From the
Exposure of cells to hydrogen peroxide
(H
Reactive by-products of oxygen, superoxide anion radicals
(O
``Oxidative stress''
refers to imbalances between the production and disposal of oxygen
radicals (Gerschman et al., 1954; Sies, 1991). Oxidative
stress has been associated with aging (Harman, 1991), carcinogenesis
(Cerutti, 1985), and diverse clinical situations such as
Alzheimer's disease (Luft, 1994; Yan, 1994) and cell damage due
to ischemia-reperfusion (González-Flecha et al., 1993).
Oxidative stress is also exploited as a cytotoxic weapon during
phagocytosis (Babior, 1991). H
Genetic responses to
oxidative stress occur in bacteria (Demple, 1991), yeast (Jamieson,
1994), and mammalian cell lines (Amstad et al., 1994; Keyse
and Tyrrel, 1989; Schulze-Osthoff and Baeuerle, 1994). Escherichia
coli cells possess a specific defense against peroxides mediated
by the transcriptional activator OxyR and another against superoxide,
controlled by the two-stage soxRS system (Hidalgo and Demple,
1995). The OxyR regulon includes catalase-hydroperoxidase I, encoded by
katG, a NADPH-dependent alkyl hydroperoxidase, encoded by
ahpFC, glutathione reductase, encoded by gorA, a
protective DNA binding protein, encoded by dps, and several
other genes (Hidalgo and Demple, 1995). The expression of these genes
is elevated in E. coli exposed to 5-100 µM
H
Despite
intensive study of adaptive responses of bacteria to oxidative stress
as cited above, no systematic analysis of the effects of growth state
or the physiological threshold of oxidative stress required to trigger
these responses has been reported. Imlay and Fridovich (1991a)
estimated a steady-state O
For growth, strains were inoculated into LB broth
(Miller, 1992) containing the appropriate antibiotic and incubated at
37 °C for 12-16 h with gentle shaking (200 rpm). The
saturated cultures were diluted 100-fold into fresh LB and incubated
for the indicated times at 37 °C with shaking at 200 rpm in flasks
of volume 10-20-fold greater than the culture. Antibiotics were
used at the following concentrations (in µg/ml): ampicillin, 100;
tetracyclin, 12.5; kanamycin, 50; and chloramphenicol, 25.
To identify specific sites of the respiratory
chain at which single electrons might leak to form
O
The experiments presented here indicate that the major source
of H
Interestingly, the calculated rate of
H
Mutational inactivation
of different components of the respiratory chain also affected the rate
of H
H
Mutational changes in
the composition of the respiratory chain prompted complementary changes
in catalase activity. It is worth noting that, even in the strains with
a decreased rate of H
It is interesting to note that the magnitude of the
H
Exponentially growing E. coli AB1157
cells were incubated with FCCP, rotenone, or antimycin for 10 min,
washed, resuspended in PBS, and assayed for H
Exponentially growing bacteria were
assayed for H
Overnight cultures of BGF931 in LB were diluted 1:100 in fresh LB
and incubated at 37 °C. Cultures grown for 1, 2, and 3 h were
treated with the concentration of H
We are grateful to Dr. Robert Gennis (University of
Illinois) for providing us with strains GR70N, GO103, GO104, MWC190,
MWC215, and MWC232; Dr. Gisela Storz (National Institute of Child
Health and Human Development) for providing plasmid pAQ24; and Dr.
Nancy Kleckner (Harvard University) for providing bacteriophage
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
O
) mediates adaptive responses or oxidative
damage, depending on the magnitude of the challenge. Determining the
threshold for peroxide-mediated oxidative stress thus requires
quantitation of the changes in endogenous H
O
production. The intracellular steady-state concentrations of
H
O
were measured in intact Escherichia coli under different conditions. Compounds that block electron
transport at NADH dehydrogenase (rotenone) or between ubiquinone and
cytochrome b (antimycin) showed that univalent reduction of
O
can occur at these sites in vivo to form
superoxide anion (O
), in agreement with
reports for mammalian mitochondria. Mutational inactivation of
different components of the respiratory chain showed that
H
O
production also depended on the energy
status of the cell and on the arrangement of respiratory chain
components corresponding to particular growth conditions. Production
rates for O
and H
O
were linearly related to the number of active respiratory chains
that reached maximal values during exponential growth. In the strains
defective in respiratory chain components, catalase activity was
regulated to compensate for changes in the H
O
production rates, which maintained intracellular
H
O
at 0.1-0.2 µM during
aerobic growth over a wide range of cell densities. The expression of a
katG`::lacZ fusion (reporting transcriptional control of the
catalase-hydroperoxidase I gene) was increased by H
O
given either as a pulse or as a steady production. This response
not only depended on the type and severity of the stimulus but was also
strongly influenced by the growth phase of the cells.
), hydrogen peroxide
(H
O
), and hydroxyl radicals (HO), are derived
from sequential univalent reductions of molecular oxygen. These agents
are produced continuously in aerobically growing cells (Chance et
al., 1979). In eukaryotic cells, the respiratory chain and
cytochrome P-450 seem to be the significant intracellular sources of
O
(Chance et al., 1979).
H
O
is produced by the superoxide
dismutase-catalyzed dismutation of O
in
mitochondria and in the cytosol and by flavin oxidases in peroxisomes
(Chance et al., 1979). Intracellular
O
and H
O
are kept
at acceptably low concentrations by the action of antioxidant enzymes
such as superoxide dismutase, catalase, and other peroxidases (Chance
et al., 1979; Sies, 1991).
O
can be excreted
by the acatalasic bacterium Streptococcus sanguis in amounts
sufficient to prevent the growth of other organisms (Holmberg and
Hallander, 1973). In plants, hydrogen peroxide from an oxidative burst
in pathogen-infected cells may act as a signal for the induction of
resistance in adjacent cells (Levine et al., 1994).
Environmental agents such as ionizing or near-UV radiations or numerous
compounds that generate intracellular O
(e.g. paraquat, plumbagin, and menadione) can cause oxidative
stress (Sies, 1991; Kappus and Sies, 1981).
O
(Demple and Halbrook, 1983; Storz et
al., 1990; Demple, 1991; Hidalgo and Demple, 1995).
concentration
of
10
M in E. coli by
measuring the rate of superoxide production in isolated membranes. We
present here a study of the physiological production and disposal of
H
O
in intact E. coli, measured by the
rapid equilibration of intracellular H
O
(which
passes freely through membranes) (Chance et al., 1979) with
the surrounding medium. We have also analyzed intracellular sources of
oxygen radicals, the effect of different types of oxidative stress on
the steady-state H
O
concentration, and the
growth-dependent variation in the extent of oxyR-regulated
H
O
response.
Reagents
Antimycin A, ampicillin, rotenone,
D-glucose, bovine cytochrome c type III,
tetracycline, NADH, succinate, carbonyl cyanide
p-trifluoromethoxyphenylhydrazone
(FCCP)(
)
, scopoletin, bovine serum albumin,
horseradish peroxidase type VI, glucose oxidase, bovine superoxide
dismutase, and bovine catalase were purchased from Sigma.
Bacterial Strains and Growth Conditions
lists the strains used in these studies. The bacteriophage
RS45[ bla`-`lacZ lacYA
]
(Simons et al., 1987) was used to insert the katG`::lacZ fusion into chromosomal DNA by recombination in MC4100 carrying
plasmid pAQ24 (katG`::lacZ) (Tartaglia et al., 1989).
The construction was carried out as described by Simons et
al.(1987). Briefly, a culture of MC4100/pAQ24 was infected with a
RS45 lysate; recombination between
RS45 and the plasmids
within the homologous lacZYA-bla region yielded a
[
(katG`::lacZ)] fusion and a still
incomplete bla gene. Recombinant phages were screened and
identified by their Lac
phenotype (blue plaques on LB
agar supplemented with 40 µg/ml
5-bromo-4-chloro-3-indol-
-D-galactopyranose). A
plaque-purified isolate,
AQ24, was integrated into the
att
site in the chromosome of the Lac
strains RK4936 and TA4112. The resulting lysogens, BGF931 and
BGF933, were identified by their Lac
Amp
phenotype.
Hydrogen Peroxide and Superoxide Anion
Measurements
Intracellular concentrations of
HO
and H
O
production
rates were measured as described previously (González-Flecha and
Demple, 1984) by the horseradish peroxidase-scopoletin method (Boveris,
1994). Antimycin and rotenone, inhibitors of electron transport, and
FCCP, an uncoupler, were used at final concentrations of 1, 0.5, and
0.2 µM, respectively. Bacterial suspensions (10
cells ml
in phosphate-buffered saline (PBS))
were incubated with the indicated compound for 10 min at 0-4
°C, washed twice with fresh PBS, and assayed as described
(González-Flecha and Demple, 1994). The rates of
H
O
production were expressed as
µM/s by assuming a cellular volume of 3.2
10
liter (Imlay and Fridovich, 1991a). Superoxide
anion production was measured in isolated membranes prepared by a
standard procedure (Imlay and Fridovich, 1991a). The rate of
O
production was measured by following
the superoxide dismutase-sensitive rate of cytochrome c reduction at 550 nm (
-
= 21
mM
cm
) (Boveris, 1984).
The reaction mixture consisted of 50 mM potassium phosphate
buffer (pH 7.4), 20 µM cytochrome c, 100
µM NADH, and
0.2 mg/ml of membrane protein, with or
without 50 units of bovine copper-zinc superoxide dismutase.
Enzymatic Activities
-Galactosidase activity
in sodium dodecyl sulfate-CHCl
-treated cells was determined
as described by Miller(1992). Two different approaches were used to
assay the total catalase concentration of the cells. Catalase activity
in cell lysates was determined as described previously
(González-Flecha and Demple, 1994) and normalized to either the
protein content of the lysate or the number of cells extracted. Protein
concentration in the extracts was measured by the method of Lowry
(Lowry et al., 1951) using bovine serum albumin as standard.
All the measurements were carried out in a Perkin-Elmer Lambda 3A
UV/Vis spectrophotometer. In the second approach, catalase activity was
measured by following the rate of elimination of H
O
by cells suspended in PBS containing 2 mM
H
O
. The H
O
concentrations in the supernatants were determined by the
horseradish peroxidase-scopoletin method. NADH-cytochrome c reductase activity was measured in bacterial membrane preparations
(0.3-0.5 mg protein/ml) by following the superoxide
dismutase-insensitive cytochrome c reduction as described
above but using 50 µM cytochrome c in the
presence of 50 units of bovine copper-zinc superoxide dismutase
(Trumpower and Simmons, 1979).
Hydrogen Peroxide and Glucose/Glucose Oxidase
Treatments
Overnight cultures of BGF931 and BGF933 were diluted
1:100 in fresh LB and grown for 1, 3, and 7 h. For the pulse-type
treatment with HO
, cultures at the indicated
times were treated with different concentrations of
H
O
, and the
-galactosidase activity
directed by katG`::lacZ was followed for 30 min after addition
of H
O
. In the case of treatment with
glucose/glucose oxidase to generate a continuous flux of
H
O
, cultures at the indicated times were
supplemented with 10 mM glucose, and various amounts of
glucose oxidase, and
-galactosidase activity were followed for 60
min after addition of glucose oxidase.
Statistics
Results are indicated as the mean value
of four independent experiments ± S.E. Statistical significance
of differences was analyzed by ANOVA, followed by Dunnett's test
for other comparisons (Winer, 1971).
Hydrogen Peroxide Generation and Elimination in E.
coli
The metabolic production of hydrogen peroxide in intact
bacteria was evaluated initially in exponentially growing AB1157 after
suspension of the cells in fresh PBS free of
HO
. The H
O
concentration measured in the extracellular medium increased with
time, reaching a plateau at 0.15 ± 0.01 µM after
about 5 min for a cell density of 10
cells ml
(Fig. 1A). Bacteria suspended at 10- or 100-fold
lower densities approached the same plateau, but with slower kinetics
(Fig. 1A). Cells resuspended in PBS initially containing
1.5 µM H
O
rapidly destroyed the
extracellular H
O
and again reached a plateau
concentration of
0.15 µM (Fig. 1B).
The combined results correspond to the equilibration between intra- and
extracellular H
O
and indicate an intracellular
steady-state concentration of H
O
of 0.15
µM in exponentially growing E. coli.
Figure 1:
Hydrogen
peroxide production and elimination by E. coli.
HO
concentration was measured in the
extracellular medium of AB1157 incubated in PBS supplemented with 0
(A) and 1.5 µM (B)
H
O
. A, cell densities after
resuspension were 10
cells ml
(opentriangles), 10
cells ml
(opencircles), 10
cells
ml
(filledtriangles), and no
cells (filledcircles). B, cell density
after resuspension was 10
cells
ml
.
Intracellular Sources of Hydrogen Peroxide
The
contribution of cytosolic enzymes to the total production of
HO
in intact E. coli was estimated by
using the uncoupler of electron transport FCCP, which reduces to
negligible levels the rate of H
O
production
associated with mitochondrial respiratory chain (Boveris and Chance,
1973). Addition of FCCP to E. coli AB1157 decreased the
H
O
steady-state concentration and production
rate to about one-seventh the value of untreated cells ().
Thus, the production of oxygen free radicals by the respiratory chain
in intact bacteria accounts for most of the H
O
generation.
, inhibitors of electron transport were
used. The maximal rate of O
production by
autoxidation of the flavin-semiquinone of NADH-dehydrogenase (FMNH) was
measured by supplementing E. coli membrane preparations with
NADH and the site I inhibitor rotenone (Boveris and Chance, 1973).
Similarly, the maximal production of O
by
ubisemiquinone (UQH) autoxidation was measured by supplementing E.
coli membranes with NADH and the site II inhibitor antimycin
(Boveris and Chance, 1973). The latter rate was assumed to represent
the sum of the rates of O
production at
UQH plus FMNH. The effect of these inhibitors on
O
and H
O
production was also tested for intact bacteria by following the
generation of H
O
by the cells ().
As described previously for mitochondria (Turrens and Boveris, 1980),
both rotenone and antimycin showed a biphasic effect on
H
O
production peaking at a concentration of 0.5
µM for rotenone and 1 µM for antimycin, when
each compound blocked
50% of the electron flux through the
respiratory chain, as evaluated by the NADH-cytochrome c reductase activity (data not shown). Using these concentrations,
both the rate of O
production in E.
coli membranes and the rate of H
O
production in intact bacteria were increased 1.5-2-fold
after rotenone treatment, and 2-3-fold after antimycin treatment
(). These results indicate that both NADH dehydrogenase
and ubiquinone have significant potential to leak electrons to form
O
in E. coli.
Hydrogen Peroxide and Superoxide Production during
Aerobic Growth
The rate of HO
production
during aerobic growth in rich medium showed a biphasic profile with a
progressive increase during exponential growth, followed by a decrease
after cessation of cell growth (Fig. 2). To estimate the
contribution of oxygen free radicals produced at the respiratory chain
level to the total oxygen free radical production, we studied the
relationship between the rate of O
production and the number of active respiratory chains. The
O
production by isolated membranes was
linearly related to the number of respiratory chain units/cell,
estimated by the NADH-cytochrome c reductase activity
(Fig. 3).
Figure 2:
Hydrogen peroxide production during
aerobic growth. Overnight cultures of E. coli AB1157 were
diluted 1:100 in fresh LB and incubated at 37 °C. At different time
points samples were taken to measure OD (opencircles) and assay H
O
production
rate (filledcircles).
Figure 3:
Superoxide anion production as a function
of the number of respiratory chain units in isolated
membranes.
Effect of Respiratory Chain Mutations on Intracellular
H
It has been reported
that mutants defective in the biosynthesis of ubiquinone or menaquinone
are also defective in the ability to induce the synthesis of catalases
during aerobic growth (Hassan and Fridovich, 1978). To test the
consequences of specific defects in NADH dehydrogenase or cytochrome
oxidase on the production of HO
Generation
O
by E.
coli, a series of isogenic strains with mutations affecting these
components was assayed for H
O
steady-state and
catalase concentrations. The rate of H
O
production by GO103 (deficient in cytochrome oxidase d)
was significantly lower than the rate of H
O
production by either GO104 (deficient in cytochrome oxidase
o) or the parental strain GR70N (I). The
production of H
O
was also significantly lower
in strains MWC215 (deficient only in NADH dehydrogenase 2) and MWC232
(deficient in NADH dehydrogenase 1 and 2), and significantly higher in
strain MWC190 (deficient in NADH dehydrogenase 1), compared with the
control strain (I). In all the strains studied, the
catalase concentration varied directly with the rate of
H
O
production. This balance kept the
steady-state concentration of H
O
within a
2-fold range for all the strains (I) and reflects a kind
of homeostasis for reactive oxygen.
Growth Phase-dependent Triggering of a Response to
H
The above results suggested an almost
continuous response of E. coli to different physiological
rates of HO
O
generation. We therefore used a
strain (BGF931) carrying a katG`::lacZ operon fusion to
examine whether such a continuous response could be observed at the
level of transcription of the catalase gene. We first examined the
response of the reporter fusion to acute ``pulse-type''
exposures. The initial H
O
concentrations
required for maximal induction of oxyR-dependent katG transcription varied with the growth phase and were, respectively,
15,
25, and
100 µM for 1-h, 2-h, and 3-h
cultures (Fig. 4). The 7-h culture had a somewhat higher initial
expression of katG`::lacZ but displayed a < 2-fold
induction by H
O
over the range 2
µM to 1 mM (Fig. 4D). These
H
O
concentrations provided about the same
H
O
:catalase initial ratio for the 1-3-h
cultures (), so that the H
O
concentration remained above the physiological value for
15
min (data not shown).
Figure 4:
Induction of katG`::lacZ by acute
HO
exposure. Overnight cultures of BGF931 in LB
were diluted 1:100 in fresh LB and incubated at 37 °C for 1, 2, 3,
or 7 h. At the indicated times, samples were treated with the indicated
concentration of H
O
and incubated for a further
30 min. At 5, 10, 20, and 30 min, samples were taken and assayed for
-galactosidase activity. No increase in
-galactosidase
activity was observed in the
oxyR strain BGF933 (not
shown). Values in the figure correspond to maximal induction, obtained
10 min after H
O
addition.
To evaluate regulation of katG expression in cells subjected to a ``ramp-type''
stimulus, we subjected E. coli BGF931 to a constant and
defined external flux of HO
provided by
glucose/glucose oxidase. The rates of H
O
production were chosen from Fig. 2to yield 2-fold
increases in both the rate of production and the intracellular
concentration of H
O
at any given time. A 2-fold
increase was chosen to mimic the elevated H
O
generation observed between hours 2 and 3 of the growth curve
(Fig. 2), which is associated with the induction of katG during exponential growth. As in the case of the pulse-type
stimuli, the effective concentration of H
O
depended on the catalase concentration in the cells, and on the
growth phase. Catalase activity in lag-phase cells destroyed only 18%
of the H
O
produced by glucose/glucose oxidase
(Fig. 5A). In exponentially growing cells 90% of the
external flux was destroyed by intracellular catalase
(Fig. 5B). Despite this difference, the 2-fold increases
in H
O
production significantly induced
katG`:lacZ expression during both the lag and the exponential
phase (Fig. 6). In contrast, katG`:lacZ was hardly
induced during early stationary phase by the 2-fold increase in the
H
O
production rate (Fig. 6).
Figure 5:
Time course of HO
production by the glucose/glucose oxidase system. Growth conditions
were as in Fig. 4. At 1 h (A) and 3 h (B), glucose
(Glc) was added to a final concentration of 10 mM and
followed by 0.4 mg/ml (A) or 10 mg/ml (B) of glucose
oxidase (GO) to generate H
O
at a flux
equal to the intracellular rate (Fig. 2). The amount of
H
O
in the culture medium was measured at the
indicated times.
Figure 6:
Induction of katG expression by
extracellular fluxes of HO
.Experimental
conditions were as described in Fig. 5. Values in the figure correspond
to maximal induction, obtained at 15, 45, and 60 min after glucose
oxidase (GO) addition to the 1-h (0.4 mg/ml), 3-h (10 mg/ml),
and 7-h (60 mg/ml) cultures, respectively. No increase in
-galactosidase activity was observed in BGF933 (not
shown).
O
in intact E. coli is probably
the respiratory chain, which can account for as much as
87% of the
total H
O
production. Similar to eukaryotic
mitochondria, the leakage of single electrons from the bacterial
respiratory chain was observed at the NADH dehydrogenase and ubiquinone
sites. Accordingly, changes both in the number of respiratory chain
units/cell and in the composition of the electron transport chain
affected the rate of hydrogen peroxide production. Our results showed
that the rate of H
O
production changes
dramatically during aerobic growth and was linearly related to the
number of respiratory chain units/cell estimated by the specific
activity of NADH-cytochrome c reductase. Bacteria seem to cope
with this changing generation of oxygen radicals by elevating the
expression of antioxidant functions, represented here by the
katG-encoded catalase. Indeed, mutational suppression of
catalase-hydroperoxidase I, but not of catalase-hydroperoxidase II,
increased intracellular H
O
concentrations to
0.3 µM, which demonstrates a role for
catalase-hydroperoxidase I as a defensive enzyme.
(
)
O
production in exponentially growing E.
coli approached values similar to the rate of production in
mammalian cells (
4 µM s
)
(González-Flecha et al., 1993). This production
followed the theoretical 2:1 stoichiometry for superoxide:hydrogen
peroxide (2O
+ 2H
H
O
+ O
), which
indicated that most of the H
O
generation in
E. coli arises as a by-product of
O
generation.
O
production. Elimination of either or both
NADH dehydrogenases (in strains MWC190, MWC215, or MWC232) decreased
the rate of H
O
production. NADH dehydrogenase 1
is a multisubunit complex homologous in structure and function to the
eukaryotic complex I (Meinhardt et al., 1989). This enzyme
contains four iron-sulfur clusters and FMN and couples its reaction to
the generation of proton-motive force (Matsushita et al.,
1987). NADH dehydrogenase 2, in contrast, contains FAD and no iron
(Hayashi et al., 1989), and its function is not related to
proton translocation (Matsushita et al., 1987). Both
dehydrogenases would be likely to react with O
to generate
superoxide. The results presented here show that the inactivation of
either NADH dehydrogenase 1 or NADH dehydrogenase 2 decreased the rate
of H
O
production by only 25-30%. These
results contrast with those of Imlay and Fridovich (1991b), in which
the residual O
production in membranes
isolated from a strain lacking NADH dehydrogenase 2 was only 8% of that
for the parental strain. However, in intact bacteria lacking the NADH
dehydrogenases, reducing equivalents can and evidently do still enter
the respiratory pathway at the ubiquinone level via alternative
dehydrogenases, such as succinate dehydrogenase or lactate
dehydrogenase (Ingledew and Poole, 1984).
O
production was also affected by changing the energy status of
E. coli by directing the electron flux through components with
higher or lower energetic efficiency. In isolated mitochondria
H
O
production strongly depends upon the energy
status; an ``energized'' condition (state 4, with slow
O
consumption and ADP phosphorylation) corresponds to a
highly reduced steady state for the respiratory carriers and to a
relatively high H
O
generation; a
``de-energized'' condition (state 3, with fast O
consumption and ATP production) corresponds to a highly oxidized
steady state for the respiratory chain components and to a relatively
low H
O
production (Boveris and Chance, 1973).
In our experiments, mutants utilizing the so-called
``coupled'' components of the respiratory chain (NADH
dehydrogenase 1 in strain MWC215 and cytochrome bo in strain
GO103) (Calhoun et al., 1993) appear to be in a de-energized
condition, with the respiratory carriers largely in the oxidized state
and generating H
O
at a relatively low rate. In
contrast, the mutant strain GO104 utilizing the ``uncoupled''
cytochrome d (Calhoun et al., 1993) seems to be in an
energized state with highly reduced electron transport carriers. This
observation could extend to the growth of bacteria on different carbon
sources or other conditions that determine different patterns of
``coupled-uncoupled'' components.
O
production (GO103,
MWC215, and MWC232), catalase expression was regulated to keep the
intracellular H
O
steady-state concentration at
0.1-0.2 µM. These results suggest that at least some
basal induction of the oxyR regulon occurs even without
exogenous H
O
stress, triggered by values that
exceed the 0.1-0.2 µM physiological value. This
represents a sensitivity
25-fold greater than previously associated
with oxyR-dependent catalase induction, which occurred in
response to a pulse-type exposure to 5 µM
H
O
(Demple and Halbrook, 1983). A key
difference is likely to be the more constant H
O
generation by respiration leakage, as in the case of the
ramp-type oxidative stress, which might exert a cumulative effect by
constantly activating OxyR protein. We have shown here that pulse-type
stimuli may provide an increased H
O
concentration for only
15 min. A critical question for
understanding the difference between these two situations is the
half-life of activated OxyR protein, which has not been determined. A
long half-life would favor more dramatic inducing effects of a modest
H
O
pulse (1-5 µM) than would
a short half-life. Important factors that could remove activated OxyR
might be proteolysis and direct reversal of the activated form, which
is thought to be an oxidized protein (Storz et al., 1990).
O
response not only depends on the magnitude
and type of stimulus but also on the growth phase, being maximal at
logarithmic phase and almost negligible during early stationary phase.
This observation is in good agreement with the reported resistance of
bacteria to H
O
in stationary phase cultures
(Jenkins et al., 1988; Hengge-Aronis. 1993) and with the dual
regulation of katG (Ivanova et al., 1994) and dps and oxyS (Altuvia et al., 1994) by both oxyR and by the stationary phase regulator rpoS. We do not
know if rpoS regulation merely supersedes that by oxyR in stationary phase cells, or whether the activity of OxyR may be
less under those conditions.
Table:
Bacterial strains used in this study
Table:
Effect of
respiratory chain blockers on HO
production in
intact E. coli and O
production in
membrane preparations
O
or O
production and catalase
activity. ND, not determined.
Table:
Hydrogen peroxide metabolism in
respiratory chain mutants
O
and catalase concentrations. Cyt
d
and Cyt bo
,
deficient in cytochrome oxidases d and o,
respectively; NDH-1
and NDH-2
,
deficient in NADH dehydrogenases 1 and 2, respectively.
Table:
Elimination of HO
by
intracellular catalase in pulse-type models of oxidative stress
O
required
for maximal induction of the katG`::lacZ fusion, and the
concentration of H
O
in the extracellular medium
was followed.
[H
O
]
, initial
concentration of H
O
; elimination
t
, half-life of H
O
in
the extracellular medium; [Catalase]
,
catalase concentration before the addition of H
O
RS45. We also thank Drs. Elena Hidalgo, Rafael Rodriguez Ariza,
and Tatsuo Nunoshiba for helpful discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.