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Journal of Bacteriology, December 2001, p. 7173-7181, Vol. 183, No. 24
Department of Microbiology, University of
Illinois, Urbana, Illinois 61801
Received 9 July 2001/Accepted 20 September 2001
Hydrogen peroxide is generated during aerobic metabolism and is
capable of damaging critical biomolecules. However, mutants of
Escherichia coli that are devoid of catalase typically
exhibit no adverse phenotypes during growth in aerobic media. We
discovered that catalase mutants retain the ability to rapidly scavenge
H2O2 whether it is formed internally or
provided exogenously. Analysis of candidate genes revealed that the
residual activity is due to alkyl hydroperoxide reductase (Ahp).
Mutants that lack both Ahp and catalase could not scavenge
H2O2. These mutants excreted substantial
amounts of H2O2, and they grew poorly in air.
Ahp is kinetically a more efficient scavenger of trace
H2O2 than is catalase and therefore is likely
to be the primary scavenger of endogenous H2O2.
Accordingly, mutants that lack Ahp accumulated sufficient hydrogen
peroxide to induce the OxyR regulon, whereas the OxyR regulon remained
off in catalase mutants. Catalase still has an important role in
wild-type cells, because the activity of Ahp is saturated at a low
(10 Virtually all aerobic
organisms contain enzymes that convert superoxide and hydrogen peroxide
to innocuous products. The ubiquity of these scavenging enzymes
suggests that exposure to O2 The fitness of the catalase mutants did not reflect invulnerability to
H2O2. Low concentrations of
exogenous H2O2
(ca. 30 µM) are sufficient to inhibit the growth of
E. coli. Although the growth-blocking cell lesions have not
yet been identified, H2O2
can oxidize thiols, which may inactivate enzymes that have active-site
sulfhydryl residues. Methionine sulfoxide adducts (21) and
protein carbonyls (5) may also be products of enzyme oxidation. In addition,
H2O2 can inactivate the
exposed [4Fe-4S] clusters of aconitase B and fumarase B (S. M. Varghese, S. Korshunov, and J. A. Imlay, unpublished data).
Finally, reactions between H2O2 and intracellular iron
generate hydroxyl radicals, which in turn attack DNA. Micromolar
concentrations of H2O2 are
therefore mutagenic.
Thus, the robust performance of catalase mutants seemed to imply that
these amounts of H2O2 are
not normally generated during aerobic growth. There are presently no
data that firmly establish the amount of
H2O2 that is formed as a
by-product of metabolism. No enzymes in E. coli, other than
SOD, generate H2O2 as a
deliberate, stoichiometric product. However, in vitro studies have
shown that H2O2 can be
formed by the adventitious oxidation of redox enzymes by molecular oxygen.
The main sources of H2O2
inside the cell are probably flavoenzymes, both because they are
abundant and because flavins are amenable to the univalent electron
transfer reaction that initiates the production of superoxide and
H2O2 (6, 18).
The rates at which some flavoenzymes generate these species have been
measured in vitro, and extrapolations suggest that they may form about 10 µM H2O2
s In this study, we discovered that mutants which are devoid of catalase
still scavenge physiological concentrations of
H2O2 as rapidly as do
wild-type cells. By identifying and eliminating the remaining
scavenging enzyme, alkyl hydroperoxide reductase, we were able to
directly quantify endogenous
H2O2 production and demonstrate that it is enough to poison scavengerless cells.
Chemicals, enzymes, and media.
Cumene hydroperoxide,
5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), horseradish peroxidase
(type II), hydrogen peroxide, o-dianisidine, o-nitrophenyl-
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.24.7173-7181.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Alkyl Hydroperoxide Reductase Is the Primary
Scavenger of Endogenous Hydrogen Peroxide in Escherichia
coli
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
5 M) concentration of H2O2. In
contrast, catalase has a high Km, and
it therefore becomes the predominant scavenger when
H2O2 concentrations are high. This arrangement
is reasonable because the cell cannot provide enough NADH for Ahp to
rapidly degrade large amounts of H2O2. In sum,
E. coli does indeed generate substantial
H2O2, but damage is averted by the scavenging
activity of Ahp.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
and H2O2 is an inevitable
part of the aerobic lifestyle and that these species can damage cells.
It follows that organisms that cannot scavenge them will fare poorly in
an aerobic habitat, and in 1971 McCord et al. proposed that some
obligate anaerobes may be unable to grow in air at least in part
because they lack sufficient levels of scavenging enzymes
(17). This reasoning also suggested that aerobes would be
much less oxygen tolerant if they lacked superoxide dismutase (SOD) and
catalase. In 1985 this prediction was partly affirmed by studies of
sodA sodB mutants of Escherichia coli
(3). These strains suffered elevated rates of DNA damage, could not catabolize nonfermentable carbon sources, and did not grow at
all without extensive amino acid supplements. However, contrary to
expectation, mutants that lacked catalase grew as well as their
wild-type parents and exhibited no increase in mutation rate (14,
29).
1 in vivo (18). Gonzalez-Flecha
and Demple reported evidence of endogenous
H2O2 production by E. coli (7). If these rates are correct, then the lack
of phenotype of catalase mutants seems difficult to explain.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-D-galactopyranoside,
plumbagin, and potassium cyanide were purchased from Sigma. Coomassie
protein reagent was obtained from Pierce. Bovine liver catalase (20 mg/ml) was from Boehringer Mannheim, and Amplex red (AR) was from
Molecular Probes. Dimethyl sulfoxide (DMSO) was purchased from Fisher.
Water for buffers was purified with a Labconco Water Pro PS system
using deionized water as the feedstock.
Growth conditions and strains. Cultures were routinely grown at 37°C. Aerobic cultures were grown in shaking flasks; anaerobic cultures were grown in a Coy chamber (Coy Laboratory Products, Inc.) under 85% N2-10% H2-5% CO2. The optical densities (OD) of all cultures were measured at 600 nm.
The strains used in this study were derived from E. coli K-12 and are listed in Table 1; isogenic strains were used in all experiments. Mutations were introduced into strains via P1 transduction (19). To avoid the outgrowth of suppressed strains, the katG, katE, oxyR, and Tn10-linked ahp mutations were transduced and selected under anaerobic conditions. The presence of katG null mutations was confirmed by an o-dianisidine assay for hydroperoxidase I (HPI) activity (below). katE mutants failed to form bubbles on plates when colonies were overlaid with a drop of 30% H2O2. Mutants lacking oxyR were identified by their inability to induce HPI when anaerobic, exponentially growing cultures at 0.1 OD in LB medium were exposed to 60 µM H2O2 for 45 min. Mutations in gshA were confirmed by measurements of intracellular thiol levels using DTNB (13).
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H2O2 detection. In the presence of H2O2, horseradish peroxidase (HRP) oxidizes AR to the fluorescent product resorufin. One milligram of AR was dissolved in 0.78 ml of DMSO, and 0.75 ml of this solution was then diluted into 18 ml of 50 mM potassium phosphate (KPi, pH 7.8) to generate a 200 µM stock solution. This solution was shielded from light. HRP was dissolved in 50 mM KPi (pH 7.8) to 0.02 mg/ml. To measure H2O2, 0.45 ml of sample was mixed with 0.25 ml of AR and 0.25 ml of HRP. Fluorescence was then measured in a Shimadzu RF Mini-150 fluorometer and converted to H2O2 concentration using a curve obtained from standard samples. Note that a small amount of H2O2 is generated by the dye/HRP detection system itself; this amount was accounted for by the standard curve.
H2O2 scavenging by whole cells. Cultures were grown aerobically for at least four generations to 0.1 to 0.3 OD. Cells were pelleted in a microcentrifuge, washed twice, and resuspended in room temperature phosphate-buffered saline (PBS) at an OD of 0.1. H2O2 was added to the appropriate final concentration (see figure legends). At intervals, 0.45-ml aliquots were removed, diluted when necessary, and assayed immediately for H2O2 content by the AR/HRP method.
LB medium was used for most experiments. However, in experiments designed to measure the scavenging of trace H2O2, cultures were grown in minimal A glucose medium containing 0.5 mM each of the 20 L-amino acids. Cells were then washed and resuspended into fresh medium containing only 0.02% glucose and 0.05 mM amino acids, so that metabolism was active and could provide Ahp with reductants, yet the amount of H2O2 generated by the medium was minimal. When cyanide was included in the medium, fluorescence development was permitted to proceed for 10 min before a final reading was made, since the carryover of cyanide inhibits the activity of HRP.Measurement of H2O2 accumulation in cell cultures. To detect H2O2 formation by drug-treated cells, we exposed log-phase cultures (0.2 OD) in LB medium to 300 µM plumbagin, an amount that is sufficient to consume 8 µM oxygen per min in a cyanide-resistant (nonrespiratory) fashion. After 16 min, cells were removed by centrifugation, and the residual H2O2 in the medium was assayed by the HRP/o-dianisidine assay (18).
H2O2 can be detected with greater sensitivity in defined medium using the AR/HRP assay. Cells were grown anaerobically overnight in minimal A medium containing 0.2% glucose, diluted to 0.01 OD, and grown anaerobically for four generations. These log-phase cells were then subcultured to 0.05 OD in fresh aerobic minimal A medium containing 0.05% glucose. At 0.05 and 0.1 OD, the culture medium was assayed for H2O2. Hydrogen peroxide levels were also determined in sterile medium that was incubated at 37°C for an equivalent time. The low glucose concentration was used in order to minimize H2O2 production by salt-catalyzed glucose autooxidation (below).H2O2 production rates.
In order to
monitor continuously the intracellular formation of
H2O2, extracellular
H2O2 levels were measured.
Cells were first grown overnight in minimal A 0.2% glucose medium
containing 0.5 mM each of the 20 amino acids. Most cultures were then
diluted to 
0.001 OD and grown aerobically to an OD of 0.1;
however, JI377 was grown only to an OD of 0.05 in order to measure
H2O2 production before
growth was significantly inhibited. Cells were then washed in fresh
medium containing only 0.02% glucose and 0.05 mM amino acids,
resuspended at an OD of 0.1 in the same medium, and incubated with
shaking at either 25 or 37°C. Glucose was added to the minimal A
salts immediately before use. Aliquots were removed at intervals, and
their H2O2 content was
measured. The rate of H2O2
production was normalized to the cytoplasmic volume of the suspended
cells, using a standard ratio of 0.47 µl of internal volume per 1 ml
of 1.0 OD of E. coli (10).
Total catalase activity. Aerobic exponential-phase cells were harvested at 0.5 OD, washed in cold 50 mM KPi, resuspended in 1/4 the original volume, and lysed by passage through a French press. Extracts were centrifuged at 13,000 × g for 20 min to remove cell debris and then stored on ice. Catalase activity was measured in a 1-ml reaction mixture containing 50 µl of extract, 1 mM H2O2, and PBS (pH 7.3). At various time points, 10 µl was removed, diluted 1:500 in PBS, and assayed for H2O2 by the AR/HRP procedure.
HPI assay. Cultures were grown aerobically for five to six generations in LB medium. Chloramphenicol was added, and cultures were incubated for 5 min. Cells were then washed in PBS containing chloramphenicol, and cell pellets were frozen on dry ice. Within an hour the cells were resuspended in 1/10 the culture volume of 10 mM KPi buffer (pH 6.4) and lysed by sonication. Debris was centrifuged out at 13,000 × g for 20 min. HPI activity was assayed by the o-dianisidine method (18).
-Galactosidase assay.
Studies of
katG::lacZ expression used a 
RS45
(katG::lacZ) lysogen. Cultures were
grown overnight under anaerobic conditions, diluted to an OD of 0.01 in
LB, and grown anaerobically to an OD of 0.1. For measurement of
anaerobic expression, chloramphenicol was added and cultures were
harvested. For measurement of aerobic expression, cultures were shifted
into air and grown with vigorous shaking to an OD of 0.3 to 0.4. Where
indicated, 13,000 U of catalase was added every 15 min. Repeated
additions were necessary because catalase rapidly loses activity in
cell cultures.
-Galactosidase activity was assayed in a 1.2-ml reaction mixture
consisting of 0.2 ml of ONPG
(o-nitrophenyl--D-galactopyranoside, 4 mg/ml), extract, and Z-buffer (19) at 28°C. Change in
absorption over time was monitored at 420 nm. Protein concentrations of
all extracts were determined using a Coomassie dye-based assay by Pierce. All assays were performed on duplicate samples, and the values
were then averaged.
Growth experiments.
Care was taken to determine whether the
poor aerobic growth of the Ahp
Kat strain (JI377) was due to endogenous
H2O2 or
H2O2 that was chemically generated by autooxidation of the medium. Low-peroxide medium, which
contains 0.02 µM H2O2,
was prepared by adding filter-sterilized glucose to anaerobic minimal A
salts immediately before inoculation. During aerobic incubation at
37°C, this medium accumulates 0.04 µM
H2O2 per h. JI377 was
inoculated from an anaerobic overnight culture to 0.01 OD in anaerobic,
low-peroxide medium and grown anaerobically to 0.1 OD. Cells were then
subcultured into the same medium and shifted into air. Growth was
monitored during the first 2 h, during which the vast majority of
H2O2 present in the medium
was generated by the cells.
Disk diffusion.
Standing overnight cultures in LB medium
were diluted to 0.005 OD in aerobic LB and then grown to an OD of
0.1. Cultures were diluted 1:10, and 100 µl was spread on LB
plates. Round sterile filters (1-cm diameter) were placed in the center
of the plates and spotted with 25 µl of 3%
H2O2. Plates were incubated
at 37°C overnight. The distance from the edge of the disk to the edge of the growth zone was measured. This experiment was performed in
triplicate; mean values are reported.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Catalase is not the primary scavenger of low-level H2O2. Inside bacteria, antibiotics such as paraquat, plumbagin, and juglone are cyclically reduced by redox enzymes and oxidized by molecular oxygen, thereby generating superoxide and, upon its dismutation, hydrogen peroxide. The rate at which H2O2 is made inside drug-treated E. coli can be determined by measurement of nonrespiratory oxygen consumption in the presence of cyanide. However, we were unable to detect H2O2 accumulating in the medium of plumbagin-treated wild-type cells, despite the fact that oxygen consumption measurements indicated that the amount of H2O2 should have been easily within our detection limits (data not shown).
E. coli contains two catalases, and we anticipated that these were responsible for scavenging the H2O2. However, when the experiments were repeated with strain UM1, which has point mutations in both of the catalase structural genes (katG and katE), H2O2 was again undetectable. By adding 1.5 µM H2O2 to the bacterial culture, we found that UM1 degraded H2O2 as rapidly as did its wild-type parent (data not shown). HPI, the catalase encoded by katG, exhibits weak NAD(P)H-dependent peroxidase activity in vitro, and the katG17 mutation that is present in UM1 eliminates catalase but not peroxidase activity. Therefore, we wondered whether the residual peroxidase activity of HPI was responsible for the scavenging activity of UM1. True null mutations of both katE and katG were transduced into the wild-type strain MG1655, and the H2O2 scavenging measurements were repeated. This catalase double mutant had no detectable catalase or HPI peroxidase activity in vitro, but it still scavenged 1.5 µM H2O2 approximately as rapidly as its wild-type parent (Fig. 1). Therefore, E. coli must have another means of efficiently scavenging H2O2.
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), JI367
(katG katE, 
), JI370 (ahpCF, *), JI377
(ahpCF katG katE, 
), JI374 (ahpCF
katG, 
), and JI372 (ahpCF katE, 
) were
grown aerobically in LB and resuspended in PBS at an OD of 0.1. H2O2 was added at a final concentration of 1.5 µM. At various time points after addition of
H2O2, the H2O2
concentration was measured as described in Materials and Methods.
Ahp is the source of scavenging in a catalase null mutant.
Other mechanisms of scavenging were considered. Some 
-ketoacids,
such as pyruvate, can be excreted into the medium of glucose-fed cells,
and these can chemically reduce
H2O2 in an oxidative
decarboxylation reaction (4). In separate work we have
observed that both the respiratory cytochrome o and
cytochrome d oxidases have weak peroxidase activities
(A. Nguyen and J. A. Imlay, submitted for publication). Finally, alkyl hydroperoxide reductase (Ahp) has been shown to accept
H2O2 as a substrate in
vitro (22).
HPI induction can compensate for loss of Ahp. We wished to learn which enzyme, Ahp or HPI, was the predominant scavenger in wild-type cells. Because the ahpCF and katG single mutants each scavenged H2O2 at the same rate as did wild-type cells (Fig. 1), we inferred that one of the enzymes might be induced to compensate for the absence of the other. Both ahpCF and katG are positively regulated by the OxyR regulon, and a mutation in the constitutive scavenger might cause intracellular H2O2 to accumulate until OxyR activated the expression of the other, ultimately restoring wild-type levels of H2O2 scavenging.
We observed that an ahpCF mutant contained sevenfold more total catalase (including both HPI and HPII) than did wild-type cells. The o-dianisidine peroxidase activity, which specifically reflects the HPI titer, was increased 10-fold. Similarly, the-galactosidase activity of an aerobic RS45
katG::lacZ lysogen was increased
10-fold when the ahpCF null mutation was introduced, rising
from 0.04 ± 0.01 to 0.39 ± 0.05 -galactosidase U/mg. In contrast, the introduction of katG (LC80), katE
(HDO3), or both katG and katE (LC84) mutations
did not increase -galactosidase activity at all (0.04 ± 0.01, 0.03 ± 0.01, and 0.04 ± 0.01 U/mg, respectively). These
results agreed with the observations of Rosner and Storz
(27) and Ritz et al. (25a).
Induction of katG in the ahpCF mutant was blocked
by an oxyR mutation (Table 2).
To ensure that induction of the OxyR regulon was not due to a
suppressor mutation secondary to the ahpCF mutation, the
ahpCF null allele was retransduced into the
katG::lacZ lysogen under anaerobic
conditions. Once again, the fusion was induced when the transductant
was cultured in aerobic medium (data not shown). These data demonstrate
that in wild-type cells Ahp rather than catalase is the primary
scavenger of an endogenous inducer of the OxyR regulon.
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Ahp and HPI have discrete roles in scavenging
H2O2.
Mutants that contain only one or the
other scavenger exhibited very different dose-response curves (Fig.
2). Whole cells that contained only Ahp
scavenged low concentrations of
H2O2 very effectively, exhibiting a half-maximal rate when the extracellular concentration of
H2O2 was about 5 µM. The
activity became saturated when extracellular H2O2 exceeded 20 µM (Fig.
2). In contrast, HPI-expressing cells were not saturated by even
millimolar concentrations of
H2O2, consistent with its
Km of 5.9 mM (9). The
dose-response curves suggest that the primary role of HPI might be to
scavenge higher concentrations of
H2O2, against which Ahp is
ineffective.
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Ahp+ mutant scavenged 0.1 µM
H2O2 slightly more rapidly
than did an HPI+ Ahp
mutant, despite the fact that the latter strain has 10-fold-induced levels of HPI (Fig. 3, left panel). Thus,
in wild-type E. coli most scavenging of low-dose
H2O2 must be done by Ahp.
Conversely, the HPI Ahp+
strain was poor at degrading 150 µM
H2O2, unlike the
HPI+ Ahp and
HPI+ Ahp+ strains (Fig. 3,
right panel). Although Ahp can be inactivated by
H2O2 in vitro
(11), its poor activity in vivo seemed not to stem
from this problem, since normal scavenging activity was observed
when the 150 µM H2O2 was
washed away and the cells were exposed to 1.5 µM
H2O2 (data not shown). The
Ahp mutant scavenged 150 µM
H2O2 twice as rapidly as
did the wild type, because HPI+ was induced
(30).
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Calculation of endogenous H2O2 production
during aerobic growth.
Substantial
H2O2 accumulated in the
medium of Ahp Kat
cultures. The extracellular concentration of
H2O2 rose to 1.8 µM
during aerobic growth in minimal 0.05% glucose medium when cells were cultured for a single generation, from 0.05 to 0.10 OD600. In contrast, the
H2O2 concentrations of
wild-type (MG1655), Kat (JI367), and
Ahp (JI370) cultures were below our detection
limit of 0.04 µM H2O2. The fact that the H2O2
concentration was so low in Ahp cultures was
initially surprising, given that sufficient
H2O2 was present to
activate the OxyR protein. However, subsequent work indicated that the
concentration of H2O2
inside these cells may have been substantially higher than that outside
them (30).
Ahp strains have
<5% of the scavenging activity of wild-type cells, so that virtually
all of the H2O2 that enters
or is formed within these cells diffuses out without being scavenged (30). For this reason the measurement of excreted
H2O2 is a valid proxy for
measurement of endogenous
H2O2 formation. Using this
strain, we quantitated the rate at which E. coli generates H2O2 (Fig.
4). Measurements were made after
exponentially growing cells were suspended in fresh glucose/amino acids
medium at 37°C (and do not necessarily apply in other media). The
rate of H2O2 formation,
normalized to cell volume, was 14 µM
H2O2/s at 37°C. This
result is consistent with earlier predictions (Discussion).
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Inability to scavenge endogenous H2O2
causes a growth defect.
It has long been suspected that aerobic
metabolism generates enough
H2O2 to damage cells that
cannot scavenge it. No growth defects were apparent in the
ahpCF mutant; we presume this is because the cell
compensates sufficiently for the loss of ahpCF by inducing
katG. However, the Ahp
Kat strain grew poorly in all aerobic media
that were tested (Fig. 5). Growth slowed
progressively over time, and when cells were repeatedly subcultured,
growth often stopped entirely (Fig. 5B). This may reflect the continual
accumulation of damage in the cell. Growth was particularly poor on
defined media that lacked amino acids (data not shown). Wild-type
growth was restored when catalase was added to the medium (Fig. 5C) or
when the mutant was cultured anaerobically (not shown).
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), JI370
(ahpCF, Kat strain grew
more poorly than did its wild-type parent. Our measurements over this
period confirmed that the
H2O2 found in this culture was generated primarily by the cells rather than by glucose oxidation. Thus, aerobic E. coli generates enough
H2O2 to debilitate the cell. In previous studies of catalase mutants, the toxicity of endogenous H2O2 was
obscured by the scavenging action of Ahp.
The necessity for care in these experiments was underscored by the
observation that medium that had been stored on the bench often
contained micromolar amounts of
H2O2, and dilution of even wild-type cells into stored medium was sufficient to transiently induce
the OxyR regulon (data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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H2O2 may be the sole physiological substrate of Ahp. The results of this study indicate that in exponentially growing E. coli Ahp is responsible for the degradation of low concentrations of hydrogen peroxide. Previously, Ahp was primarily associated with the detoxification of organic hydroperoxides. However, the range of organic peroxides that are good substrates for the enzyme indicates that its active site can accommodate virtually any ROOH, so it is not surprising that it reacts with HOOH as well. Earlier genetic studies confirmed that Ahp provides cellular resistance to organic hydroperoxides but found little role in resistance to H2O2. This result is now understandable: because catalases cannot degrade organic hydroperoxides, Ahp is likely to be the only enzyme in E. coli with that catalytic ability. At the same time, the role of Ahp in H2O2 decomposition was obscured because the widely employed disk inhibition assays confront cells with high H2O2 concentrations that are more efficiently decomposed by catalases.
It is not clear whether E. coli must ever detoxify organic hydroperoxides in nature. Lipid hydroperoxides were suggested to be the physiological substrates for Ahp, but E. coli lacks the polyunsaturated fatty acids that appear to be necessary for lipid peroxidation (1), and we are not aware of any study that has documented the recovery of peroxidized lipids from this bacterium. It is possible that organic hydroperoxides are pseudosubstrates for an enzyme whose only role in nature is the decomposition of hydrogen peroxide.Why does E. coli use multiple scavengers?
In
general, all peroxidases will be inferior to catalases at scavenging
high concentrations of H2O2 because peroxidases
can turn over only as quickly as the cell can provide a reductant to
them. Catalases escape this restriction. For example, the HPI catalase
of Ahp E. coli degraded 500 µM
H2O2 at a rate of 7 × 107
molecules cell1 s1. To achieve the same
rate using Ahp would require an equivalent amount of NADH, which
substantially exceeds the rate at which metabolism generates NADH.
(Glucose-saturated cells can generate enough NADH to respire 6 × 106 molecules of oxygen cell1
s1.) The disparity is even greater when one considers
decomposition of higher concentrations of H2O2.
Therefore, catalases allow cells to degrade high concentrations of
H2O2 far more quickly than would peroxidases alone.
1 s1 (9);
that of Ahp has not been reported, but the data of Niimura et al.
(22) imply that it is at least 8 × 106 M1
s1. The implication is that at least 10-fold
more molecules of HPI need to be synthesized to provide the scavenging
activity provided by Ahp. A second disadvantage to catalase may stem
from the fact that enzymes that require reactions with two molecules of
substrate to complete a catalytic cycle can have difficulty at low
substrate concentrations, when an intermediate state is long-lived.
Compound I, the divalently oxidized catalase intermediate, can be
unstable and in some circumstances may reversibly deactivate
(9), which would further diminish the ability of catalase
to scavenge trace amounts of
H2O2. Peroxidases may
comprise an evolutionary solution to this problem.
The kinetic efficiency of HPII
(kcat/Km = 1.3 × 106 M1
s1) (23), the stationary-phase
catalase, is similar to that of HPI. It seems reasonable that E. coli would increase its catalase titer in stationary phase, when
there may not be sufficient NADH for Ahp to remain an effective
scavenger. Gonzalez-Flecha and Demple reported that both catalases are
induced as growth slows (8). It is not clear why
scavenging efficiency could not be maintained by induction of HPI
alone. For now, the benefit to E. coli of having two
catalases instead of one remains obscure.
The compensatory interactions that we observed between catalase and Ahp
synthesis have been observed previously in a wide range of bacteria. In
Xanthomonas campestris, Bacteroides fragilis, and
Pseudomonas aeruginosa, mutations in ahp cause
catalase induction (20, 24, 26); this may be true in
Bacillus subtilis as well (2). Conversely,
katG mutations in Mycobacterium tuberculosis select for promoter upmutations in ahp (31). It
seems likely that the division of labor found in E. coli,
wherein Ahp scavenges low levels of
H2O2 and catalase scavenges
high levels, is widespread.
Mechanisms of H2O2 formation and cell damage. The existence of a strain unable to scavenge the H2O2 it produces has enabled us to measure the rate at which H2O2 is formed. In earlier studies in vitro, we determined that the respiratory chain was likely to be the primary source of H2O2 during aerobic growth on glucose, largely because of the autooxidizability of the NADH dehydrogenase II (18). This enzyme adventitiously transfers electrons to oxygen from its reduced flavin in a direct second-order chemical reaction. Other flavoenzymes can do so as well, and in circumstances where these other enzymes are especially abundant, they may be the predominant sources of superoxide and H2O2.
The rate at which H2O2 is likely to be formed in vivo can be predicted from the in vitro data. We found that 9 molecules of H2O2 were formed per 10,000 electrons flowing through the NADH dehydrogenase II (18). We do not know the fraction of the respiratory flux that flows through this enzyme during growth on glucose, but given that exponentially growing cells consume about 3.2 mM O2/s, one extrapolates that the rate of H2O2 formation could be up to 12 µM/s. This is close to the rate determined in vivo in this study (14 µM/s). Efforts to test the sources of H2O2 production in vivo are under way. Gonzalez-Flecha and Demple inferred rates of H2O2 production by intact, wild-type cells (ca. 1 to 2 µM/s) that are lower than those that we have reported (8). However, we observed rates similar to theirs when we suspended cells in room temperature buffer, as they did (3 µM/s). It is not surprising that H2O2 is most rapidly formed when metabolism is active and temperatures are high enough to overcome the activation energy (18). There was a procedural difference, however. Their calculation was based on measurements of steady-state concentrations of extracellular H2O2 in suspensions of cells: by assuming that catalase was the predominant scavenger of H2O2, they used measurements of its activity to calculate the rate of H2O2 production. In our experiments we found that H2O2 did not accumulate extracellularly due to the action of Ahp. These differences may reflect the different conditions under which the experiments were performed. Inside growing cells, the steady-state concentration of H2O2 depends on the rates of its formation and of its dissipation. In this study we quantified the rate at which H2O2 is formed when E. coli is cultured under a particular set of growth conditions. In the accompanying work (30), we measured the processes that consume H2O2 and estimated the internal H2O2 concentration.| |
ACKNOWLEDGMENTS |
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We are grateful to Gigi Storz, Peter Loewen, Bruce Demple, Bob Gennis, and Jim Slauch for providing strains and discussion that helped us in this study, and we thank Holly Oliver for assistance with strain constructions.
This work was supported by grant GM49640 from the National Institutes of Health.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, University of Illinois, Urbana, IL 61801. Phone: (217) 333-5812. Fax: (217) 244-6697. E-mail: jimlay{at}uiuc.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, December 2001, p. 7173-7181, Vol. 183, No. 24
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.24.7173-7181.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Panek, H. R., O'Brian, M. R.
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(2004). Diversion of the Metabolic Flux from Pyruvate Dehydrogenase to Pyruvate Oxidase Decreases Oxidative Stress during Glucose Metabolism in Nongrowing Escherichia coli Cells Incubated under Aerobic, Phosphate Starvation Conditions. J. Bacteriol.
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Djaman, O., Outten, F. W., Imlay, J. A.
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Verneuil, N., Sanguinetti, M., Le Breton, Y., Posteraro, B., Fadda, G., Auffray, Y., Hartke, A., Giard, J.-C.
(2004). Effects of the Enterococcus faecalis hypR Gene Encoding a New Transcriptional Regulator on Oxidative Stress Response and Intracellular Survival within Macrophages. Infect. Immun.
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Sayed, A. A., Williams, D. L.
(2004). Biochemical Characterization of 2-Cys Peroxiredoxins from Schistosoma mansoni. J. Biol. Chem.
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(2004). A new activity for an old enzyme: Escherichia coli bacterial alkaline phosphatase is a phosphite-dependent hydrogenase. Proc. Natl. Acad. Sci. USA
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Budanov, A. V., Sablina, A. A., Feinstein, E., Koonin, E. V., Chumakov, P. M.
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Seki, M., Iida, K.-i., Saito, M., Nakayama, H., Yoshida, S.-i.
(2004). Hydrogen Peroxide Production in Streptococcus pyogenes: Involvement of Lactate Oxidase and Coupling with Aerobic Utilization of Lactate. J. Bacteriol.
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Pauwels, F., Vergauwen, B., Van Beeumen, J. J.
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Cha, M.-K., Hong, S.-K., Lee, D.-S., Kim, I.-H.
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Cha, M.-K., Kim, W.-C., Lim, C.-J., Kim, K., Kim, I.-H.
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Brenot, A., King, K. Y., Janowiak, B., Griffith, O., Caparon, M. G.
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Choi, J., Choi, S., Choi, J., Cha, M.-K., Kim, I.-H., Shin, W.
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Pericone, C. D., Park, S., Imlay, J. A., Weiser, J. N.
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Chevallet, M., Wagner, E., Luche, S., van Dorsselaer, A., Leize-Wagner, E., Rabilloud, T.
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Vergauwen, B., Pauwels, F., Van Beeumen, J. J.
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Bandyopadhyay, P., Byrne, B., Chan, Y., Swanson, M. S., Steinman, H. M.
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Michan, S., Lledias, F., Hansberg, W.
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Kashima, Y., Ishikawa, K.
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Klotz, M. G., Loewen, P. C.
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Pauwels, F., Vergauwen, B., Vanrobaeys, F., Devreese, B., Van Beeumen, J. J.
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(2003). Complete Genome Sequence of the Ammonia-Oxidizing Bacterium and Obligate Chemolithoautotroph Nitrosomonas europaea. J. Bacteriol.
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Cussiol, J. R. R., Alves, S. V., Antonio de Oliveira, M., Netto, L. E. S.
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Park, S., Imlay, J. A.
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Baker, L. M. S., Poole, L. B.
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Vergauwen, B., Pauwels, F., Vaneechoutte, M., Van Beeumen, J. J.
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Olczak, A. A., Seyler, R. W. Jr., Olson, J. W., Maier, R. J.
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Helmann, J. D., Wu, M. F. W., Gaballa, A., Kobel, P. A., Morshedi, M. M., Fawcett, P., Paddon, C.
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Comtois, S. L., Gidley, M. D., Kelly, D. J.
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Messner, K. R., Imlay, J. A.
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Hahn, J.-S., Oh, S.-Y., Roe, J.-H.
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Woodmansee, A. N., Imlay, J. A.
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Wallecha, A., Munster, V., Correnti, J., Chan, T., van der Woude, M.
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Fuangthong, M., Helmann, J. D.
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