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Journal of Bacteriology, June 1999, p. 3833-3836, Vol. 181, No. 12
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Role for the oxyS Gene in Regulation of
Intracellular Hydrogen Peroxide in Escherichia
coli
Beatriz
González-Flecha1,* and
Bruce
Demple2
Physiology Program, Department of
Environmental Health,1 and Division of
Toxicology, Department of Cancer Cell
Biology,2 Harvard School of Public Health,
Boston, Massachusetts 02115
Received 25 January 1999/Accepted 8 April 1999
 |
ABSTRACT |
Intracellular hydrogen peroxide is regulated in Escherichia
coli by OxyR in response to the metabolic production of
H2O2. Here, we show that the untranslated
oxyS RNA controlled by OxyR has a role in this regulation.
The oxyS transcript appears to affect the metabolic output
of H2O2 rather than the removal of H2O2 by catalases-hydroperoxidases.
| |
TEXT |
The intracellular steady-state
concentration of hydrogen peroxide (H2O2)
depends on its rates of generation and dismutation. The major source of
superoxide (O2
) and
H2O2 in Escherichia coli cells is
the respiratory chain, which accounts for as much as 90% of the total
production of H2O2 (10, 12). Even
though the metabolic generation of H2O2 varies >10-fold during growth, E. coli cells maintain the
intracellular concentration of hydrogen peroxide within a narrow range
(0.20 ± 0.05 µM). This regulation is accomplished by a
continuous variation in the degree of activation of the OxyR protein,
which in turn governs transcription of katG, the gene
encoding catalase-hydroperoxidase I (catalase-HP-I) (8).
Although catalase-HP-I constitutes the major detoxification system for
endogenous H2O2 in exponential-phase E. coli cultures (8, 14), other regulated activities are
involved in controlling H2O2 levels:
oxyR strains have a higher concentration of
H2O2 and are more susceptible to exogenous
oxidative stress than strains mutated only in katG
(8).
In addition to catalase-HP-I, OxyR controls the expression of seven to
eight other proteins in Salmonella typhimurium and E. coli (4, 11, 19). One of these, the
ahpFC-encoded alkyl hydroperoxide reductase, was reported to
react with H2O2 in vitro (20), but
the activity did not contribute measurably to the regulation of the
H2O2 concentration in vivo (8).
Another member of the OxyR regulon, oxyS, was recently
reported to have posttranscriptional regulatory functions
(1). Strains overexpressing oxyS had a reduced
rate of spontaneous and H2O2-induced
mutagenesis, and cells carrying a deletion had near-wild-type
resistance to challenge with exogenous H2O2
(1). These results prompted us to evaluate the possible role
of oxyS in H2O2 homeostasis.
Bacterial strains and experimental procedures.
The strains of
E. coli used in this study are listed in Table
1. Strains BGF416 and BGF420 were
constructed by transduction (18) of the
oxyS2::Cm allele from strain GS035 into strains AB1157 and BGF611, respectively. Plasmid poxyS has the oxyS
gene under the control of the tac promoter in a multicopy
vector; even without induction by
isopropyl-
-D-thiogalactoside, the level of the
oxyS transcript expressed from poxyS is similar to the level
seen in H2O2-treated wild-type E. coli (1). Plasmid psyxO has the gene in the reverse
orientation. Cells were inoculated into Luria-Bertani (LB) broth
(18) containing the appropriate antibiotic and incubated
overnight at 37°C with gentle shaking (100 rpm). For experimental
measurements, the saturated cultures were diluted 100-fold into fresh
LB broth and incubated at 37°C for 3 h (optical density at 600 nm, ~1). Antibiotics were used at the following concentrations (in
micrograms per milliliter): tetracycline, 12.5; streptomycin, 50;
chloramphenicol, 25; and ampicillin, 100. The intracellular
concentration of H2O2 was assessed with
peroxidase-mediated scopoletin oxidation as previously described (9). Total catalase activity was assayed by monitoring the disappearance of H2O2 at 240 nm in cell
homogenates as described previously (8, 23) and normalized
to the protein concentration determined with bovine serum albumin as
the standard (15). The rate of H2O2
production was calculated from the experimental values for
H2O2 and catalase concentrations as previously
described (3, 8). The rate of O2
production was measured in membrane preparations by monitoring the
superoxide dismutase (SOD)-sensitive rate of cytochrome c reduction at 550 nm (
reduced 
oxidized = 21 mM1 cm1)
(2, 12). The reaction mixtures consisted of 50 mM potassium phosphate buffer (pH 7.4), 20 µM cytochrome c, 100 µM
NADH, and membrane protein (~0.2 mg/ml), with or without 50 U of
bovine CuZn SOD. The rate of respiration by cell cultures was measured with a Clark-type electrode (5). Three-hour cultures were
placed in the chamber of an oxygraph, and O2 concentration
was monitored for 5 to 10 min. Values are expressed in nanoatoms of
O2 per minute per 106 cells. The temperature
was 37°C. The cellular sensitivity to H2O2
was assessed on plates. Three-hour cultures were plated on LB plates,
and a filter disk (10-mm diameter, Whatman no. 1) was soaked with 150 mmol of H2O2 in water and placed in the center of the plate. After 12 to 16 h, the diameter of growth inhibition was measured (11). The frequency of spontaneous mutations to rifampin resistance was measured by plating on LB plates containing 125 µg of rifampin per ml; Rifr colonies were scored after
24 h of incubation at 37°C (11) and normalized to the
number of cells plated (8).
Regulation of intracellular concentrations of hydrogen
peroxide.
The level of H2O2 in the
oxyS-deficient strain BGF416 (in exponential phase) was
~2-fold higher than the level measured for the parental
oxyS+ strain (Fig.
1A), consistent with regulation mediated
by oxyS. This increase was similar to that observed for a
katG strain and less than that for a oxyRS
strain (Fig. 1A). We therefore tested whether mutations in both
oxyS and katG would act synergistically to
elevate the intracellular H2O2 concentration. A
oxyS katG double mutant (BGF420) had an ~3-fold
increase in H2O2 concentration (Fig. 1A), which
shows that katG and oxyS play independent roles in the OxyR-dependent regulation of H2O2.
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FIG. 1.
Effect of genetic deficiency in oxyS or
katG on the intracellular concentration of hydrogen peroxide
or catalase activity. (A) Steady-state H2O2
concentrations in intact cells. (B) Total catalase activity in cell
extracts. Values are the means of four to six independent
experiments ± SEMs. Strain abbreviations: WT1, AB1157
(oxyRS+ katG+); oxyS,
BGF416 (oxyR+ oxyS2::Cm);
katG, BGF611 (oxyRS+
katG17::Tn10); oxyS katG, BGF420
(oxyR+ oxyS2::Cm
katG17::Tn10); WT2, RK4936
(oxyRS+ katG+); oxyR,
TA4112 [(oxyRS-btuB)3].
|
|
Increased steady-state concentrations of H
2O
2
can result from decreases in the rate of its decomposition or increases
in the
rate of H
2O
2 production. The
catalase-HP-I is the major H
2O
2-decomposing
activity in exponentially growing
E. coli (
8,
14), and it
is possible that
oxyS regulates
katG by a posttranscriptional
mechanism (
1)
distinct from the transcriptional activation
of
katG
mediated by OxyR. However, the deletion of
oxyS did not
alter the total catalase activity in either the wild-type or the
katG background (Fig.
1B). As previously reported, the
katG mutant
strain had an ~70% lower catalase activity
due to the lack of
a functional catalase-HP-I (the remainder is the
katE-encoded
enzyme [
8,
14]).
Since
oxyS did not seem to affect expression of the primary
H
2O
2 scavenging activity, we hypothesized that
oxyS might influence
the cellular generation of
H
2O
2, most of which arises from the
dismutation
of O
2 by SOD (
10). We therefore
tested the
oxyS,
katG, and
oxyRS strains for changes in the rate of O
2
production. The rates of superoxide anion production in membrane
preparations (
12) increased 6-fold in the
oxyS
strain and 2.5-fold
in the
oxyRS strain compared to their
wild-type counterparts
(Table
2).
Expression of
oxyS from the multicopy plasmid poxyS
(
1) complemented the
oxyS phenotype by
preventing the increased
superoxide production (Table
2). In the
wild-type strain, the
rate of O
2 production
was not changed significantly by poxyS, the vector
plasmid, or a
plasmid with
oxyS in the reverse orientation (psyxO
[
1]) (Table
2 and data not shown).
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TABLE 2.
Effect of oxyS mutation on the rates of
H2O2 production in intact cells and
O2 production in
isolated membranesa
|
|
Unexpectedly, there seemed to be a small decrease in
O
2 production in the
katG strain
BGF611, but this was not statistically
significant (Table
2). However,
this strain is
oxyR proficient,
and it may be that there is
diminished O
2 production due to
OxyR-dependent induction of
oxyS as a result
of the
increased H
2O
2 concentration in this strain
(Fig.
1A).
Indeed, forcing the increased expression of
oxyS
by itself with
poxyS was sufficient to decrease
O
2 production in BGF611 as well as in the
oxyRS strain TA4112 (Table
2).
The rates of H
2O
2 production were calculated
from the experimental values for the steady-state
H
2O
2 concentration and the
total catalase
concentration (
3,
8). Deletion of
oxyS in
strain
BGF416 increased H
2O
2 production 1.7-fold,
compared to
a 6-fold increase in O
2
generation in this strain (Table
2). We have not determined
whether
H
2O
2 production is decreased by poxyS in the
oxyS strain.
In principle, the 2:1
O
2-H
2O
2 stoichiometry
of superoxide dismutation (
3) would predict
a maximal
threefold increase in H
2O
2 generation in the
oxyS strain.
However, reactions other than SOD can
consume O
2 (
7), and such reactions
may contribute to this difference.
The lack of a significant increase
in H
2O
2 production in the
oxyRS strain (TA4112) (Table
2) could also be related to non-SOD pathways
consuming
superoxide.
The metabolic production of O
2 and
H
2O
2 in
E. coli depends on the
number of active respiratory chain units per cell and on
the energetic
state of the chains, which can be altered by directing
the electron
flux through components with higher or lower energetic
efficiency
(coupled versus uncoupled components) (
10,
22).
Therefore,
changes in growth conditions or in the proportions
of coupled and
uncoupled components would determine the rate of
free radical
production at the respiratory chain. The effect of
oxyS on
the metabolic production of superoxide and hydrogen peroxide
reported
here could be due to this type of regulation. Three of
the eight
oxyS-regulated genes reported so far,
fhlA,
gadB, and
uhpT, are involved in
energy metabolism, although they have not
been related directly to
electron transport processes. The
fhlA gene encodes a
transcriptional activator of the hydrogenase pleiotropic
operon
(
hypABCDE) (
16,
17). The
gadB gene
encodes a glutamic
acid decarboxylase (
21), and
uhpT encodes the sugar phosphate
transporter (
6,
13). Perhaps
oxyS represses some energy pathways
that
leak more O
2 and are deleterious to cells
under oxidative
stress.
To test the hypothesis that
oxyS deletion affects the energy
metabolism of the cells, we measured the rates of respiration
in intact
wild-type and
oxyS cells. Oxygen uptake by exponentially
growing cells was significantly increased in the
oxyS
strain
(mean rate of O
2 consumption ± standard error
of the mean [SEM]
for three independent experiments, 0.77 ± 0.02 nanoatoms of O
2/min/10
6 cells; wild type,
0.51 ± 0.06 nanoatoms of O
2/min/10
6
cells), and this effect was largely suppressed by introduction
of the
multicopy plasmid poxyS (0.63 ± 0.07 nanoatoms of
O
2/min/10
6 cells). As seen for the rate of
O
2 production (Table
2), expression of
additional
oxyS from poxyS
in the wild-type strain may
slightly decrease the rate of respiration
(from 0.51 ± 0.06 to
0.44 ± 0.04 nanoatoms of O
2/min/10
6
cells). We therefore conclude that
oxyS does affect cellular
respiration. More-detailed studies will be required to delineate
the
mechanism underlying this
regulation.
We previously reported that twofold increases in the rate of production
of H
2O
2 are enough to trigger a substantial
OxyR-dependent
transcription of
katG (
10) and
that twofold increases in the
steady-state concentration of
H
2O
2 significantly increased the
frequency of
spontaneous mutation (
8). We therefore tested
whether the
lack of
oxyS or
oxyS and
katG resulted
in phenotypic
changes. We measured the frequency of spontaneous
mutation (a
sensitive marker of oxidative DNA damage
[
8]) and the cellular
sensitivity to exogenous
H
2O
2 in the various strains.
Compared
to the wild-type strain (AB1157), both parameters were
unchanged
in the
oxyS strain (BGF416), but
there were significant increases
in spontaneous mutation (2.8-fold) and
H
2O
2 sensitivity (1.5-fold)
in the
oxyS katG strain (BGF420) (Table
3). Multicopy
oxyS had
an
antimutagenic effect in the
oxyS and
katG
strains and possibly
in the
oxyRS+ strain (Table
3). Multicopy
oxyS in the double mutant strain
BGF420
(
oxyS katG), decreased the frequency of spontaneous
mutation
to almost the same value as obtained the
katG
strain without poxyS
(Table
3). Interestingly, multicopy
oxyS did not complement the
mutator phenotype of the
oxyRS strain (Table
3). This result
suggests that, in
addition to
oxyS, either
katG or some other
OxyR-dependent activity is critical for limiting mutagenesis by
endogenous H
2O
2. Alternatively,
oxyS
in poxyS is not regulated
in response to oxidative stress
(
1), so the level of the RNA
does not adjust to changing
H
2O
2 production.
Multicopy
oxyS did not significantly alter the sensitivity
to hydrogen peroxide in any of the strains listed (data not shown),
in
agreement with the lack of regulation of
katG by
oxyS (Fig.
1B).
Our results show an
oxyS-dependent regulation of the
intracellular production of oxygen free radicals. In this way, the
oxyS pathway and the OxyR-dependent induction of
catalase-HP-I would
provide two independent and complementary
mechanisms to limit
the levels of H
2O
2 during
aerobic growth and possibly under oxidative
stress. Elimination of
either pathway is still compatible with
almost normal aerobic growth,
but elimination of both
oxyS and
katG produces a
oxyRS-like phenotype with significant increases
in the
frequency of spontaneous mutations and sensitivity to
H
2O
2.
Defining the mechanism(s) by which
oxyS limits O
2 production in
respiring
E. coli may reveal general pathways to
avoid
oxidative
damage.
| |
ACKNOWLEDGMENTS |
We are grateful to members of the laboratory for discussions. We
thank G. Storz for providing us with strain GS035 and plasmids poxyS
and psyxO.
This work was supported by grants from the National Institutes
of Health (CA37831 to B.D. and P30 ES00002 to J. Brain).
B.G.-F. acknowledges the generous support of a fellowship from the
Francis Families Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Physiology
Program, Department of Environmental Health, 665 Huntington Ave.,
Boston, MA 02115. Phone: (617) 432-1277. Fax: (617) 432-0014. E-mail: bgonzale{at}hsph.harvard.edu.
| |
REFERENCES |
| 1.
|
Altuvia, S.,
D. Weinstein-Fischer,
A. Zhang,
L. Postow, and G. Storz.
1997.
A small, stable RNA induced by oxidative stress: role as a pleiotropic regulator and antimutator.
Cell
90:43-53[Medline].
|
| 2.
|
Boveris, A.
1984.
Determination of the production of superoxide radicals and hydrogen peroxide in mitochondria.
Methods Enzymol.
105:429-435[Medline].
|
| 3.
|
Chance, B.,
H. Sies, and A. Boveris.
1979.
Hydroperoxide metabolism in mammalian organs.
Physiol. Rev.
59:527-605[Free Full Text].
|
| 4.
|
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].
|
| 5.
|
Estabrook, R. W.
1967.
Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios.
Methods Enzymol.
10:41-47.
|
| 6.
|
Friedrich, M. J., and R. J. Kadner.
1987.
Nucleotide sequence of the uhp region of Escherichia coli.
J. Bacteriol.
169:3556-3563[Abstract/Free Full Text].
|
| 7.
|
Gardner, P. R., and I. Fridovich.
1992.
Inactivation-reactivation of aconitase in Escherichia coli. A sensitive measure of superoxide radical.
J. Biol. Chem.
267:8757-8763[Abstract/Free Full Text].
|
| 8.
|
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].
|
| 9.
|
Gonzálex-Flecha, B., and B. Demple.
1994.
Intracellular generation of superoxide as a by-product of Vibrio harveyi luciferase expressed in Escherichia coli.
J. Bacteriol.
176:2293-2299[Abstract/Free Full Text].
|
| 10.
|
Gonzalez-Flecha, B., and B. Demple.
1995.
Metabolic sources of hydrogen peroxide in aerobically growing Escherichia coli.
J. Biol. Chem.
270:13681-13687[Abstract/Free Full Text].
|
| 11.
|
Greenberg, J. T., and B. Demple.
1988.
Overproduction of peroxide-scavenging enzymes in Escherichia coli suppresses spontaneous mutagenesis and sensitivity to redox-cycling agents in oxyR-mutants.
EMBO J.
7:2611-2617[Medline].
|
| 12.
|
Imlay, J. A., and I. Fridovich.
1991.
Assay of metabolic superoxide production in Escherichia coli.
J. Biol. Chem.
266:6957-6965[Abstract/Free Full Text].
|
| 13.
|
Island, M. D.,
B.-Y. Wei, and R. J. Kadner.
1992.
Structure and function of the uhp genes for the sugar phosphate transport system in Escherichia coli and Salmonella typhimurium.
J. Bacteriol.
174:2754-2762[Abstract/Free Full Text].
|
| 14.
|
Loewen, P. C.,
J. Switala, and B. L. Triggs-Raine.
1985.
Catalases HPI and HPII in Escherichia coli are induced independently.
Arch. Biochem. Biophys.
243:144-149[Medline].
|
| 15.
|
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr, and R. J. Randall.
1951.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:265-275[Free Full Text].
|
| 16.
|
Lutz, S.,
A. Jacobi,
V. Schlensog,
R. Bohm,
G. Sawers, and A. Bock.
1991.
Molecular characterization of an operon (hyp) necessary for the activity of the three hydrogenase isoenzymes in Escherichia coli.
Mol. Microbiol.
5:123-135[Medline].
|
| 17.
|
Maier, T.,
A. Jacobi,
M. Sauter, and A. Böck.
1993.
The product of the hypB gene, which is required for nickel incorporation into hydrogenases, is a novel guanine nucleotide-binding protein.
J. Bacteriol.
175:630-635[Abstract/Free Full Text].
|
| 18.
|
Miller, J. H.
1972.
Experiments in molecular genetics.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 19.
|
Morgan, R. W.,
M. F. Christman,
F. S. Jacobson,
G. Storz, and B. N. Ames.
1986.
Hydrogen peroxide-inducible proteins in Salmonella typhimurium overlap with heat shock and other stress proteins.
Proc. Natl. Acad. Sci. USA
83:8059-8063[Abstract/Free Full Text].
|
| 20.
|
Niimura, Y.,
L. B. Poole, and V. Massey.
1995.
Amphibacillus xylanus NADH oxidase and Salmonella typhimurium alkyl-hydroperoxide reductase flavoprotein components show extremely high scavenging activity for both alkyl hydroperoxide and hydrogen peroxide in the presence of S. typhimurium alkyl-hydroperoxide reductase 22-kDa protein component.
J. Biol. Chem.
270:25645-25650[Abstract/Free Full Text].
|
| 21.
|
Smith, D. K.,
T. Kassam,
B. Singh, and J. F. Elliott.
1992.
Escherichia coli has two homologous glutamate decarboxylase genes that map to distinct loci.
J. Bacteriol.
174:5820-5826[Abstract/Free Full Text].
|
| 22.
|
Trumpower, B. L., and R. B. Gennis.
1994.
Energy transduction by cytochrome complexes in mitochondrial and bacterial respiration: the enzymology of coupling electron transfer reactions to transmembrane proton translocation.
Annu. Rev. Biochem.
63:675-716[Medline].
|
| 23.
|
Visick, J. E., and S. Clarke.
1997.
RpoS- and OxyR-independent induction of HPI catalase at stationary phase in Escherichia coli and identification of rpoS mutations in common laboratory strains.
J. Bacteriol.
179:4158-4163[Abstract/Free Full Text].
|
Journal of Bacteriology, June 1999, p. 3833-3836, Vol. 181, No. 12
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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