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Journal of Bacteriology, June 1999, p. 3833-3836, Vol. 181, No. 12
Physiology Program,
Received 25 January 1999/Accepted 8 April 1999
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.
The intracellular steady-state
concentration of hydrogen peroxide (H2O2)
depends on its rates of generation and dismutation. The major source of
superoxide (O2 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:
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
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
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
ABSTRACT
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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).
oxyR strains have a higher concentration of
H2O2 and are more susceptible to exogenous
oxidative stress than strains mutated only in katG
(8).
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).
TABLE 1.
Bacterial strains and plasmids of E. coli used
in this study
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.
View larger version (35K):
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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].
by SOD (10). We therefore
tested the oxyS, katG, and oxyRS strains for changes in the rate of O2
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 O2 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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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 O2 production due to
OxyR-dependent induction of oxyS as a result of the
increased H2O2 concentration in this strain
(Fig. 1A). Indeed, forcing the increased expression of oxyS
by itself with poxyS was sufficient to decrease
O2 production in BGF611 as well as in the
oxyRS strain TA4112 (Table 2).
The rates of H2O2 production were calculated
from the experimental values for the steady-state
H2O2 concentration and the total catalase
concentration (3, 8). Deletion of oxyS in strain
BGF416 increased H2O2 production 1.7-fold,
compared to a 6-fold increase in O2
generation in this strain (Table 2). We have not determined whether
H2O2 production is decreased by poxyS in the
oxyS strain. In principle, the 2:1
O2-H2O2 stoichiometry
of superoxide dismutation (3) would predict a maximal
threefold increase in H2O2 generation in the
oxyS strain. However, reactions other than SOD can
consume O2 (7), and such reactions
may contribute to this difference. The lack of a significant increase
in H2O2 production in the oxyRS strain (TA4112) (Table 2) could also be related to non-SOD pathways consuming superoxide.
The metabolic production of O2 and
H2O2 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 O2 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 O2 consumption ± standard error
of the mean [SEM] for three independent experiments, 0.77 ± 0.02 nanoatoms of O2/min/106 cells; wild type,
0.51 ± 0.06 nanoatoms of O2/min/106
cells), and this effect was largely suppressed by introduction of the
multicopy plasmid poxyS (0.63 ± 0.07 nanoatoms of
O2/min/106 cells). As seen for the rate of
O2 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 O2/min/106
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 H2O2 are enough to trigger a substantial
OxyR-dependent transcription of katG (10) and
that twofold increases in the steady-state concentration of
H2O2 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
H2O2 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
H2O2 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 H2O2. Alternatively, oxyS
in poxyS is not regulated in response to oxidative stress
(1), so the level of the RNA does not adjust to changing
H2O2 production.
|
oxyRS-like phenotype with significant increases in the
frequency of spontaneous mutations and sensitivity to
H2O2. Defining the mechanism(s) by which
oxyS limits O2 production in
respiring E. coli may reveal general pathways to avoid
oxidative damage.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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* 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.
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