.
|
Toledano, M. B.,
I. Kullik,
F. Trinh,
P. T. Baird,
T. D. Schneider, and G. Storz.
1994.
Redox-dependent shift of OxyR-DNA contacts along an extended DNA-binding site: a mechanism for differential promoter selection.
Cell
78:897-909[Medline].
|
| 29.
|
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].
|
| 30.
|
Tsaneva, I. R., and B. Weiss.
1990.
soxR, a locus governing a superoxide response regulon in Escherichia coli K-12.
J. Bacteriol.
172:4197-4205[Abstract/Free Full Text].
|
| 31.
|
Wu, J., and B. Weiss.
1991.
Two divergently transcribed genes, soxR and soxS, control a superoxide response regulon of Escherichia coli.
J. Bacteriol.
173:2864-2871[Abstract/Free Full Text].
|
| 32.
|
Zheng, M.,
F. Åslund, 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, May 1999, p. 2759-2764, Vol. 181, No. 9
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Zeller, T., Li, K., Klug, G.
(2006). Expression of the trxC Gene of Rhodobacter capsulatus: Response to Cellular Redox Status Is Mediated by the Transcriptional Regulator OxyR. J. Bacteriol.
188: 7689-7695
[Abstract]
[Full Text]
-
Liu, X., Kim, K., Leighton, T., Theil, E. C.
(2006). Paired Bacillus anthracis Dps (Mini-ferritin) Have Different Reactivities with Peroxide. J. Biol. Chem.
281: 27827-27835
[Abstract]
[Full Text]
-
Johnson, J. R., Clabots, C., Rosen, H.
(2006). Effect of Inactivation of the Global Oxidative Stress Regulator oxyR on the Colonization Ability of Escherichia coli O1:K1:H7 in a Mouse Model of Ascending Urinary Tract Infection. Infect. Immun.
74: 461-468
[Abstract]
[Full Text]
-
Olsen, K. N., Larsen, M. H., Gahan, C. G. M., Kallipolitis, B., Wolf, X. A., Rea, R., Hill, C., Ingmer, H.
(2005). The Dps-like protein Fri of Listeria monocytogenes promotes stress tolerance and intracellular multiplication in macrophage-like cells. Microbiology
151: 925-933
[Abstract]
[Full Text]
-
Panek, H. R., O'Brian, M. R.
(2004). KatG Is the Primary Detoxifier of Hydrogen Peroxide Produced by Aerobic Metabolism in Bradyrhizobium japonicum. J. Bacteriol.
186: 7874-7880
[Abstract]
[Full Text]
-
Li, K., Hein, S., Zou, W., Klug, G.
(2004). The Glutathione-Glutaredoxin System in Rhodobacter capsulatus: Part of a Complex Regulatory Network Controlling Defense against Oxidative Stress. J. Bacteriol.
186: 6800-6808
[Abstract]
[Full Text]
-
Park, K.-J., Kang, M.-J., Kim, S. H., Lee, H.-J., Lim, J.-K., Choi, S. H., Park, S.-J., Lee, K.-H.
(2004). Isolation and Characterization of rpoS from a Pathogenic Bacterium, Vibrio vulnificus: Role of {sigma}S in Survival of Exponential-Phase Cells under Oxidative Stress. J. Bacteriol.
186: 3304-3312
[Abstract]
[Full Text]
-
Hulsmann, A., Rosche, T. M., Kong, I.-S., Hassan, H. M., Beam, D. M., Oliver, J. D.
(2003). RpoS-Dependent Stress Response and Exoenzyme Production in Vibrio vulnificus. Appl. Environ. Microbiol.
69: 6114-6120
[Abstract]
[Full Text]
-
Michan, C., Manchado, M., Pueyo, C.
(2002). SoxRS Down-Regulation of rob Transcription. J. Bacteriol.
184: 4733-4738
[Abstract]
[Full Text]
-
Ferreira, A., O'Byrne, C. P., Boor, K. J.
(2001). Role of {sigma}B in Heat, Ethanol, Acid, and Oxidative Stress Resistance and during Carbon Starvation in Listeria monocytogenes. Appl. Environ. Microbiol.
67: 4454-4457
[Abstract]
[Full Text]
-
Zheng, M., Wang, X., Doan, B., Lewis, K. A., Schneider, T. D., Storz, G.
(2001). Computation-Directed Identification of OxyR DNA Binding Sites in Escherichia coli. J. Bacteriol.
183: 4571-4579
[Abstract]
[Full Text]
-
Landini, P., Volkert, M. R.
(2000). Regulatory Responses of the Adaptive Response to Alkylation Damage: a Simple Regulon with Complex Regulatory Features. J. Bacteriol.
182: 6543-6549
[Full Text]
-
Manchado, M., Michán, C., Pueyo, C.
(2000). Hydrogen Peroxide Activates the SoxRS Regulon In Vivo. J. Bacteriol.
182: 6842-6844
[Abstract]
[Full Text]
-
Prieto-Alamo, M.-J., Jurado, J., Gallardo-Madueno, R., Monje-Casas, F., Holmgren, A., Pueyo, C.
(2000). Transcriptional Regulation of Glutaredoxin and Thioredoxin Pathways and Related Enzymes in Response to Oxidative Stress. J. Biol. Chem.
275: 13398-13405
[Abstract]
[Full Text]
-
Geissmann, T. A., Teuber, M., Meile, L.
(1999). Transcriptional Analysis of the Rubrerythrin and Superoxide Dismutase Genes of Clostridium perfringens. J. Bacteriol.
181: 7136-7139
[Abstract]
[Full Text]
-
Monje-Casas, F., Jurado, J., Prieto-Alamo, M.-J., Holmgren, A., Pueyo, C.
(2001). Expression Analysis of the nrdHIEF Operon from Escherichia coli. CONDITIONS THAT TRIGGER THE TRANSCRIPT LEVEL IN VIVO. J. Biol. Chem.
276: 18031-18037
[Abstract]
[Full Text]
Top
Abstract
Introduction
