Journal of Bacteriology, March 1999, p. 1939-1943, Vol. 181, No. 6
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
SodA and Manganese Are Essential for Resistance to
Oxidative Stress in Growing and Sporulating Cells of
Bacillus subtilis
Takashi
Inaoka,1
Yoshinobu
Matsumura,1,2,* and
Tetsuaki
Tsuchido1,2
Department of Biotechnology, Faculty of
Engineering,1 and High-Technology
Research Center,2 Kansai University, Suita
564-8680, Japan
Received 8 September 1998/Accepted 12 January 1999
 |
ABSTRACT |
We constructed a sodA-disrupted mutant of
Bacillus subtilis 168, BK1, by homologous recombination.
The mutant was not able to grow in minimal medium without Mn(II). The
spore-forming ability of strain BK1 was significantly lower in
Mn(II)-depleted medium than that of the wild-type strain. These
deleterious effects caused by the sodA mutation were
reversed when an excess of Mn(II) was used to supplement the medium.
Moreover, the growth inhibition by superoxide generators in strain BK1
and its parent strain was also reversed by the supplementation with
excess Mn(II). We therefore estimated the Mn-dependent
superoxide-scavenging activity in BK1 cells. Whereas BK1 cells have no
detectable superoxide dismutase (Sod) on native gel, the
superoxide-scavenging activity in crude extracts of BK1 cells grown in
Mn(II)-supplemented LB medium (10 g of tryptone, 5 g of yeast
extract, and 5 g of NaCl per liter) was significantly detected by
the modified Sod assay method without using EDTA. The results obtained
suggest that Mn, as a free ion or a complex with some cellular
component, can catalyze the elimination of superoxide and that both
SodA and Mn(II) are involved not only in the superoxide resistance of
vegetative cells but also in sporulation.
| |
TEXT |
Reactive oxygen species, such as
superoxide anion (O2
) and hydrogen peroxide
(H2O2), are formed as a consequence of partial reduction of oxygen in the cellular compartment. These reactive oxygen
species have been known to cause severe damage to DNA, RNA, proteins,
and lipids (10, 17, 27). Therefore, most aerobic organisms
have potent systems to protect themselves from this damage. Superoxide
dismutase (Sod; EC 1.15.1.1) catalyzes the dismutation of
O2 to oxygen and H2O2
(13), and catalase and peroxidase prevent the intracellular
accumulation of toxic H2O2 (15).
Bacillus subtilis is an aerobic bacterium and forms an
endospore which is highly resistant to a variety of stresses, such as
oxidants, heat, and UV light. We have recently reported that this
bacterium possesses one detectable Sod (SodA), which is present in both
vegetative cells and spores, and also that this enzyme is predicted to
protect cells from oxidative stress during growth and sporulation
(18). Casillas-Martinez and Setlow (6) have recently demonstrated that SodA, KatX, alkyl hydroperoxide reductase, and MrgA play no role in spore resistance to heat and oxidants. In this
study, we characterized a B. subtilis sodA mutant and found
that a SodA-independent defensive system against oxidative stress is
mediated substantially by manganese in B. subtilis.
Construction of a sodA mutant of B. subtilis.
To investigate the function of SodA in B. subtilis, we constructed a sodA-disrupted mutant by the
following method. A recombinant plasmid which contained the
sodA gene disrupted by the insertion of a chloramphenicol
acetyltransferase-encoding (cat) gene derived from pMSG-CAT
was constructed, designated
pBR-sodA::cat, and introduced into
cells of B. subtilis 168 (trpC2). Some
chloramphenicol-resistant colonies which appeared on an agar plate of
LB medium (10 g of tryptone, 5 g of yeast extract, 5 g of
NaCl per liter) containing 5-µg/ml chloramphenicol were selected. It
was confirmed by Southern blotting analysis that sodA was
expectably replaced with sodA::cat in
the chromosomal DNAs of some chloramphenicol-resistant mutants (data
not shown). One of these sodA::cat
mutants was designated BK1.
Growth ability of the B. subtilis sodA mutant.
Although no difference in the growth rate at 37°C in LB medium was
seen between the wild-type strain and strain BK1, the final cell mass
of the latter reached only half of that of the former (Fig.
1C, open symbols). In Spizizen salts
medium [14 g of K2HPO4, 6 g of
KH2PO4, 2 g of
(NH4)2SO4, 1 g of sodium
citrate, 0.2 g of MgSO4 · 7H2O, and
5 g of glucose per liter] supplemented with 20 mg of
L-tryptophan per liter, the growth of strain BK1 was markedly depressed (Fig. 1A).
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FIG. 1.
Manganese requirement for the growth of strain BK1.
Overnight cultures of the wild-type and BK1 strains were grown in LB
medium not supplemented with metal were diluted 1:50 in Spizizen salts
medium containing different kinds of metal ion at 0.1 mM (A and B) or
in LB medium containing 0.1 mM EDTA (C) or 1 µM CdCl2
(D). Cell growth was monitored by measuring OD650. (A)
Effect of MnCl2 (solid symbols) on the growth of the wild
type (squares) and strain BK1 (circles). Control values (open symbols)
are also shown. (B) Effects of MnCl2 ( ),
FeSO4 ( ), Ca(NO3)2 ( ),
CuSO4 ( ), and ZnCl2 (×) on the growth of
strain BK1. (C and D) Effects of EDTA and CdCl2 (solid
symbols), respectively, on the growth of the wild type (squares) and
strain BK1 (circles). Control values (open symbols) are also shown.
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An Escherichia coli sodA sodB double mutant lacking
cytoplasmic Sods has been reported to require branched-chain amino
acids for growth in minimal medium (5). Unlike E. coli, however, strain BK1 was not able to recover the ability to
grow, even in minimal medium supplemented with Casamino Acids at final
concentrations of 0.02 to 0.2% (data not shown). But unexpectedly,
this growth depression was found to be completely reversed by
supplementation with MnCl2 at concentrations higher than 1 µM (Fig. 1A). No such drastic restoring effect was observed with the
other metals tested [FeSO4, CuSO4,
ZnCl2, and Ca(NO3)2], although
Fe(II) at 0.1 mM had a slight effect (Fig. 1B). To confirm this
manganese requirement of strain BK1, we compared the sensitivities of
both strains of B. subtilis to EDTA as a divalent-cation
chelator and Cd(II), which is a competitive inhibitor of Mn(II) uptake
(21). Addition of EDTA at 0.1 mM or Cd(II) at 1 µM to the
culture caused a remarkable delay in the growth of strain BK1 (Fig. 1C
and D). No such phenomena were observed with the wild-type strain under
identical conditions. These results indicate that manganese is
necessary for the SodA-deficient strain to grow.
Effect of Mn(II) supplementation on oxidant sensitivity.
We
examined the effects of oxidants on the cell growth (growth test) and
viability (viability test) of strain BK1. In the growth test, cells
were grown in LB medium until the optical density at 650 nm
(OD650) of the culture reached 0.3 and then paraquat, menadione, or H2O2 at various concentrations
was added to the culture. The subsequent cell growth was monitored. In
the viability test, cells grown in LB medium were harvested at the
mid-log or stationary phase (cultivated for 8 h), washed with 50 mM Tris-HCl buffer (pH 7.0), and resuspended in the same buffer at a
density of approximately 108 cells/ml. The suspension was
treated with paraquat or H2O2 at appropriate
concentrations for 15 min at 37°C. The viable cells were counted on
an LB agar plate after incubation for 12 to 18 h at 37°C.
Growing cells of strain BK1 were more sensitive to paraquat, menadione,
and H2O2 than were those of the wild-type
strain (Fig. 2). Similar results were
obtained in the viability test (data not shown). Since the ability of
strain BK1 to grow in minimal medium was restored by supplementation
with MnCl2, Mn(II) is suggested to protect B. subtilis cells against oxidative stress. We therefore tested
whether Mn(II) influences cellular sensitivity to some oxidants or not.
Indeed, supplementation of the growth medium with 0.1 mM
MnCl2 markedly relieved cells from growth inhibition by
paraquat and menadione (Fig. 2A and B). In the case of
H2O2 at 200 µM, on the other hand,
MnCl2 caused transient cell lysis after exposure. This
phenomenon may result from the repression of katA, one of
the peroxide regulon genes (8), by Mn(II).
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FIG. 2.
Effect of manganese on sensitivity to various oxidants.
Overnight cultures of the wild type and strain BK1 grown in LB medium
were diluted 1:50 with fresh LB medium with (solid symbols) or without
(open symbols) 0.1 mM MnCl2. Cultures were incubated to an
OD650 of 0.3 (at 0 h), and then paraquat (A),
menadione (B), or H2O2 (C) was added (0 h) to
the medium at the following concentrations: 50 (circles), 100 (triangles), or 200 (diamonds) µM paraquat; 2 µM menadione
(circles), and 200 µM H2O2 (circles) for the
wild-type strain and 5 (circles), 20 (triangles), or 50 (diamonds) µM
paraquat; 0.1 µM menadione (circles); and 200 µM
H2O2 (circles) for strain BK1. Untreated
control values (squares) are also shown.
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We further examined the effect of Mn(II) on oxidant sensitivity by
using the viability test. However, no supplementation effect of
MnCl2 at 0.1 mM was observed either in growth medium or
during paraquat treatment. It should be noted that the effect of Mn(II) on oxidant sensitivity was observed only in growing cells.
Effects of SodA and Mn(II) on sporulation.
Cells of strain BK1
normally sporulated in Schaeffer's sporulation medium consisting of
0.8% nutrient broth (Difco), 27 mM KCl, 2 mM MgSO4
· 7H2O, 1 mM Ca(NO3)2 · 4H2O, 0.1 mM MnCl2 · 4H2O, and 1 µM FeSO4 · 7H2O (data not
shown), suggesting that SodA is not essential for sporulation under
conventional sporulation conditions. However, in Mn(II)-depleted
Schaeffer's sporulation medium in which MnCl2 was replaced
with a corresponding amount of FeSO4, the sporulation
frequency of strain BK1 was markedly lower than that of the wild-type
strain (Table 1). This result suggests that strain BK1 requires Mn(II) not only for vegetative growth but also
for sporulation at a concentration higher than that needed by the
wild-type strain.
Furthermore, even in Schaeffer's sporulation medium originally
containing 0.1 mM MnCl2, the sporulation frequencies of
both strain BK1 and the wild-type strain bearing vector plasmid
pHY300PLK (Takara) were affected by the addition of paraquat at the
time of entry into the sporulation phase (time zero) (Fig.
3A). Addition of paraquat at a
concentration of 5 µM, which did not affect vegetative growth,
inhibited the sporulation of the wild-type strain, and more
importantly, the sporulation of strain BK1 was profoundly depressed at
an even lower concentration (1 µM). Overproduction of SodA from
pHY-sod, which was derived from pHY300PLK and contained the
complete sodA gene, restored viability during sporulation and the ability to sporulate after the addition of paraquat at time
zero in both strain BK1 and the wild-type strain (Fig. 3B). Addition of
paraquat at 2 h of incubation or later did not affect the
sporulation frequency (data not shown). These results suggest that an
elevated level of O2 may inhibit an early
event in the sporulation process and also that Mn(II), as well as SodA,
may relieve the toxicity of such an oxidative milieu for sporulating
cells.
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FIG. 3.
Inhibition of sporulation by paraquat. The wild type
(wt) and strain BK1 carrying pHY300PLK (A) or pHY-sod (B)
were sporulated in Schaeffer's sporulation medium containing 25 µg
of tetracycline per ml. Paraquat was added at the concentrations
indicated at the time of entry into the sporulation phase (0 h), and
the culture was incubated for a further 48 h with shaking. Spore
counts (solid bars) on LB agar plates obtained after heat treatment at
80°C for 15 min (left axis) and viable cell counts (open bars) are
shown. The sporulation frequencies of the wild-type strain (squares)
and strain BK1 (circles) are also plotted (right axis).
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Manganese-dependent O2-scavenging
activity in B. subtilis.
Manganese is known to have a
Sod-like activity in vitro, and several Mn(II) complexes and manganous
porphyrins catalyze the elimination of O2
(2, 4, 9, 11, 19). Furthermore, Mn(II) suppresses the
methionine and lysine auxotrophic phenotypes of a Saccharomyces cerevisiae SOD1 mutant lacking intracellular Cu, Zn-Sod (7, 22) and functions as a substitute for Sod in Lactobacillus
plantarum (1). We therefore measured the Mn-dependent
O2-scavenging activity in crude extracts of
BK1 cells. Although the Sod assay, which is based upon the inhibition
of cytochrome c reduction (26), is usually
carried out with 50 mM potassium phosphate buffer (pH 7.8) containing
0.1 mM EDTA as previously described (18),
non-EDTA-supplemented 50 mM Tris-HCl buffer (pH 7.8) was used here for
the measurement of Mn(II)-dependent O2-scavenging activity. Whereas BK1 cells had
no detectable Sod protein on native gel, the
O2-scavenging activity in crude extracts of
BK1 cells grown in LB medium supplemented with 0.1 mM MnCl2
was significantly detectable in this modified assay system and was
approximately 60% of that of the wild-type strain (Table
2). No such detectable
O2-scavenging activity was observed in crude
extracts of BK1 cells grown in the absence of MnCl2 (data
not shown). The activity in BK1 cells was diminished by addition of
EDTA or dialysis, in contrast to that in wild-type cells (Table 2).
After boiling for 10 min, the activities remaining in the supernatants
of BK1 and wild-type cells were approximately 85 and 55% of the total
O2-scavenging activity, respectively (Table
2). These results suggest that Mn(II) itself or in a complex with some
cellular component is involved in the cellular defense against
O2 stress and therefore can substitute for
Sod. However, the finding that this Mn effect was observed only with
growing cells is a puzzle. Mn(II) alone or in a complex might not be
recycled as an O2 scavenger in nongrowing
cells. Alternatively, Mn(II) might form a functional complex with some
cellular component depending upon cell growth.
As other possibilities for the manganese effect, Mn(II) might function
in vivo as a stabilizer of some critical
O2-sensitive protein(s) such as
glutamine synthetase, which contains Mn and is known to be sensitive to
oxidative stress (23, 24), or as a cofactor of some
regulator protein which is involved in oxidative stress resistance. To
understand the physiological role of manganese, detailed chemical and
genetic studies are necessary.
In E. coli, some [4Fe-4S] cluster-containing proteins are
known to be to O2 generator or oxygen
sensitive (12, 14, 20, 25). Supplementation with Fe has been
reported to restore the ability of a Sod-deficient strain of E. coli to grow in minimal medium. This Fe effect is suggested to be
the reactivation of such inactivated proteins (3). In this
report, we demonstrate that Fe(II) partially restores the growth of a
sodA mutant in minimal medium, and therefore, this Fe(II)
effect may resemble that observed with a Sod-deficient strain of
E. coli.
From the finding that the sporulation frequency of the mutant markedly
decreased, compared with that of the wild-type strain, both under
stress conditions and in Mn-depleted medium, it is suggested that cells
in the early sporulation phase and/or premature spores are sensitive to
O2 and also that both SodA and Mn(II) protect
sporulating cells from oxidative stress. It is still unclear whether
the resistance of mature spores to oxidative stress directly reflects
the O2-scavenging ability of those spores.
Henriques et al. (16) have recently reported that the spores
of a sodA mutant of B. subtilis was not able to
mature sufficiently. We presume that the spores of a Sod-deficient
strain produced in the absence of Mn(II) may be sensitive to oxidative stress.
In conclusion, our findings indicate that not only SodA but also Mn(II)
possesses a functional role in the cellular defense against
O2 and that these two systems function in
both growing and sporulating cells. This is the first paper reporting
that Mn(II) is an additional key factor in the cellular defense against
oxidative stress in B. subtilis.
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ACKNOWLEDGMENTS |
We thank Ryoko Nakamura and Naoki Hirayama for technical assistance
in part of the experiments.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biotechnology, Kansai University, Yamate-cho, Suita 564-8680, Japan. Phone: 81-6-6368-1121. Fax: 81-6-6388-8609. E-mail:
ymatsu{at}ipcku.kansai-u.ac.jp.
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Journal of Bacteriology, March 1999, p. 1939-1943, Vol. 181, No. 6
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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