From the Departamento de Bioquímica y
Biología Molecular, Universidad de Córdoba,
14071-Córdoba, Spain and the
Medical Nobel Institute for
Biochemistry, Department of Medical Biochemistry and Biophysics,
Karolinska Institute, S-171 77, Stockholm, Sweden
Received for publication, December 27, 2000, and in revised form, January 31, 2001
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ABSTRACT |
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Escherichia coli has two aerobic
ribonucleotide reductases encoded by the nrdAB and
nrdHIEF operons. While NrdAB is active during aerobiosis,
NrdEF is considered a cryptic enzyme with no obvious function. Here, we
present evidence that nrdHIEF expression might be important
under certain circumstances. Basal transcript levels were
dramatically enhanced (25-75-fold), depending on the growth-phase and
the growth-medium composition. Likewise, a large increase of >100-fold
in nrdHIEF mRNA was observed in bacteria lacking Trx1
and Grx1, the two main NrdAB reductants. Moreover, nrdHIEF
expression was triggered in response to oxidative stress, particularly
in mutants missing hydroperoxidase I and alkyl-hydroperoxide reductase
activities (69.7-fold) and in cells treated with oxidants (up to
23.4-fold over the enhanced transcript level possessed by cells grown
on minimal medium). The mechanism(s) that triggers nrdHIEF
expression remains unknown, but our findings exclude putative global
regulators like RpoS, Fis, cAMP, OxyR, SoxR/S, or RecA. What we have
learned about nrdHIEF expression indicates strong differences between its regulation and that of the
nrdAB operon and of genes coding for components of both
thioredoxin/glutaredoxin pathways. We propose that E. coli
might optimize the responses to different stimuli by co-evolving the
expression levels for its multiple reductases and electron donors.
Ribonucleotide reductases
(RRases)1 provide the
building blocks for DNA biosynthesis in all living organisms. RRases
are grouped into three major classes (class I, II, and III) based on
the mechanisms they use for radical generation and on their structural
differences. Class I is subdivided further into two subclasses (Ia and
Ib) based mainly on allosteric regulation but also on involvement of
auxiliary proteins (recently reviewed in Ref. 1). Escherichia coli has the coding potential for three different RRases. The NrdAB (class Ia) is active during aerobiosis, NrdDG (class III) is strictly anaerobic, and NrdEF (class Ib) is thought to be a cryptic
enzyme with no obvious function (1).
Class I enzymes receive the electrons required for the reduction of
ribose from small proteins, thioredoxins (Trxs) and glutaredoxins (Grxs), with two redox-active cysteine thiols, which by
dithiol-disulfide interchange reduce an acceptor disulfide in the
active center of RRase. Trxs and Grxs are kept in a reduced form by
NADPH, which reduces the redoxin either via the flavoprotein
thioredoxin reductase or via the flavoprotein glutathione reductase and
the ubiquitous tripeptide GSH (2). Escherichia coli
contains two thioredoxins (Trx1 and Trx2), three glutaredoxins (Grx1,
Grx2, and Grx3), and a novel redoxin (called NrdH) with
thioredoxin-like activity but glutaredoxin-like amino acid sequence
(2-5). Grx1 and Trx1 are the two main hydrogen donors of the E. coli NrdAB enzyme (3, 4, 6, 7). NrdH is a more specific hydrogen
donor for the NrdEF than for the NrdAB enzyme, whereas the opposite is
the case for Grx1 (5). Trx1 and Trx2 are hydrogen donors for NrdAB but
not for NrdEF (3, 5, 8).
The nrdA and nrdB genes that code for the NrdAB
class Ia reductase constitute a tightly regulated transcription unit
that does not include the gene for either Trx or Grx. Expression of nrdAB genes is cell cycle-regulated and increases when DNA
synthesis is inhibited (1). Regulation of nrdAB expression
in E. coli has been shown to be very complex; multiple
cis-acting positive regulatory sites identified upstream of
the nrdAB promoter appear to control, independently but in
concert, the cell cycle-dependent transcription and the
response to inhibition of DNA synthesis (9). Recent data from our group
add further complexity to the nrdAB regulation,
demonstrating a tight and inverse relation between expression of
nrdAB and that of genes coding for components of both
glutaredoxin and thioredoxin pathways (10). Interestingly, we have
observed that induction ratios of nrdAB transcription by
hydroxyurea (RRase inhibitor) are similar to the increments in
nrdAB basal expression of mutants lacking both Trx1 and
Grx1. Therefore, we have postulated that operation of the NrdAB enzyme in the absence of its two main reductants must lead to
disturbances in deoxyribonucleotide production sensed as those caused
by hydroxyurea.
The nrdE and nrdF genes that code for the NrdEF
class Ib reductase form a conserved operon where the promoter is
followed by four genes (11). Genes nrdH, coding for the
NrdH-redoxin, and nrdI, coding for a protein with a
stimulatory effect on ribonucleotide reduction, are present upstream of
nrdEF (5). It has been reported that the E. coli
transcription unit is not expressed in sufficient amounts to support
growth under normal laboratory conditions. Neither inhibitors of DNA
replication nor DNA damages induce its expression. Thus far, only
hydroxyurea has been shown to stimulate the expression of
nrdEF genes (11).
We have quantitated the in vivo transcription of genes
encoding for components of both glutaredoxin and thioredoxin pathways by means of a novel multiplex reverse transcription-polymerase chain
reaction (RT-PCR) approach (10, 12). In this protocol, all target
genes, a housekeeping gene (also named control gene, reference gene, or
internal standard), and one external standard 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
standard. Relative expressions of the targets to the reference (or to
the external standard) are measured. Lately (13), we have
experimentally demonstrated that our methodology fulfills all
theoretical requirements for precise quantification of both induction
and repression of gene transcription. Because of the PCR amplification
step, our method displays a much higher sensitivity than those of
current techniques for mRNA quantitation, like Northern blotting or
primer extension analyses. Here, we used this sensitive experimental approach to identify those growth conditions and stress circumstances under which expression of nrdHIEF genes might be required in
E. coli.
Bacterial Strains and Growth Conditions--
All bacterial
strains were E. coli K-12. UC5710 (arg56, nad113,
araD81, Treatments--
E. coli cells from an overnight
culture in LB broth were centrifuged and 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 presence of hydroxyurea, hydrogen
peroxide (H2O2), paraquat (PQ), t-butylhydroperoxide (tBOOH), 4-nitroquinoline 1-oxide
(4NQO), or
N-methyl-N'-nitro-N-nitrosoguanidine
for a fixed time period. The cells were then rapidly cooled to 0 °C
for total RNA purification.
Primers--
Primers (listed in Table I) were designed with the
Oligo 6.1.1/98 (Molecular Biology Insights Inc., Plymouth, MN) program in order to obtain the highest specificity and performance in multiplexed PCRs. Target genes code for the NrdH-redoxin
(nrdH), the NrdI stimulatory protein (nrdI), and
the R1E (nrdE) and R2F (nrdF) subunits of
E. coli class Ib RRase. As described previously (10, 12),
the gapA gene, which codes for
D-glyceraldehyde-3-phosphate dehydrogenase, was used as an
internal standard. The internal standard normalizes variations in RNA
extraction, reverse transcription, and PCR amplification among samples.
To evaluate the extent of putative variations in the expression of the
reference gene, an external standard was included, as described (10).
The external standard was an in vitro synthesized RNA
fragment encoded by the CYP1A gene from Liza
aurata. This external standard has no homology with the E. coli genome.
Multiplex RT-PCR for in Vivo Quantification of nrdHIEF
Transcription--
RNA purification and cDNA synthesis were as
described (10). At least two independent RNA preparations were isolated
for each experimental condition, each RNA sample being retrotranscribed on three separated occasions. PCR amplification of cDNA was carried out using all of the primer pairs listed in Table I or using just those
for the nrdH and nrdE target genes, the
gapA reference gene, and the external standard. In this last
case, the multiplex PCR amplification was performed in a mixture
(25-µl final volume) containing MPCR buffer 3 (Maxim Biotech Inc.,
San Francisco, CA) supplemented with 1 mM
MgCl2, a 250 µM concentration of each dNTP, 1 µl of cDNA solution, 1.25 units of AmpliTaq Gold DNA polymerase, and primers at the following concentrations: 0.07 µM
(nrdH), 0.09 µM (nrdE), 0.04 µM (gapA), and 0.14 µM (external
standard). After incubation at 95 °C for 12 min (for activation of
DNA polymerase), 26 cycles of PCR were performed. Each cycle consisted
of 1 min of denaturation at 94 °C and 45 s of annealing and
enzymatic primer extension at 70 °C. These multiplex PCR conditions
were optimized as detailed (13) to ensure that the amplifications were
in the exponential phase and that the efficiencies remained constant in
the course of the PCR. Reaction products were quantified as described
previously (12). Differences among PCR outcomes were normalized by
comparing the fluorescence intensity of each band to that resulting
from gapA amplification (internal standard). The levels of
gapA in reference to the external standard remained essentially equal among the strains and experimental conditions investigated in this work. Consequently, changes detected with reference to the housekeeping gapA gene were accurately
attributed to variations in the expression levels of the target genes
under analysis. Samples for comparison of different experimental
conditions or different bacterial strains were handled in parallel.
Data are the means ± S.E. from n Quantification of the nrdHIEF Operon Expression--
In a prior
study (11), expression of nrdEF genes was quantified by a
competitive RT-PCR, in which the competitor had an internal deletion of
99 base pairs and the reaction products were resolved in ethidium
bromide-agarose gels and analyzed by densitometry. Several points at
which errors can occur by using such a methodology include differences
in amplification efficiencies because of the great size difference
between the competitor and the target, possible heteroduplex formation,
and the need to compensate for differences in fragment label
incorporation. When measuring rare species of mRNA, which requires
a high number of PCR cycles, the control of those problems is of
maximal importance, even for relative quantification (e.g.
see Refs. 22-24). In this work, by taking advantage of the high
sensitivity, accuracy, and reproducibility of our multiplex RT-PCR
procedure (13), we monitored variations in basal levels of
nrdHIEF expression and in response to several stress
conditions. To achieve this objective, we first confirmed that
transcription from the nrdHIEF promoter normally takes place in wild-type cells and increases upon hydroxyurea exposure (11).
UC5710 cells were, thus, exposed to increasing concentrations of
hydroxyurea (varying from 1 to 30 mM) for 5 min.
Thereafter, the expression of all of the genes that constitute the
nrdHIEF operon was examined by using the primer pair set in
Table I. Basal levels of nrdH,
nrdI, nrdE, and nrdF gene expression
were readily detected and increased upon hydroxyurea treatment
(data not shown). As an average, an induction level of 12.4 ± 2.5-fold (relative to untreated bacteria) was observed at the minimal
dose assayed of 1 mM. It is worth noting that in the prior
study by Jordan et al. (11), such an induction level was
observed under much more acute treatment conditions (bacteria grown in
the presence of 50 mM hydroxyurea). Since we do not detect
differences in the number of times that expression of each
nrd gene was increased by hydroxyurea, the multiplex PCR was
simplified in further experiments by amplifying just two
(nrdH and nrdE) of the four genes included in the
nrdHIEF transcription unit (in addition to the internal and
external standards).
Effects of Growth Conditions on Basal Levels of nrdHIEF
Expression--
The expression profiles of nrdH and
nrdE genes throughout the growth curve of wild-type UC5710
cells in rich LB medium are shown in Fig.
1. Maximal expression levels were
observed at the initial stages of exponential growth
(A600
Notable variations in gene expression were observed also with respect
to the composition of the growth medium (Fig. 1). Thus, bacteria
growing on M9 minimal medium with glucose had from 2-fold (A600 = 0.2) to 75-fold
(A600 = 0.7) higher levels of nrdH
and nrdE transcripts than did bacteria growing on rich LB
medium alone. Since carbon sources control intracellular cAMP levels
(27), we examined next the effects on nrdH and
nrdE expression, of the addition of glucose to the LB
nutrient medium (to 2 g/liter), and of replacement of glucose by
lactose as the sole carbon source in the M9 minimal medium. No
dependence on carbon source was observed (data not shown).
As indicated above, -fold variations were identical for both
nrdH and nrdE genes; nrdH data will
henceforth be considered representative of the entire
nrdHIEF transcription unit.
Effects of Deficiencies in Thioredoxin and Glutaredoxin/GSH
Pathways or in Antioxidant Enzymes on Basal Levels of nrdHIEF
Expression--
In addition to the classic function of acting as
reductants for RRase, both the Trx and Grx/GSH pathways are required to
maintain the low thiol-disulfide redox potential of the bacterial
cytoplasm (20, 28). On the other hand, catalase, alkyl hydroperoxide reductase, and superoxide dismutase activities maintain the
steady-state concentrations of peroxides and superoxide beneath their
respective toxicity thresholds (29). The influence of missing various
components of these systems on basal levels of nrdHIEF
expression is studied in Table II.
Significant increments in the steady-state levels of nrdHIEF
transcript were detected in mutants compromised either in both the Trx-
and Grx/GSH-reducing pathways or in the removal of endogenous oxidants.
Of particular note is the large up-regulation (>30-fold) quantified in
bacteria lacking Trx1 in combination with either Grx1 (UC827) or GSH
(UC1358) or missing both catalase hydroperoxidase I and alkyl
hydroperoxide reductase activities (UC4101).
Since OxyR can be activated either by challenge with an oxidant such as
H2O2 or directly by a change in the cellular
thiol-disulfide status caused by the inactivation of the two Trx- and
Grx/GSH-reductive pathways (20, 28), it seemed possible that the
transcriptional induction of nrdHIEF that we report in Table
II was due to activation of OxyR. This possibility was investigated by
studying the effects of oxyR mutant alleles on basal levels
of gene expression in UC1358. As shown in Table II, expression of
nrdHIEF genes was modulated by mutations in the
oxyR regulatory locus, but it followed a pattern opposite to
that exhibited by most OxyR-regulated genes (10, 29). Therefore, the
high basal level of nrdHIEF message in UC1358 (32.7-fold)
was further elevated (not diminished) in its
The higher increment in basal level of nrdHIEF expression
caused by the Gene Expression Induction by Hydrogen Peroxide and
Superoxide--
To gain further information for the regulation of
nrdHIEF expression under oxidative stress conditions,
wild-type bacteria were exposed to increasing concentrations of
H2O2 or paraquat (a superoxide-generating
compound). Since OxyR and SoxR together with SoxS are key regulators of
the adaptive responses to H2O2 and superoxide
radicals, respectively (recently reviewed in Ref. 29), strains with
mutations that eliminate either OxyR (UC1342) or both SoxR and SoxS
(UC1333) were used in conjunction with wild-type bacteria (UC5710). As
shown in Fig. 2, induction of
nrdHIEF mRNA was readily seen in response to both
oxidants. Therefore, increments of 23.4- and 4.3-fold were quantified
shortly (5 min) upon exposure of wild-type cells to 100 µM H2O2 or 500 µM
PQ concentration, respectively. Inductions were preserved in the
Gene Expression Induction by Alkyl Hydroperoxide and
4NQO--
AhpC together with AhpF reduces a variety of physiologically
relevant alkyl hydroperoxides such as thymine hydroperoxide and linoleic acid hydroperoxide as well as nonphysiological model alkyl
hydroperoxides like tBOOH (32). Encouraged by the high increment in
basal level of nrdHIEF mRNA caused by the simultaneous deficiency in catalase hydroperoxidase I and alkyl hydroperoxide reductase activity (UC4101 in Table II), we examined the effect of
tBOOH treatments on expression of nrdHIEF operon (Fig.
3). In this experiment, we used a set of
primers (13) that allows us to compare the response of
nrdHIEF genes with that of genes encoding for the main
aerobic RRase (nrdAB operon) besides that of well known
components of the E. coli oxidative stress responses (like
oxyS and grxA genes) (10). While the amount of
nrdAB transcript remains basically unchanged, the
nrdHIEF transcript level was readily induced by tBOOH. As
shown in Fig. 3, the oxidative stress-responsive genes, oxyS
and grxA, gave also a clear positive response to tBOOH treatments.
4NQO is a widely studied model mutagen and carcinogen, which derives
its activity from the induction of both bulky adducts (for that,
it is often referred to as UV radiation-like) and oxidative damages in cellular DNA (33). As in the case of tBOOH, treatments with
4NQO elevated the nrdHIEF and the oxyS and
grxA transcript levels without affecting the
nrdAB expression. Since 4NQO is known to trigger the SOS
response (33), we next studied whether this compound increased
nrdHIEF expression in the absence of RecA protein. No
difference between RecA+ and RecA
In contrast to chemical oxidants,
N-methyl-N'-nitro-N-nitrosoguanidine,
which is a strong monofunctional alkylating agent that methylates
cellular DNA, resulting in multiple types of primary lesions (33), did
not affect nrdHIEF expression in wild-type E. coli (data not shown).
Previous studies have reported that suppression by the
nrdHIEF genes of the inviability of E. coli
strains defective in either the NrdAB reductase or three of its
reductants (Grx1, Trx1, and Trx2) requires a second gene copy placed in
either the chromosome or a cloning plasmid (11, 34). Based on this
genetic evidence, it is commonly accepted that the nrdHIEF
operon is underexpressed in bacteria grown at standard aerobic
conditions, thus somewhat calling into question its physiological
significance. The main goal of this study was the accurate
quantification by multiplex RT-PCR of variations in nrdHIEF
transcript levels in order to elucidate the growth conditions and
stress circumstances under which expression of nrdHIEF genes
might become important to E. coli.
Results presented in this work confirm that transcription of the
nrdHIEF operon normally takes place in E. coli
cells grown in LB medium to midexponential phase (11). Nevertheless, we present the first indication that this low basal level of
nrdHIEF mRNA can be dramatically enhanced in wild-type
bacteria as a function of the growth phase and the composition of the
growth medium: a pronounced increase (from 25- to 75-fold) in
nrdHIEF transcript could be monitored at the initial stages
of exponential growth in LB and in cells cultured in M9 minimal medium.
Of note is the additional observation of no significant
differences among the relative transcript levels for the genes of
the nrdHIEF operon, indicating for the first time that the
expression of the genes encoding the NrdEF ribonucleotide reductase is
tightly co-regulated with that of genes encoding accessory proteins,
like its specific NrdH-redoxin hydrogen donor.
We hypothesize that the strong growth phase- and growth
medium-dependent regulation of nrdHIEF
transcription might have a functional significance for wild-type cells.
It is well known that the physiology of a bacterial cell shifts between
the phases of a culture and with the quality of the growth medium and
that many of these changes are realized at the level of gene expression (35). Therefore, while the rich medium contains preformed building blocks of macromolecule synthesis, in the minimal medium the carbon backbone of the glucose molecule is rearranged through
biosynthetic pathways to generate each of the building blocks de
novo. The higher expression of nrdHIEF genes in minimal
medium might thus be indicative of the need to generate the building
blocks for DNA biosynthesis de novo from glucose.
Accordingly, a recent single experiment that used DNA arrays of the
entire set of E. coli genes to discover genomic expression
patterns has revealed that those genes with a pivotal role in central
metabolism tend to be expressed at higher levels in minimal medium than
in rich LB medium (36). Here, we verified this tendency for the
expression of the nrdHIEF operon. Many genes having a growth
phase- and growth medium-dependent regulation are under
global regulatory mechanisms like those mediated by RpoS, Fis, or the
intracellular levels of cAMP (35, 36). Our data indicate, however, that
these global regulators are not responsible for the nrdHIEF
expression pattern, thus making a difference with the nrdAB
operon, which is known to be under the positive regulation of Fis
protein (37).
The striking up-regulation of the nrdHIEF operon in
wild-type cells under certain growth conditions raises an intriguing
question: why is the inactivation of the nrdAB operon lethal
to E. coli cells in the presence of oxygen? The straight
answer to this question is that this up-regulation is conceivably
insufficient to complement the lack of the first NrdAB RRase or three
of its reductants (Grx1, Trx1, and Trx2) unless a second extra
copy of the nrdHIEF operon is placed on the bacterial
chromosome. In this context, quantification at the protein level would
be of maximal interest, since it has been postulated that translation
of nrdE message might be low because the start codon is TTG
(11).
We have also been able to show that the basal level of
nrdHIEF mRNA is dramatically increased (>100-fold) in
bacteria (UC827) simultaneously lacking Trx1 and Grx1, the two main
reductants of the NrdAB reductase. We speculate that this enormous
increment in nrdHIEF expression might be physiologically
relevant for the viability of UC827 (38). This trxA grxA
double mutant would maintain the balanced supply of
deoxyribonucleotides required for DNA synthesis by triggering the
transcription of the operons (nrdAB and nrdHIEF)
(Ref. 12; this work) that code for both aerobic RRases and for the NrdH
reductant. In this respect, it is worth noting that (i) NrdH, the
specific reductant of the NrdEF enzyme, is also a functional hydrogen
donor for the NrdAB reductase (5) and (ii) although the other two known
reductants for NrdAB remain in trxA grxA mutant cells, one
(Grx3) is highly inefficient (4) and the other (Trx2) has a low level
of expression under normal aerobic conditions (3, 34).
Oxidative stress is an unavoidable by-product of aerobic life.
Oxidative stress is caused by exposure to H2O2,
superoxide anion and hydroxyl radical, which in turn damage proteins,
nucleic acids, and cell membranes, producing detrimental molecules like alkyl hydroperoxides (29, 32). In this paper, we present evidence that
nrdHIEF expression is triggered in E. coli when
confronting oxidative stress. Imbalances in the intracellular
pro-oxidant/antioxidant ratio were produced either by exposing the
cells to different chemical oxidants (H2O2, PQ,
tBOOH, and 4NQO) or by loss of major antioxidant defenses (catalase,
superoxide dismutase, and alkyl hydroperoxide reductase activities). In
both situations, large increments in nrdHIEF transcript
levels were quantified. Of particular note is the vast up-regulation
observed in bacteria missing both catalase hydroperoxidase I and alkyl
hydroperoxide reductase (69.7-fold) and, in general, in bacteria
exposed to the oxidants. In this latter situation, the increments (up
to 23.4-fold) were observed shortly (5 min) after oxidant exposure and
over the already enhanced basal levels of nrdHIEF mRNA
displayed by cells grown on minimal medium.
What might an enhanced nrdHIEF expression do in the E. coli oxidative stress response? An answer to this question could
be to increase the free radical scavenging capacity of cells by
increasing the NrdH protein level. In this respect, it is worth noting
that Trx is a highly efficient antioxidant with a role in protecting E. coli against oxidative stress (39-41); NrdH with a redox
potential of The mechanism by which nrdHIEF expression is triggered under
oxidative stress conditions remains elusive, but data reported indicate
that the presence of reactive oxygen species must be sensed by
regulators that are distinct from both OxyR and SoxR/S. Contrary to
what we have learned in this work about nrdHIEF expression, the expression of the nrdAB operon that codes for the main
class I reductase was not induced by oxidative stress, in agreement with previous results (10). Interestingly, however, genes that code for
two (Grx1 and Trx2) out of the five known NrdAB reductants, together
with those that code for the enzymes (glutathione reductase and
thioredoxin reductase) that regenerate their reduced forms, are part of
the OxyR regulon. These findings suggest that E. coli might
optimize the responses to different stress situations by co-evolving
the expression levels for multiple RRases and reductants. In this
respect, it is worth noting that while DNA-damaging agents that induce
the SOS response produce in general an overexpression of
nrdAB genes, these agents have no effect on
nrdHIEF expression (Ref. 11; this work).
In short, this report strongly suggests that nrdHIEF
expression might be important under specific physiological
circumstances. Findings presented here open numerous ways for future
studies. One challenge will be the construction of a mutant
lacking the entire nrdHIEF transcription unit in order
to elucidate further compensations among the expression of both aerobic
RRases and their reductants under either normal or stressed conditions.
The multiplex RT-PCR approach will be of relevance in these coming experiments. Nevertheless, quantifications at the protein level will be
also necessary in order to unravel the relationships between mRNA
production and protein synthesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(uvrB-bio)) was considered the parental wild
type (14). UC1342 (
oxyR::kan),
UC1394 (oxyR2), UC1333
(
sox-8::cat), UC827 (
trxA
grxA::kan), UC1358 (
trxA gshA),
UC1363 (
trxA gshA
oxyR::kan), UC1395 (
trxA
gshA oxyR2), UC1101 (pKM101), UC498 (katG
katE::Tn10 pKM101), and UC628
(((sodA::Mud PR13)25 (sodB-kan)1-
2) pKM101) have been previously described (10, 15, 16). UC499 (katG17::Tn10), UC4910
(
ahpCF::kan), UC1049 (katG
katE::Tn10), and UC4101
(katG17::Tn10
ahpCF::kan) were constructed by
P1-mediated transduction. Successful transfer of the
katG17::Tn10 mutation was confirmed by
assaying catalase hydroperoxidase I activity, as described (17).
Successful transfer of the
ahpCF::kan deletion was confirmed by
screening for no DNA amplification with specific primers (18). Strains
with the
oxyR::kan or
sox-8::cat mutant allele do not
induce the expression of genes regulated by OxyR or SoxR/S upon
exposure to H2O2 or paraquat, respectively
(10). Strains carrying the oxyR2 allele exhibit constitutive
high levels of OxyR-regulated gene expression (10). Strains with the
trxA and with either the
grxA::kan or the gshA mutant
allele exhibit undetectable levels of Trx1 and of Grx1 or GSH,
respectively (15). Double katG katE or sodA sodB
mutants retain <1% of the wild-type catalase or superoxide dismutase
level, respectively (16). Double katG ahpCF mutants are
known to have increased intracellular levels of peroxides (19, 20).
Plasmid pKM101 carries the mucAB genes, which make bacteria
more susceptible to SOS-dependent mutagenesis (21).
Bacteria were grown in Luria-Bertani (LB) nutrient broth or M9 minimal
medium (glucose at 2 g/liter), as described (10).
6 independent
multiplexed PCR amplifications. Statistical comparisons were done by a
hierarchical analysis of variance with SAS software (Statistical
Analysis System, version 6.03). Data presented here as relative
expression ratios do not provide any indication of the mechanism that
contributes to the dynamic control of a particular mRNA
concentration, whether it is the rate of transcription initiation or
the rate of transcript turnover.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PCR primer characteristics
0.2); then gene expression decreased rapidly, reaching 25-fold lower levels as the culture continued growing from midexponential to stationary phase
(A600
0.4). Fis and RpoS (also named
S or
38) are two regulatory proteins with
acute growth phase-dependent expression in LB medium (25,
26). To determine the hypothetical influence of Fis and RpoS on the
nrdH and nrdE transcript levels during the course
of cell growth in LB, both fis::767 and
rpoS::Tn10 mutants were analyzed in
comparison with otherwise isogenic wild-type cells. Data obtained (not
shown) indicate that neither the presence of Fis nor the presence of
RpoS affects the expression of nrH and nrdI genes
in E. coli.
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Fig. 1.
Effect of growth phase and growth medium on
basal levels of gene expression. Wild-type cells (UC5710) from an
overnight culture in LB broth were centrifuged and diluted 100-fold
into 50 ml of fresh LB broth or M9 minimal medium. Bacteria were then
incubated at 37 °C with shaking at 150 rpm. Samples were collected
at regular time intervals and frozen with liquid nitrogen. Culture
growth was monitored by measuring A600
(OD600). The growth rate was 28 min on the rich LB
medium and 48 min on M9 minimal medium. The mean values of the
fluorescence signal of the nrdH (left) or
nrdE (right) target sequence relative to that of
gapA (internal standard) were plotted as a function of the
A600 value. Bacteria grown in LB broth are
indicated by the line, and bacteria grown in M9 minimal
medium are indicated by lightly shaded
bars (A600 of 0.2 and 0.7).
Error bars, S.E.
Basal levels of gene expression in bacteria defective in Trx and
Grx/GSH pathways or in antioxidant enzymes
oxyR::kan null mutant derivative
(UC1363), where a 54.4-fold increase in the amount of
nrdHIEF transcript was quantified. On the contrary, a
decrease (not an increase) in the steady-state level of UC1358 was
observed in the strain (UC1395) that carries the oxyR2
constitutive mutation. These results indicated that OxyR is not
directly involved in the nrdHIEF up-regulation reported in
Table II.
oxyR::kan null allele
might therefore be attributed to the underexpression in UC1363 of
OxyR-regulated genes involved in antioxidant defense, like those coding
for catalase hydroperoxidase I and alkyl hydroperoxide reductase (10,
18, 29). A similar (while opposite) argument might explain the lower
increment quantified in bacteria with the oxyR2 constitutive
mutation. Likewise, the difference between UC827 and UC1358 with
respect to nrdHIEF expression can be explained by
differences in the expression levels of OxyR-regulated genes. The
strain (UC1358) with higher levels of antioxidant defenses (10)
displayed the lower increment in nrdHIEF/gapA ratio (Table II).
oxyR and
soxR/S mutant strains, indicating
that neither OxyR (in agreement with the results above) nor SoxR/S is
involved in the oxidative stress stimulation of nrdHIEF
expression. In fact, as expected from data in Table II, bacteria
compromised in the removal of H2O2
(
oxyR) or superoxide (
soxR/S) exhibited
higher (not lower) levels of induction compared with the otherwise
isogenic control strain. Therefore, the increased level of
H2O2 induction in the OxyR mutant and that of
PQ induction in the SoxR/S mutant is expected because of the
inability of these bacteria to induce both katG and
ahpCF (18) or sodA (30) transcription upon
H2O2 or PQ exposure, respectively. The
increased level of PQ induction in the OxyR mutant is additionally
explained by the spontaneous and superoxide dismutase conversion of
O
View larger version (17K):
[in a new window]
Fig. 2.
Induction of gene expression by
H2O2 and PQ. UC5710 (wild type), UC1342
( oxyR::kan), and UC1333
(
sox-8::cat) cells were treated (see
"Experimental Procedures") for 5 min with
H2O2 or PQ at the indicated concentrations. The
mean values of the fluorescence signal of the nrdH target
sequence (considered representative of the entire nrdHIEF
transcription unit) relative to that of gapA (internal
standard) were plotted for untreated and H2O2-
or PQ-treated bacteria. Error bars represent S.E.
To facilitate the comparison among different bacterial strains, data
were divided by that of untreated UC5710. These -fold increments are
given in parentheses. Only statistically significant
increments are indicated.
View larger version (20K):
[in a new window]
Fig. 3.
Induction of gene expression by tBOOH and
4NQO. UC5710 (wild type) cells were exposed (see "Experimental
Procedures") to increasing concentrations of tBOOH or 4NQO (varying
from 3 to 300 µM) for 5 min. The set of primers (see Ref.
13) used in these experiments considers the nrdE and
nrdA sequences as representative of the entire
nrdHIEF and nrdAB transcription units,
respectively. The mean values of the fluorescence signal of each target
sequence were referred to that of gapA (internal standard).
To facilitate the comparison among different genes, data from treated
bacteria were divided by those from the corresponding untreated
samples. These -fold increments were plotted as a function of the log
values of tBOOH or 4NQO concentrations. Significant increments are
indicated by solid symbols.
cells was
observed (data not shown), indicating that this effect is not
SOS-dependent.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
248.5 mV is as potent a reductant as Trx (5).
Furthermore, an enhanced ribonucleotide reduction capacity should be
advantageous under oxidative stress conditions, since reactive oxygen
species escaping from antioxidant defenses can inflict much oxidative damage on DNA (42).
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FOOTNOTES |
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* This work was supported by Dirección General de Enseñanza Superior Grant PB98-1627, by Junta de Andalucía Group CVI 0187, and by Swedish Cancer Society Grant 961.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 predoctoral fellowship from Junta de Andalucía.
¶ Recipients of postdoctoral contracts from the Ministerio de Educación y Cultura.
** To whom correspondence and reprint requests should be addressed: Dept. de Bioquímica y Biología Molecular, Campus de Rabanales, edificio C-6, planta 2a, Carretera Madrid-Cádiz Km 396-a, Universidad de Córdoba, 14071-Córdoba, España. Tel.: 34-957-218695; Fax: 34-957-218688; E-mail: bb1pucuc@uco.es.
Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.M011728200
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ABBREVIATIONS |
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The abbreviations used are: RRase, ribonucleotide reductase; Trx, thioredoxin; Grx, glutaredoxin; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; PQ, paraquat; tBOOH, t-butyl hydroperoxide; 4NQO, 4-nitroquinoline 1-oxide.
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