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Journal of Bacteriology, March 1999, p. 1875-1882, Vol. 181, No. 6
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
In Vivo Role of Catalase-Peroxidase in
Synechocystis sp. Strain PCC 6803
Martin
Tichy and
Wim
Vermaas*
Department of Plant Biology and Center for
the Study of Early Events in Photosynthesis, Arizona State
University, Tempe, Arizona 85287-1601
Received 28 September 1998/Accepted 13 January 1999
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ABSTRACT |
The katG gene coding for the only catalase-peroxidase
in the cyanobacterium Synechocystis sp. strain PCC 6803 was
deleted in this organism. Although the rate of
H2O2 decomposition was about 30 times lower in
the 
katG mutant than in the wild type, the strain had a
normal phenotype and its doubling time as well as its resistance to
H2O2 and methyl viologen were indistinguishable from those of the wild type. The residual
H2O2-scavenging capacity was more than
sufficient to deal with the rate of H2O2
production by the cell, estimated to be less than 1% of the maximum
rate of photosynthetic electron transport in vivo. We propose that catalase-peroxidase has a protective role against environmental H2O2 generated by algae or bacteria in the
ecosystem (for example, in mats). This protective role is most apparent
at a high cell density of the cyanobacterium. The residual
H2O2-scavenging activity in the
katG mutant was a light-dependent peroxidase activity. However, neither glutathione peroxidase nor ascorbate peroxidase accounted for a significant part of this
H2O2-scavenging activity. When a small
thiol such as dithiothreitol was added to the medium, the rate of
H2O2 decomposition in the
katG mutant increased more than 10-fold, indicating that
a thiol-specific peroxidase, for which thioredoxin may be the
physiological electron donor, is present. Oxidized thioredoxin is
likely to be reduced again by photosynthetic electron transport.
Therefore, under laboratory conditions, there are only two enzymatic
mechanisms for H2O2 decomposition present in
Synechocystis sp. strain PCC 6803. One is catalyzed by a
catalase-peroxidase, and the other is catalyzed by thiol-specific peroxidase.
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INTRODUCTION |
Active oxygen species, including
superoxide (O2
), hydrogen peroxide
(H2O2), and the hydroxyl radical (OH·), are
byproducts of both aerobic respiration and oxygenic photosynthesis in
all organisms that carry out these processes. Because of the relatively high reactivity of active oxygen species with proteins and membranes, efficient scavenging is important to prevent photooxidative damage to
the organism (1). The work described in this paper focuses on cyanobacteria, which can carry out photosynthesis and respiration simultaneously.
The major site of O2 production in the
photosynthetic electron transport chain is at the reducing side of
photosystem I (PS I): particularly under conditions when NADPH
utilization is suboptimal and NADP levels are low, O2
rather than NADP may occasionally accept an electron from PS I
(1). This phenomenon is known as the Mehler reaction, which
is a major electron transfer route in the presence of the herbicide
methyl viologen (MV). This herbicide, also known as paraquat,
efficiently accepts an electron from PS I and reduces oxygen to
O2, thus serving as a potent inhibitor of
growth of photosynthetic organisms in the light. Respiratory
dehydrogenases of mitochondria and bacteria also have been shown to be
important sources of O2 and
H2O2 (12, 28).
O2 is efficiently scavenged by superoxide
dismutase (SOD); the activity of the enzyme is sufficient to limit
O2-induced damage except when MV is present
and large amounts of O2 are produced
(29). There are three types of SOD. All cyanobacteria contain an Fe-containing SOD (FeSOD) in the cytosol; some also contain a Mn-containing form of SOD (MnSOD) that is associated with
thylakoid membranes (6, 36). In addition, Cu/ZnSOD activity also has been reported in a cyanobacterium (7), even though initially Cu/ZnSOD was thought to be specific for eukaryotes. According to CyanoBase (12a), the genome of the
cyanobacterium Synechocystis sp. strain PCC 6803 contains
only one recognizable gene for SOD, sodB, which codes for
FeSOD, suggesting that FeSOD is the sole
O2-scavenging enzyme in
Synechocystis sp. strain PCC 6803.
H2O2 is the most stable of active oxygen
species. However, the most reactive and destructive active oxygen
species, OH·, can be formed from H2O2 in the
presence of O2 and selected metal ions such
as Fe2+ (13). The best-documented source of
H2O2 in chloroplasts is O2 generated by the Mehler reaction and
disproportionated by SOD. A significant amount of
H2O2 was also shown to be produced upon illumination of relatively intact PS II membranes (14, 38), suggesting that H2O2 can be formed by partial
oxidation of water at the donor side of PS II, at least in vitro.
Early reports indicated the presence of two major hydrogen
peroxide-scavenging activities in cyanobacteria, catalase and
ascorbate peroxidase (34, 35). Catalase activity has
been found in all cyanobacterial species tested (21). The
Synechococcus sp. strain PCC 7942 enzyme responsible for
catalase activity has been purified and characterized, and the
corresponding gene has been cloned (23, 24). The gene
sequence, enzymatic activity, and resistance to 3-amino-1,2,4-triazole,
an inhibitor of eukaryotic catalase, provided evidence that the
catalase in fact is a catalase-peroxidase, a member of the family of
prokaryotic enzymes exhibiting both catalase and peroxidase activities
(23, 24).
Ascorbate peroxidase is also a major H2O2
scavenger in chloroplasts. However, in cyanobacteria, the concentration
of ascorbate was found to be 250-fold lower than in chloroplasts
(41), and an ascorbate-specific peroxidase activity has not
yet been found (1). Indeed, the Synechocystis sp.
strain PCC 6803 genome sequence does not contain an ascorbate
peroxidase gene. Despite the fact that several cyanobacteria (including
Synechocystis sp. strain PCC 6803, Anabaena
variabilis, and Anabaena cylindrica) have been found to
exhibit a significant peroxidase activity (21), the enzyme
itself and its physiological donor remain to be determined.
Cyanobacteria contain millimolar concentrations of glutathione,
but no glutathione peroxidase activity has been detected
(34), even though two open reading frames
(slr1171 and slr1992 according to CyanoBase
[12a]) with significant similarity to glutathione peroxidase genes are present in the Synechocystis sp. strain
PCC 6803 genomic sequence. Glutathione peroxidase is widespread among bacteria, plants, and animals and is involved in the detoxification of
lipid hydroperoxides and of H2O2. The enzyme is
well conserved among all organisms, with the active site containing
selenocysteine (11).
Recently, another protein exhibiting peroxidase activity has been found
to be ubiquitously distributed among prokaryotes and eukaryotes
(3, 9). This enzyme originally was called a thiol-specific antioxidant protein (8) and was later shown to be a
peroxidase that reduces H2O2 and alkyl
hydroperoxides with the use of hydrogens provided by sulfhydryl groups
of small thiols (9). This protein was the first peroxidase
that uses thioredoxin as the immediate hydrogen donor to be identified
and was thus named thioredoxin-dependent peroxide reductase (TPx)
(9).
In the present study, we have deleted the katG gene coding
for the catalase-peroxidase in Synechocystis sp. strain PCC
6803 and have characterized the resulting mutant to help determine the
physiological role of catalase-peroxidase. Moreover, upon elimination
of the overwhelming catalase activity of catalase-peroxidase, the rates
of H2O2 production and of remaining scavenging
activity could be measured in vivo.
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MATERIALS AND METHODS |
Growth conditions.
The wild type and mutants of
Synechocystis sp. strain PCC 6803 were grown in liquid BG-11
medium (27) at 30°C at 40 microeinsteins/m2 · s on a rotary shaker. Solid
media were supplemented with 1.5% (wt/vol) agar, 0.3% (wt/vol) sodium
thiosulfate, and 10 mM TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic
acid]-NaOH buffer (pH 8.2). Filter-sterilized media or solid media
without sodium thiosulfate were used where indicated. Sodium
thiosulfate is an efficient H2O2 scavenger,
and freshly autoclaved BG-11 medium without this compound was found to
contain up to 5 µM H2O2 (data not shown).
Enzyme assays.
Catalase activity was determined
spectrophotometrically by monitoring the rate of
H2O2 decomposition at 240 nm by using an extinction coefficient for H2O2 of 43.6 M1 · cm1 (23). For lower
concentrations of H2O2 (<1 mM), the rate of O2 production was determined polarographically with a
Clark-type electrode. Both measurements were performed with whole cells
in 25 mM HEPES-NaOH buffer (pH 7.0) at 25°C. The rate of
H2O2 decomposition by the katG
strain was determined as a decrease in H2O2
concentration in the medium as a function of time. For this purpose,
H2O2 concentrations were determined through
oxidation of Fe2+ in the presence of xylenol orange
(37). Ascorbate peroxidase activity was determined as a
decrease in the absorbance of ascorbate at 290 nm upon addition of cell
homogenate and H2O2 (21). The activity of glutathione peroxidase was assayed spectrophotometrically as oxidation of NADPH in a conjugated reaction with glutathione reductase (39).
Continuous measurements of H2O2 production and
excretion by whole cells upon illumination were carried out by the
scopoletin fluorescence method (26) using a SPEX Fluoromax
spectrofluorometer. Scopoletin is oxidized by
H2O2 in the presence of peroxidase; a decrease
in the scopoletin fluorescence is a measure of
H2O2 produced. The assay mixture contained 40 µl of scopoletin (20 mg/liter), 160 U of fungal peroxidase (Fluka),
and 3 ml of cells (optical density at 730 nm [OD730],
0.5). Mixing of the sample was provided by a magnetic stirrer. The
temperature was maintained at 30°C during illumination by a
constant-temperature cuvette holder. Scopoletin was excited by
350-nm-wavelength light, and fluorescence was measured at 460 nm. The
sample was illuminated from the top with orange actinic light
(570-nm-cutoff filter) provided by an incandescent bulb (50 to 150 microeinsteins/m2 · s) or a slide projector lamp
(1,500 to 2,500 microeinsteins/m2 · s). The
steady-state H2O2 production by whole cells
varied by a factor of 2 to 3, depending on the growth stage of the
culture and the method used for sample preparation. Reproducible
results (within 30% of the average) were obtained with cells that had been grown on BG-11 plates without thiosulfate for 5 to 8 days and that
were resuspended into 25 mM HEPES-NaOH buffer (pH 7.0) 30 min before
the measurement.
Oxygen evolution.
Oxygen evolution was measured by using
samples directly taken from liquid cultures (OD730 of the
cultures, 0.5 to 0.8). Measurements were performed with a Clark-type
electrode in the presence of 1 mM K3Fe(CN)6 and
0.1 mM dimethyl-p-benzoquinone. HEPES-NaOH buffer (pH 7.0)
was added to a final concentration of 25 mM. The temperature was
25°C, and the light intensity was 4,000 microeinsteins/m2 · s. The light was passed through
an orange filter cutting off light with wavelengths of <570 nm before
reaching the sample. The chlorophyll (Chl) a concentration
was about 2 µg/ml as determined by absorption at 663 nm after Chl
extraction from cells in 100% methanol.
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RESULTS |
Deletion of the katG gene.
The katG
gene from Synechocystis sp. strain PCC 6803 (accession no.
D83990), which codes for catalase-peroxidase, was identified by its
high level of similarity to other bacterial catalase-peroxidases (71%
identity with the protein from Synechococcus sp. strain PCC 7942 [accession no. D61378] and 64% identity with that from Bacillus stearothermophilus [accession no. M29876]). No
other catalase gene copy is apparent in Synechocystis
sp. strain PCC 6803. The katG gene and its flanking
regions were cloned, and katG interruption and deletion
constructs were made by introducing a chloramphenicol resistance marker
(1.5 kb) from pACYC184. In the katGi interruption mutant,
the marker was introduced into the HpaI site 748 bp
downstream from the translation start site of the 2,262-bp-long
katG gene. In the construct made to create the
katG deletion mutant, the region between the
SmaI site 63 bp upstream of the translation start site
and the HpaI site 748 bp downstream of the translation
start site was replaced by the marker. Segregation of the mutants
was checked by PCR (Fig. 1).
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FIG. 1.
PCR amplification of the katG region of
genomic DNA from various Synechocystis sp. PCC 6803 strains.
PCR amplification was carried out with genomic DNA from the
katGi interruption mutant (lane 1) and compared to that of
the wild type (lane 2). DNA from the katG deletion mutant
(lane 3) was also compared to that of the wild type (lane 4). Two
forward primers were used: the one for lanes 1 and 2 hybridized to the
region 920 bp upstream of the katG start codon, and the one
used for lanes 3 and 4 hybridized to the region 290 bp downstream of
the katG initiation codon. The reverse primer hybridized to
the region 1,350 bp downstream of the katG start codon. The
size of the chloramphenicol marker was 1.5 kb; the size of the deletion
in the katG strain was 0.8 kb. The sizes of the DNA
markers (in kilobases) are indicated on the left.
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Characterization of the katG mutant.
The
katG deletion mutant and the katGi
interruption mutant exhibited no catalase activity, confirming that
catalase-peroxidase is the only catalase present in
Synechocystis sp. strain PCC 6803. The characteristics
of both mutant strains are summarized in Table 1. The photoautotrophic growth rate and
the PS II oxygen evolution capacity of the katG and
katGi mutants were indistinguishable from those of the wild
type under the conditions tested. However, both strains differed
greatly from the wild type in their
H2O2-scavenging capacity. At 50 µM
H2O2 in darkness, the initial rate of
H2O2 decomposition was about 30 times higher in
the wild type than in both mutants (Table 1). The
H2O2 decay in the wild type was measured
polarographically as O2 evolution, which reflects catalase activity only, whereas the total
H2O2-scavenging capacity (residual peroxidase
activity) in the mutants was determined by colorimetric detection of
the remaining H2O2. In the wild type,
H2O2 conversion was too rapid to be measured
satisfactorily by colorimetric detection. However, at 50 µM
H2O2 in the wild type, the catalase activity accounted for most of the H2O2 scavenging,
because 80 to 90% of the added H2O2 led to the
formation of oxygen (data not shown). Since both mutants had identical
properties, only results for the katG strain will be
reported here.
Since growth characteristics of the
katG mutant were
indistinguishable from those of the wild type under normal laboratory
conditions, a competition experiment was performed to compare
the
growth rates or viabilities of both strains under more extreme
conditions. The
katG mutant and wild-type strains were
mixed
and exposed to various conditions: (i) 5 h at 25°C and
2,500 microeinsteins/m
2 · s; (ii) 10 days at 10°C
and 50 microeinsteins/m
2 · s; (iii) 10 days at 5°C
in the dark; (iv) 10 days at 30°C and
50 microeinsteins/m
2 · s (control). The ratio of viable
wild-type to
katG mutant
cells was measured before and
after growth under the various conditions
by comparing the number of
CFU on plates without (wild-type plus
katG mutant cells)
and with (
katG mutant cells) chloramphenicol.
Interestingly, no significant change in the
wild-type/
katG mutant
cell ratio was found under any of
these conditions (data not shown),
indicating that the absence of the
katG gene has no physiological
effect under the conditions
tested.
The results of these experiments imply that generated
H
2O
2 is efficiently scavenged even in the
katG mutant or that H
2O
2 toxicity
is not very high in
Synechocystis sp. strain PCC 6803.
To
test this in vivo, H
2O
2 production and growth
rates of the
wild type and the
katG mutant were compared
after the addition
of different concentrations of MV. The herbicide MV
is reduced
by the acceptor side of PS I in a one-electron transfer and
is
efficiently oxidized by oxygen, forming superoxide, which is
converted
to H
2O
2 in a SOD-catalyzed reaction.
Under various conditions,
a clear difference in the rates of
H
2O
2 diffusion into the medium
was observed
between the wild type and the
katG mutant, indicating
a
decreased H
2O
2-scavenging capacity of the
katG mutant. As expected,
increases in both the light
intensity and the MV concentration
increased
H
2O
2 production (Table
2). However, when lower MV concentrations
were tested, at which both the wild type and the
katG
mutant
could grow, no significant difference between the growth rates
of the two strains was found (Fig.
2).
The cells of both strains
died at 1 µM MV, and the maximal permitted
concentration for growth
at a light intensity of 50 microeinsteins/m
2 · s for both strains was 0.5 µM
(data not shown). At this MV
concentration, the amount of
H
2O
2 that accumulated in the growth
medium
remained below the detection limit in our experiments (i.e.,
<0.2
µM), even in the
katG mutant. The fact that
H
2O
2 did not
accumulate in the medium indicates
that the remaining peroxidase
activity in the
katG mutant
is sufficient to decompose all H
2O
2 produced by
the cell.
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TABLE 2.
H2O2 production by whole cells as
a function of illumination intensity and concentration of MV
(H2O2 was detected in the medium)
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FIG. 2.
Growth curves of the wild type and the
katG mutant. Cells were grown photoautotrophically at a
light intensity of 40 microeinsteins/m2 · s without
MV (circles) or at 80 microeinsteins/m2 · s with 0 (diamonds), 1 (squares), or 2 (triangles) µM MV that was added at
time zero. Open symbols, wild type; closed symbols, the
katG strain.
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Since the previous experiment did not indicate a role of
catalase-peroxidase in protection against internally produced
H
2O
2,
protection against externally added
H
2O
2 was tested. In this experiment,
the
viabilities of the wild type and the
katG mutant were
compared
after incubation with increasing concentrations of
H
2O
2. Cell
cultures (10
4 to
10
8 cells/ml) were incubated with filter-sterilized BG-11
medium
containing increasing concentrations of
H
2O
2 for 5 h and were
then plated on BG-11
medium without glucose. The final H
2O
2
concentration
in the medium and the cell survival rate were recorded
(Fig.
3).
Filter-sterilized BG-11 medium
was used because it contained lower
concentrations of
H
2O
2 (0.5 µM) than the autoclaved media did
(3 to 5 µM). Thiosulfate present in the solid medium scavenged
any
H
2O
2 present after the 5-h experiment. In media
with low cell
concentrations (Fig.
3A and B), where the
H
2O
2 concentration did
not significantly change
during the experiment since little was
scavenged by the cells, there
was no difference in the survival
of cells between the wild-type and
katG mutant strains. At higher
cell concentrations (Fig.
3C and D), the wild-type strain survived
much better because of its
superior ability to effectively scavenge
all
H
2O
2 from the media during the 5-h incubation.
Therefore,
exposure of cells to H
2O
2
concentrations on the order of 10 µM
for several hours is toxic, and
catalase-peroxidase does not protect
single cells against the effects
of externally added H
2O
2. However,
as a
population of cells, the wild type in contrast to the
katG mutant can detoxify and thereby survive significant
concentrations
of H
2O
2.
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FIG. 3.
Effect of H2O2 on the viability
of the wild type and the katG mutant. Different
concentrations of cells were incubated for 5 h with different
concentrations of H2O2; cells were then plated
on BG-11 medium with thiosulfate and without glucose. Survival rates
(percentage of survival of control incubated without
H2O2) and final H2O2
concentrations at the end of the 5 h of incubation are
indicated.
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Ascorbate peroxidase and glutathione peroxidase activities.
In
the absence of catalase activity in the katG mutant, the
remaining H2O2-scavenging activity was assigned
to peroxidase. Known peroxidases from other photosynthetic systems
include ascorbate peroxidase (1) and glutathione peroxidase
(39) and possibly TPx (3).
Even though ascorbate peroxidase activity in cyanobacteria has been
postulated (
21,
35), no ascorbate peroxidase gene
with
similarity to plant ascorbate peroxidase genes was found
in the
Synechocystis sp. strain PCC 6803 genome. A marginal
activity
(6 µmol/mg of Chl · h with 1 mM
H
2O
2 and 0.5 mM ascorbate) was
found and was
identical for the wild type and the
katG mutant
(data not
shown). This in vitro ascorbate peroxidase activity
may originate from
some nonspecific peroxidase that naturally
has a preference for
substrates other than
ascorbate.
Regarding glutathione peroxidase, two genes for this enzyme are present
in the
Synechocystis sp. strain PCC 6803 genome, and
in
Chlamydomonas reinhardtii the enzyme can be induced by the
addition of selenite (
39). However, no selenite induction of
the H
2O
2 decomposition rate in vivo (Fig.
4B) and no glutathione
peroxidase
activity in vitro were detected in the mutant. No increase
in the
H
2O
2 decomposition rate (Fig.
4B) and no
glutathione peroxidase
activity (data not shown) were observed,
suggesting that glutathione
peroxidase is not expressed in
Synechocystis sp. strain PCC 6803.
In addition,
incubation for 1 h or growth for 3 days with 100
µM
mercaptosuccinate, which is a potent and specific inhibitor
of
the enzyme (
11), did not inhibit
H
2O
2 decomposition in the
katG mutant (Fig.
4B). Therefore, neither ascorbate
peroxidase
nor glutathione peroxidase is active in
Synechocystis sp. strain
PCC 6803 under the conditions used.
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FIG. 4.
H2O2 decomposition in the wild
type and the katG strain. (A)
H2O2 decomposition in darkness (wild type and
katG strains), in the light (katG strain),
and in the light in the absence of cells; (B) peroxidase activity of
the katG mutant in the light with 2 mg of sodium selenite
per liter or 100 µM mercaptosuccinate as indicated; (C) peroxidase
activity of the katG mutant in the presence of 1 mM DTT.
For comparison, the spontaneous decomposition rate has been indicated.
For the wild type, the decrease in H2O2
concentration was measured continuously as oxygen evolution. For the
katG strain, H2O2 was measured at
particular intervals by the peroxide assay. The initial rate of
H2O2 consumption in the presence of 50 µM
H2O2 was 750 µmol of
H2O2/mg of Chl · h in the wild
type in the dark and 25 or 50 µmol of H2O2/mg
of Chl · h for the mutant strain
incubated in the dark or light (50 microeinsteins/m2
· s), respectively.
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Thiol-dependent peroxidase activity.
Genes for
putative TPxs have been identified in the genome of
Synechocystis sp. strain PCC 6803 (sll0755, with
63.5% identity between the translated open reading frame and
peroxiredoxin from Hordeum vulgare [accession no. Z34917],
and slr1198, with 52% identity to a human peroxiredoxin
[human antioxidant protein, P30041]). To test the possibility that
the peroxidase activity observed in the katG strain in
fact reflects TPx activity, the H2O2-scavenging
capacity of the cells of the katG strain was compared
with that in the presence of 1 mM dithiothreitol (DTT) in the dark
(Fig. 4C). DTT can be substituted for thioredoxin as an electron donor
for TPx (17). Indeed, DTT was found to increase the rate of
H2O2 decomposition by about 10-fold to 300 µmol H2O2/mg of Chl · h. DTT by itself
had no effect on H2O2 stability (Fig. 4C).
Kinetics of H2O2 decomposition.
H2O2 decomposition by both the wild type and
the katG mutant in vivo followed Michaelis-Menten
kinetics (Fig. 5A and B). From the double
reciprocal graph, the apparent Kms for
H2O2 were 2.8 mM for the wild type and 70 µM
for the mutant. The Km value for the wild type
is similar to that of purified catalase-peroxidase from
Synechococcus sp. strain PCC 7942, which was found to
be 4 mM (23, 24). The Vmax for
the wild type (28,000 µmol of H2O2/mg
of Chl · h) indicates the ability of catalase-peroxidase to
efficiently convert high concentrations of
H2O2. The lower Km value
for H2O2 found in the katG mutant
is typical for peroxidase activity.
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FIG. 5.
Double reciprocal graphs of the dependence of the
H2O2 decomposition rate on the concentration of
H2O2. (A) The rate of
H2O2 decomposition in the wild type was
determined spectrophotometrically at 240 nm for
H2O2 concentrations above 1 mM or
polarographically for lower concentrations. (B and C) The rate of
H2O2 decomposition in the katG
mutant was determined as a decrease in H2O2
concentration at specific time intervals (1 to 20 min). Cells
(OD730 = 0.5) were incubated in the light at 50 µmol/m2 · s (B) or in darkness in the presence of
1 mM DTT (C). The apparent Km values of the
enzyme for H2O2 as well as the
Vmax values are indicated.
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In the
katG mutant in the presence of DTT, the apparent
Km for conversion of
H
2O
2 in the dark was 60 µM and the
Vmax was
480 µmol of
H
2O
2/mg of Chl · h (Fig.
5C). Both the
light-dependent
peroxidase reaction of the
katG mutant
(Fig.
5B) and the conversion
of H
2O
2 in the
dark in the presence of DTT (Fig.
5C) have similar
Kms and can most likely represent the activity
of the same peroxidase
enzyme when either DTT or electrons generated by
photosynthetic
electron transport are used as an electron donor. The
increase
in
Vmax by an order of magnitude in the
presence of DTT suggests
that in its absence the natural electron
donor for peroxidase
is present at a limiting concentration or is only
partially reduced
upon illumination (50 microeinsteins/m
2 · s). The results presented here
indicate that the remaining
peroxidase activity in the
katG mutant most likely originates
from
TPx.
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DISCUSSION |
Role of catalase-peroxidase.
Synechocystis sp. strain
PCC 6803 possesses a single catalase encoded by katG. The
high, constitutive activity of this enzyme is independent of the
growth phase of the organism and is not inducible by pretreatment
with H2O2 (50 µM for 1 h) (data not shown). This is similar to the situation in Synechococcus
sp. strain PCC 7942 (20) but different from that in
Escherichia coli, which has two catalases that are inducible
by two different mechanisms (18).
In this study, the
katG gene of
Synechocystis sp.
strain PCC 6803 was cloned and deleted to examine the role of
catalase-peroxidase
and other enzymes in protecting the cyanobacterium
against oxidative
stress. The
katG strain exhibited no
measurable catalase activity.
Despite this complete absence of
catalase-peroxidase, the mutant
grew normally with no apparent
difference in growth rate or tolerance
to MV from that of the wild
type.
Consistent with their decreased H
2O
2-scavenging
capacity,
katG cells were found to be more susceptible to
higher concentrations
of H
2O
2 in the medium as
they had a decreased capability to detoxify
H
2O
2 during the experiment (Fig.
3). At low
cell concentrations,
where no significant change in
H
2O
2 concentration during the course
of the
experiment was observed, no difference in sensitivity to
H
2O
2 between the wild type and the
katG mutant was found. A similar
surprising result has
been obtained for
E. coli (
22), where
diluted
cultures of wild type were as sensitive to H
2O
2
as the
catalase-deficient mutant. At high cell densities, the wild-type
cells but not the mutant ones could efficiently catabolize added
H
2O
2 and survive (
22).
The fact that at a low cell density the wild type and the
katG mutant are equally sensitive to externally added
H
2O
2 indicates
that
H
2O
2 diffusion into the cell is more rapid than
its decomposition
by catalase, resulting in similar cytoplasmic
concentrations of
H
2O
2 in the wild type and the
mutant. Easy and fast diffusion
of H
2O
2 through
biological membranes (
31) led to a proposal
that aquatic
organisms can keep their intracellular H
2O
2
concentration
low by simple diffusion of H
2O
2
produced in the cell (
12). This
would make an active
H
2O
2-scavenging system in algae and
cyanobacteria
less important than that in plant chloroplasts.
Interestingly,
carbon fixation enzymes normally known for their high
sensitivity
to H
2O
2 have been shown to be much
more H
2O
2 tolerant in algae
and cyanobacteria
than in plant chloroplasts (
33).
Our observation that catalase-peroxidase can protect cyanobacterial
cells against high concentrations of external
H
2O
2 but
is dispensable for growth under
various laboratory conditions
leads us to conclude that
catalase-peroxidase in this cyanobacterium
serves primarily in
protection against external H
2O
2. In pathogenic
bacteria (
4,
25), catalase has a protective role against
H
2O
2 generated by the host organism during
infection. It is possible
that cyanobacteria in their natural
environment are also subject
to oscillating levels of external
H
2O
2 and that cell populations
need catalase
activity for survival. For example, significant
photosynthetic
production and excretion of H
2O
2 by the alga
Ulva rigida have been implied to be of ecological
significance (
12).
High catalase activity may be
advantageous for survival, for example,
in algal mats in competition
with H
2O
2-producing algae and bacteria.
The
fact that wild-type
E. coli but not a catalase-deficient
mutant
survived and multiplied in the presence of peroxide-generating
streptococci (
22) supports this
argument.
H2O2 production in cells.
The
H2O2 production in the katG
mutant can be estimated as a combination of
H2O2 diffusion from the cells and
H2O2 decomposition by peroxidase within cells.
The maximum rate of H2O2 diffusion from mutant
cells into the medium was less than 2 µmol of
H2O2/mg of Chl · h during illumination
(2,200 microeinsteins/m2 · s) (Table 2). The
Vmax of H2O2
decomposition by peroxidase in the mutant was 480 µmol/mg of Chl
· h (Fig. 5C). Since the Km of the enzyme is
57 µM (Fig. 5C), this activity corresponds to 4.2 µmol of
H2O2/mg of Chl · h at 0.5 µM
H2O2, which is the equilibrium concentration
detected in the medium when cells are illuminated at a high light
intensity (2,200 microeinsteins/m2 · s). The rates
of diffusion and decomposition of H2O2 add up to a total H2O2 production of 6 µmol/mg of
Chl · h. This is about 1% of the maximum rate of whole-chain
photosynthetic electron transport (300 µmol of O2/mg of
Chl · h; 2 mol of H2O2 per mol of
O2). This is higher than the numbers reported for
superoxide production by respiring membranes of E. coli
(0.03 to 0.3% of the electron transport depending on the substrate
used) (15) but significantly lower than values reported for
isolated chloroplasts where acceptors of PS I electrons are in short
supply: in such systems, up to 25, 6, and 10% of electron transport
may be used to form H2O2 in Euglena
gracilis (16), in chloroplasts isolated from C. reinhardtii (32), and in chloroplasts isolated from spinach (2), respectively. Therefore, it is likely that
H2O2 production in vivo in photosynthetic
systems is significantly less than that determined from studies on
isolated organelles. This implies that O2
production in photosynthetic electron transport may be much less than
what is generally assumed.
Absence of ascorbate peroxidase and glutathione peroxidase
activity.
In our study, only marginal ascorbate peroxidase
activity was detected in cell extracts of Synechocystis sp.
strain PCC 6803 at saturating ascorbate concentrations, indicating that
at in vivo ascorbate concentrations that are estimated to be 20 to 100 µM (34), which is about 250 times lower than in
chloroplasts, ascorbate peroxidase activity does not play an
important role in H2O2 scavenging.
We did not detect any in vitro glutathione peroxidase activity in
Synechocystis sp. strain PCC 6803, despite the fact that
there are two glutathione peroxidase genes present in its genome.
As
observed in
Nostoc muscorum PCC 7119 and
Synechococcus sp.
strain PCC 6311 (
34),
peroxidase activity in
Synechocystis sp.
strain PCC 6803 was
not boosted by the addition of selenite into
growth medium, in contrast
to what was achieved in
C. reinhardtii (
39). In addition, no changes in peroxidase activity and in
the growth rate of the
katG mutant were observed upon
addition
of 100 µM mercaptosuccinate, which is a potent and specific
inhibitor
of glutathione peroxidase (
11). A similar report
regarding the
lack of mercaptosuccinate inhibition has been presented
for
Plectonema boryanum (
19). Therefore,
apparently glutathione peroxidase
is not expressed in
Synechocystis sp. strain PCC 6803 under laboratory
conditions, but it is possible that the two genes for this enzyme
are
induced under certain natural
conditions.
Thiol-specific peroxidase activity.
Although peroxidase
activity is well documented in cyanobacteria, the peroxidase itself and
its natural electron donor remain obscure. The results presented
in this paper indicate that this peroxidase activity originates from a
thiol-specific peroxidase; thioredoxin may be its natural
electron donor. In the Synechocystis sp. strain PCC
6803 genome, two genes have significant similarity to characterized
thiol-specific peroxidases.
For continued activity of the peroxidase, thioredoxin needs to be
recycled. In cyanobacteria and chloroplasts, thioredoxin
reduction is
coupled to photosynthetic electron transport via
ferredoxin:thioredoxin reductase (
3). The
ferredoxin:thioredoxin
system is responsible for light-mediated
enzyme regulation in
photosynthesis by a selective thiol redox control
(
5,
30).
If TPx indeed uses thioredoxin regenerated by
photosynthetic electron
transport as an electron donor, then under
conditions where superoxide
and consecutive
H
2O
2 production levels are highest (with
illuminated
cells or with chloroplasts with carbon fixation inhibited
and
ferredoxin reduced) (
1) TPx activity would also be
maximal,
since reduced thioredoxin is
plentiful.
Therefore, in
Synechocystis sp. strain PCC 6803, H
2O
2 at low concentrations is broken down
primarily by the peroxidase TPx.
This is the first report of
significant thiol-specific peroxidase
activity in a photosynthetic
organism. Clearly, cyanobacteria
employ a different strategy for
scavenging H
2O
2 generated by photosynthesis
than the ascorbate peroxidase pathway of chloroplasts. We suggest
that
the main function of catalase-peroxidase with its low
H
2O
2 affinity and high
Vmax is to break down
H
2O
2 entering the cell
from the environment.
This enzyme is advantageous particularly
for natural cyanobacterial
populations in competition with H
2O
2-producing
algae and
bacteria.
| |
ACKNOWLEDGMENT |
This work was supported by U.S. Department of Energy grant
DE-FG03-95ER20180.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Plant Biology, Arizona State University, Box 871601, Tempe, AZ
85287-1601. Phone: (602) 965-3698. Fax: (602) 965-6899. E-mail:
wim{at}asu.edu.
| |
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Abstract
Introduction
