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Germinability of coat-lacking spores of Bacillus megaterium
Vol.
128,
April
30,
No. 2, 1985
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1985
Pages
GERMINABILITY
OF COAT-LACKING
SPORES OF BACILLUS
728-732
MEGATERIUM
Y. Nakatani K. Tani, M. rmagawa, T. Nish:hara, and M. Kondo Department
of Environmental Chemistry and Microbiology Faculty of Pharmaceutical Sciences Osaka University 1-6 Yamadaoka, Suita, Osaka 565, Japan
Received March 4, 1985 SUMMARY: Upon treatment with acid, the germinability of both intact and coat-lacking spores of Bacillus meqaterium ATCC 19213 exhibited similar features. Namely, when the spores previously germinated by alanine in the presence of phosphate buffer were converted to H-spores by treatment with nitric acid, germination proceeded at a very low speed in a same germination medium. When H-spores converted to Ca-spores by treatment with calcium acetate and subsequently germinated, germination proceeded at a speed higher than that of native spores and occurred even in the absence of buffer. These results suggest that the site of exchangeable cations concerned with germina6ility must not exist in the coat. 0 1985 Academic Press, Inc.
INTRODUCTION: initiated
Spore
by alanine
electrolyte
(1).
facts
for
and inosine
in
when
electrolyte
suggest
the existence through
towards
germination
the
site
germination
located
in
inner rather
is
spore
coat
membrane than
C2,31.
*Abbreviations: dithiothreitol:.
spore
coat
coat
On the other role
(4).
DPA, dipicolinic
Using
728
sodium
(2).
These
upon
to clarify
which of the
studies
the initiation
SDS,
0006-291X/85 $1.50 Cowright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
recent
coat-lacking
acid;
useful
most
can be
an investigation
calcium
because hand,
in
acid
Rode and Foster that
acetate,
spores
has an effect
very
an aspect,
spore
with
Therefore, is
and suggested
has an important
the
which
calcium
these
by treatment
of calcium
the
with
Moreover,
of calcium.
From such
binding
lost.
ATCC 19213 can be
of non-specific
treated
of a component
localization
mechanism.
were
type
the binding
on the calcium
to the
was
megaterium
the presence
spores
to electrolyte-requiring
germinability geared
in Bacillus
However,
the requirement converted
germination
the studies
made
takes
part
calcium show
that
in
bound the
of germination spores,
we report
aoaecyl
sulfate;
here
DTT,
Vol.
128,
studies
No. 2, 1985
BIOCHEMICAL
conducted
participate
to determine
AND
whether
in germinability
BIOPHYSICAL
calcium
RESEARCH
binding
COMMUNICATIONS
to the
spore
coat
or not.
B. megaterium ATCC 19213 spores were grown in AND XFiTHODS: supplemented nutrient broth, except that the MnCl concentration was 0.1 mM, harvested, washed, lyophilized, and stored as pre&iously described 15). All references to spore weights are on a dry weight basis. Cation-exchanged spores were prepared as follow: spores (10 mg/ml) were incubated in a solution of 0.5% SDS-O.1 M DTT-0.1 M NaCl, pH 10.0 (6) at 31°C for 2 h, and washed 10 times by centrifugation (SDS-DTT spores); intact and SDS-DTT spores (10 mg/ml) were titrated to pH 4.0 with 0.01 N nitric acid for 120 and 30 min, respectively, and washed 4 times by centrifugation [H-spores); H-spores prepared from intact and SDS-DTT spores (10 mg/ml) were incubated in 100 mM calcium acetate at 60°C for 60 min, and washed 6 times by centrifugation [Ca-spores). Prepared spores were then lyophilized and stored in desiccator until use. Spores were heat activated at 60°C for 60 min, and then incubated in 30 mM sodium-potassium phosphate buffer CpH 8.0) containing 10 mM alanine, or in a solution of 10 mM alanine at 3O'C. At intervals, samples of germination exudate were taken by filtering the spore suspension through membrane filter. DPA content in the filtrate was determined by ultraviolet absorbance method c7). DPA content of the spores was measured by the methods of Janssen et al. C87. Spores were fixed, dehydrated, embedded, sectioned, stained, and then observed under an electron microscope as described previously (Nakatani, Y., Imagawa, M., Takubo, Y., Nishikawa, J., Nishihara, T., and Kondo. M., in submit), except that fixation with Os04 was carried out at 4'C for 48 h. MATERIALS
germinated alanine
by alanine
concentrations germinability
of N-spores with
of 11.2,
7.5,
by alanine
of that after
60 min when the mixture medium.
Hi in spores,
for
although
by the treatment
Moreover, alanine N-spores
and phosphate (Fig.
buffer
during
1). 129
and phosphate
was considered
low by exchanging
still
remained.
acetate
alone,
buffer.
without
rapid
that
cations
Ca-spores,
100 mM calcium
germination
was significantly
inosine, it
Therefore,
by alanine
the speed of DPA release
The
was less than
of alanine,
of H-spores with
60 min, could be germinated
from the spores at
100% of H-spores were
became very
germinability
to pH 4.0 for
low and the speed of DPA release
However,
of N-spores.
was used as a germination
obtained
acid
respectively.
and phosphate buffer
the response of spores to germinants with
nitric
and 5.4 nmol/mg spore,
of H-spores was considerably
one-fiftieth
buffer
titration
were
but not by
magnesium, and manganese were released
germination
germinated
in the presence of phosphate buffer,
During
alone.
120 min, calcium,
during
Spores of g. megaterium ATCC 19213 (N-spores)
AND DISCUSSION:
RESULTS
at 60°C
of Ca-spores by compared with
that
of
Vol. 128, No. 2, 1985
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0
0 0
60
30 minutes
0
30 minutes
b
60
Fig. 1. Germination of cation exchanged spores. N- (e), H- (O), and Ca[A) spores were germinated by alanine plus phosphate buffer (a) or alanine (b). DPA release was mesasured as described in the text and expressed as percent DPA released into the medium relative to the amount of DPA in the spores at 0 min.
These germination sites
that
different
results speed
suggest and
participate
judging
that
calcium
electrolyte in
when H-spores were treated
with
to
requirement
germination
from the fact
binding
that
speed
only
in
"special
germination.
and electrolyte germination
20 mM calcium
acetate
sites"
controls
Futhermore,
requirement
speed was recovered at 60°C for
60 min
Cdata not shown].
%rs
2. Electron indicate
photomicrographs 250 MI.
of intact
730
spore
(a),
and SDS-DTT spore
are
Vol. 128, No. 2, 1985
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
b (Fig. 2 -- Continued)
We carried sites"
out studies
to determine
using spore coat-lacking
consist
of spore coat,
membrane, and core
[Fig
outer 2aI.
the location
Spores of g. megaterium ATCC 19213
spores.
membrane, cortex,
germ cell
Spore coat and all
membrane could be removed by SDS-DTT treatment, spores),
consisting
of cortex,
of the "special
germ cell
wall,
100
wall,
or a part
inner
of the outer
and simple spores (SDS-DTT inner
membrane, and core,
loo-
s u al Ln
2
m
0
0
30
a
0
60
b
minutes Fig.
3.
mnd
Germination Ca[A1
of cation spores from
alanine plus phosphate buffer the
same
as
in
the
legend
of
exchanged SDS-DTT
(af or
Fig.
1.
731
60
30
minutes
SDS-DTT spores. N- (@), spores were germinated by alanine (bl, and the others
Hwere
Vol. 128, No. 2, 1985
were obtained slower
[Fig.
Zbl.
Although
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
speed of DPA release
in SDS-DTT spores compared with
germination 3) .
BIOCHEMICAL
pattern
was observed
tendency
in
between these two types of spores (Fig.
1,
When SDS-DTT spores were titrated
min, calcium,
of 15.8,
were subsequently
8.3,
converted
at 60°C for
60 min.
germination
patterns
from intact
spores (Fig.
similar
From these results, sites"
with
spores,
nitric
are not located
0.6 nmol/mg spore, to Ca-spores
with
H- and Ca-spores
to those observed
30
from spores at
respectively.
by treatment
Thus obtained
a similar
acid to pH 4.0 for
magnesium, and manganese were released
concentrations
acetate
intact
was significantly
H-spores 100 mM calcium showed
in H- and Ca-spores prepared
31. we can assert
with
confidence
that
the "special
cn the spore coat.
REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8.
Rode, L. J ., and Foster, J. W. Cl9622 Arch. Mikrobiol. 43, 201-212. 91, 1582-1588. Rode. L. J . . and Foster. J. W. Cl9661 J. Bacterial. Rode; L. J.; and Foster; J. W. Cl466i J. Bacterial. 911 1589-1593. Skomurski, J. F., and Vary, J. C. (1982] Biochim. Biophys. Acta. 731, 428-436. Nakatani, Y., Tmagawa, M., Nishihara, T., and Kondo, M. (1985) Microbial. Immunol. 29, 119-126. Vary, J. C. (1973) J. Bacterial. 116, 797-802. Lews, J. C. (1967) Anal. Biochem. 19, 327-337. Janssen, F. W., Lund, A. J., and Anderson, L. E. (1967) Science 127, 26-27.
132
128,
April
30,
No. 2, 1985
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1985
Pages
GERMINABILITY
OF COAT-LACKING
SPORES OF BACILLUS
728-732
MEGATERIUM
Y. Nakatani K. Tani, M. rmagawa, T. Nish:hara, and M. Kondo Department
of Environmental Chemistry and Microbiology Faculty of Pharmaceutical Sciences Osaka University 1-6 Yamadaoka, Suita, Osaka 565, Japan
Received March 4, 1985 SUMMARY: Upon treatment with acid, the germinability of both intact and coat-lacking spores of Bacillus meqaterium ATCC 19213 exhibited similar features. Namely, when the spores previously germinated by alanine in the presence of phosphate buffer were converted to H-spores by treatment with nitric acid, germination proceeded at a very low speed in a same germination medium. When H-spores converted to Ca-spores by treatment with calcium acetate and subsequently germinated, germination proceeded at a speed higher than that of native spores and occurred even in the absence of buffer. These results suggest that the site of exchangeable cations concerned with germina6ility must not exist in the coat. 0 1985 Academic Press, Inc.
INTRODUCTION: initiated
Spore
by alanine
electrolyte
(1).
facts
for
and inosine
in
when
electrolyte
suggest
the existence through
towards
germination
the
site
germination
located
in
inner rather
is
spore
coat
membrane than
C2,31.
*Abbreviations: dithiothreitol:.
spore
coat
coat
On the other role
(4).
DPA, dipicolinic
Using
728
sodium
(2).
These
upon
to clarify
which of the
studies
the initiation
SDS,
0006-291X/85 $1.50 Cowright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
recent
coat-lacking
acid;
useful
most
can be
an investigation
calcium
because hand,
in
acid
Rode and Foster that
acetate,
spores
has an effect
very
an aspect,
spore
with
Therefore, is
and suggested
has an important
the
which
calcium
these
by treatment
of calcium
the
with
Moreover,
of calcium.
From such
binding
lost.
ATCC 19213 can be
of non-specific
treated
of a component
localization
mechanism.
were
type
the binding
on the calcium
to the
was
megaterium
the presence
spores
to electrolyte-requiring
germinability geared
in Bacillus
However,
the requirement converted
germination
the studies
made
takes
part
calcium show
that
in
bound the
of germination spores,
we report
aoaecyl
sulfate;
here
DTT,
Vol.
128,
studies
No. 2, 1985
BIOCHEMICAL
conducted
participate
to determine
AND
whether
in germinability
BIOPHYSICAL
calcium
RESEARCH
binding
COMMUNICATIONS
to the
spore
coat
or not.
B. megaterium ATCC 19213 spores were grown in AND XFiTHODS: supplemented nutrient broth, except that the MnCl concentration was 0.1 mM, harvested, washed, lyophilized, and stored as pre&iously described 15). All references to spore weights are on a dry weight basis. Cation-exchanged spores were prepared as follow: spores (10 mg/ml) were incubated in a solution of 0.5% SDS-O.1 M DTT-0.1 M NaCl, pH 10.0 (6) at 31°C for 2 h, and washed 10 times by centrifugation (SDS-DTT spores); intact and SDS-DTT spores (10 mg/ml) were titrated to pH 4.0 with 0.01 N nitric acid for 120 and 30 min, respectively, and washed 4 times by centrifugation [H-spores); H-spores prepared from intact and SDS-DTT spores (10 mg/ml) were incubated in 100 mM calcium acetate at 60°C for 60 min, and washed 6 times by centrifugation [Ca-spores). Prepared spores were then lyophilized and stored in desiccator until use. Spores were heat activated at 60°C for 60 min, and then incubated in 30 mM sodium-potassium phosphate buffer CpH 8.0) containing 10 mM alanine, or in a solution of 10 mM alanine at 3O'C. At intervals, samples of germination exudate were taken by filtering the spore suspension through membrane filter. DPA content in the filtrate was determined by ultraviolet absorbance method c7). DPA content of the spores was measured by the methods of Janssen et al. C87. Spores were fixed, dehydrated, embedded, sectioned, stained, and then observed under an electron microscope as described previously (Nakatani, Y., Imagawa, M., Takubo, Y., Nishikawa, J., Nishihara, T., and Kondo. M., in submit), except that fixation with Os04 was carried out at 4'C for 48 h. MATERIALS
germinated alanine
by alanine
concentrations germinability
of N-spores with
of 11.2,
7.5,
by alanine
of that after
60 min when the mixture medium.
Hi in spores,
for
although
by the treatment
Moreover, alanine N-spores
and phosphate (Fig.
buffer
during
1). 129
and phosphate
was considered
low by exchanging
still
remained.
acetate
alone,
buffer.
without
rapid
that
cations
Ca-spores,
100 mM calcium
germination
was significantly
inosine, it
Therefore,
by alanine
the speed of DPA release
The
was less than
of alanine,
of H-spores with
60 min, could be germinated
from the spores at
100% of H-spores were
became very
germinability
to pH 4.0 for
low and the speed of DPA release
However,
of N-spores.
was used as a germination
obtained
acid
respectively.
and phosphate buffer
the response of spores to germinants with
nitric
and 5.4 nmol/mg spore,
of H-spores was considerably
one-fiftieth
buffer
titration
were
but not by
magnesium, and manganese were released
germination
germinated
in the presence of phosphate buffer,
During
alone.
120 min, calcium,
during
Spores of g. megaterium ATCC 19213 (N-spores)
AND DISCUSSION:
RESULTS
at 60°C
of Ca-spores by compared with
that
of
Vol. 128, No. 2, 1985
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0
0 0
60
30 minutes
0
30 minutes
b
60
Fig. 1. Germination of cation exchanged spores. N- (e), H- (O), and Ca[A) spores were germinated by alanine plus phosphate buffer (a) or alanine (b). DPA release was mesasured as described in the text and expressed as percent DPA released into the medium relative to the amount of DPA in the spores at 0 min.
These germination sites
that
different
results speed
suggest and
participate
judging
that
calcium
electrolyte in
when H-spores were treated
with
to
requirement
germination
from the fact
binding
that
speed
only
in
"special
germination.
and electrolyte germination
20 mM calcium
acetate
sites"
controls
Futhermore,
requirement
speed was recovered at 60°C for
60 min
Cdata not shown].
%rs
2. Electron indicate
photomicrographs 250 MI.
of intact
730
spore
(a),
and SDS-DTT spore
are
Vol. 128, No. 2, 1985
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
b (Fig. 2 -- Continued)
We carried sites"
out studies
to determine
using spore coat-lacking
consist
of spore coat,
membrane, and core
[Fig
outer 2aI.
the location
Spores of g. megaterium ATCC 19213
spores.
membrane, cortex,
germ cell
Spore coat and all
membrane could be removed by SDS-DTT treatment, spores),
consisting
of cortex,
of the "special
germ cell
wall,
100
wall,
or a part
inner
of the outer
and simple spores (SDS-DTT inner
membrane, and core,
loo-
s u al Ln
2
m
0
0
30
a
0
60
b
minutes Fig.
3.
mnd
Germination Ca[A1
of cation spores from
alanine plus phosphate buffer the
same
as
in
the
legend
of
exchanged SDS-DTT
(af or
Fig.
1.
731
60
30
minutes
SDS-DTT spores. N- (@), spores were germinated by alanine (bl, and the others
Hwere
Vol. 128, No. 2, 1985
were obtained slower
[Fig.
Zbl.
Although
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
speed of DPA release
in SDS-DTT spores compared with
germination 3) .
BIOCHEMICAL
pattern
was observed
tendency
in
between these two types of spores (Fig.
1,
When SDS-DTT spores were titrated
min, calcium,
of 15.8,
were subsequently
8.3,
converted
at 60°C for
60 min.
germination
patterns
from intact
spores (Fig.
similar
From these results, sites"
with
spores,
nitric
are not located
0.6 nmol/mg spore, to Ca-spores
with
H- and Ca-spores
to those observed
30
from spores at
respectively.
by treatment
Thus obtained
a similar
acid to pH 4.0 for
magnesium, and manganese were released
concentrations
acetate
intact
was significantly
H-spores 100 mM calcium showed
in H- and Ca-spores prepared
31. we can assert
with
confidence
that
the "special
cn the spore coat.
REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8.
Rode, L. J ., and Foster, J. W. Cl9622 Arch. Mikrobiol. 43, 201-212. 91, 1582-1588. Rode. L. J . . and Foster. J. W. Cl9661 J. Bacterial. Rode; L. J.; and Foster; J. W. Cl466i J. Bacterial. 911 1589-1593. Skomurski, J. F., and Vary, J. C. (1982] Biochim. Biophys. Acta. 731, 428-436. Nakatani, Y., Tmagawa, M., Nishihara, T., and Kondo, M. (1985) Microbial. Immunol. 29, 119-126. Vary, J. C. (1973) J. Bacterial. 116, 797-802. Lews, J. C. (1967) Anal. Biochem. 19, 327-337. Janssen, F. W., Lund, A. J., and Anderson, L. E. (1967) Science 127, 26-27.
132