Japan. J. Microbiol. Vol. 19(3), 187-192, 1975
Changes of Ultrastructure in Spore thiaminolyticus during Germination Kazuhito
WATABE
and
Masaomi
Coat of Bacillus and Outgrowth KONDO
Faculty of PharmaceuticalSciences,Kobe-Gakuin University,Kobe, and Faculty of PharmaceuticalSciences, Osaka University, Toyonaka (Received
for publication,
October
31, 1974)
ABSTRACT
least coat
Electron microscopic observation showed that the spore coat of Bacillus thiaminolyticus consisted four layers; a high electron dense outer spore coat layer with five prominent ridges, a middle layer including two layers of a high and a low electron density, and an inner spore coat layer
of at spore com
posing six to seven laminated layers. Rapid breakdown of the cortex and swelling of the core occurred in spores which were allowed to germinate by L-alanine for 45 min, whereas no change of surface feature was observed by scanning electron microscopy. Germination and outgrowth of spores in nutrient broth proceeded, being accompanied by morphological of the cortex and swelling of the core, the second the spore coat, and the last rupture of the spore
changes, degradation coat and
paper,
changes
of ultrastructure
of
the spore coat of B. thiaminolyticusduring germination and outgrowth are described. MATERIALSAND METHODS
The morphology of the spore coat of bacilli and clostridia has been studied ex tensively by many workers [1, 2, 9, 13, 14]. When germination has been completed and a new cell emerges, the spore coat is generally left behind as a collapsed membranous structure. The spore coat consists of two or three layers ; the coat of Bacillus megaterium spores consists of at least three characteristic components [5], and that of Bacillus cereus, Bacillus subtilis, Bacillus coagulans and Bacillus stearothermophilus spore is composed of two layers, an outer and an underlying laminated coat [11, 14]. In the previous paper [15] it was demon strated that L-alanine was incorporated mainly into a paracrystal fraction of the spore coat during an early stage of germina tion of Bacillus thiaminolyticus, suggesting that the coat layer of spore plays an important role in the initiation of germination. In the present
in three steps ; the first is a rapid breakdown of the inner layer at a prominent region emergence of a young vegetative cell.
Organism. B. thiaminolyticusMatsukawa et Misawa spores were produced on nutrient agar medium at 37 C for about 1 week as previously described [15]. Germination and outgrowth medium. For germination and outgrowth of spores, the following media were used. (i) Lyophilized spores (20 mg dry wt/10 ml) were heat activated at 65 C for 15 min and incubated in 100 ml of germination medium containing 10 mM L-alanine and 5 mM tris (hydroxymethyl)aminomethane-hydrochloride buffer(pH 7.8) at 37 C for 45 min. Germinated spores were harvested and washed by repeated cen trifugations. (ii) Heat-activated spores (40 mg dry wt/20 ml) were incubated in 200 ml of nutrient broth with shakings (120 rpm) at 37 C. Cells were harvested after incuba tion for 1, 2, and 4 hr and washed by repeated centrifugations. Figure 1 shows the time course of germination and outgrowth of spores, in which arrows indicate the times
in
Requests for reprints should be addressed to Dr. Kazuhito Watabe, Faculty of Pharmaceutical Sciences, Kobe-Gakuin University, Ikawadani-ma chi Arise, Tarumi-ku, Kobe 673, Japan. 187
K. WATABE
188
AND M. KONDO
when samples were removed for electron microscopic observation. Electron microscopy. Samples (2.0 to 5.0 ml) of spores, germinated spores, and outgrowing cells were fixed by addition of KMnO4 and phosphate buffer (pH 7.2) to a final concentration of 0.6% and 50 mM, respectively, or by adding a 4% solution of °sal to a final concentration of 1%. The KMnO4-fixed samples were kept at 2 C for 6 to 8 hr, whereas the 0s04-fixed samples
Fig.
1. of
Time
B.
were (•›) times
course
of germination
thiaminolyticus incubated or
spores. in
an
and
L-alanine
(10
in nutrient broth (•œ). Arrows when samples were removed
microscopic
outgrowth
Heat-activated
spores mM)
solution
indicate the for electron
were kept at 2 C for 3 days and then washed four times by centrifugation. Both types of cells were suspended in melted, warm agar (2%) and allowed to solidify. The resulting agar-embedded pellets were cut into little cubes (2 to 5 mm), and placed in 30% ethanol. The specimens were dehydrated through an ethanol series of 50, 70, 80, 95, and 100% and absolute acetone. Em bedding in Epon resin (Shell Oil Co., New York, N. Y.) was achieved by Luft's method [6]. Sections cut with glass knives on an LKB ultratome (L. K. B., Anerley Road, London S. E. 20) were mounted on carbon coated grids and stained with uranyl acetate [4] and lead citrate [12]. The various specimens were examined and photographed in a JEM type 120 electron microscope (JEOL, Japan) at an accelerating voltage of 120 kV. Observation of surface feature of spores or germinated spores was carried out as follows; samples were pipetted onto a circular polished-aluminum specimen stub in small droplets and allowed to air dry. The specimen was placed in a vacuum evaporator and a thick coating (40 to 50 nm) of goldpalladium (60/40 alloy) was evaporated onto a specimen at angles ranging from 35 to 40. The samples were examined in a JSM type U 3 scanning electron microscope (JEOL, Japan) at an accelerating voltage of 10 kV.
observation.
b
a
Fig.
2.
Scanning
germination
electron in
L-alanine
micrographs (10
mm),
of dormant showing
ridges
spores in
(a)
parallel
and to
germinated the
long
spores axis
of the
(b)
after
spores. •~20
45
min 000.
ULTRASTRUCTURE
OF BACTERIAL
RESULTS
Morphology of Spore Surface feature of spores envisaged by scanning electron microscopy is shown in Fig. 2-a. Ridges are seen to run parallel to the long axis of the spores, coalescing at its ends into a polygonal pore-like structure. Scanning observation also indicates an average size of 1.4 nm by 0.6 nm, with a length to width ratio of 2.3 : 1. Results observed by an electron micro scopic technique of thin sectioning of B. thiaminolylicus have previously been reported
SPORES
189
[8], but fine structure has remained obscure because of technical limitation inherent to the electron microscope. Figure 3-a and b shows cross sections of spores. A set of five ridges forms an outer most part of the spore coat. The coat of a KMnO4-fixed dormant spore (Fig. 3-a) consists of three layers ; a high electron-dense outer spore coat layer, an electron-transparent middle spore coat layer in which an electron-dense thick layer was sandwiched, and an inner spore coat layer composed of six to seven laminated components. An OsO4-fixed
cross scction
Fig.
3.
a,
fixed
ofspore
Ultrathin
with
layered
section
KMnal, spore
is illustrated
coat
of
showing
a spore a multi
structure'. •~ 75
Fig. 3. b, Ultrathin fixed with Os04, layered
spore
coat
section showing
of
000.
a spore multi
a
structure. •~ 85 000
1 AbbrCViations MC, middle GCW. germ
used
in
the
following
spore coat ; OMC, outer-middle cell wall. In all figures. markers
eleCtron spore indicate
micrograms coat ; IMC. 0.5 nm.
: C , COre ; CO, inner-middle
cortex ; OC, spore
coat
; IC,
outer
spore
coat ;
inner
spore
coat ;
190
K.
WATABE
AND M.
KONDO
in Fig. 3-b. Compared with the k MnO4fixed one, the only difference lays in the fine structure of the middle spore coat layer which is further separated by two components of a low electron-dense outer-middle and an inner-middle spore coat layer . The fine structure observed in a specimen prepared by
was disappearance of the cortex with swelling of the core (Fig. 4).
prefixation with glutaraldehyde and successive fixation with ()sal closely resembled that of the OsO4-fixed specimen described above (unpublished data) , the spore coat appearing to consist of at least four layers.
(Fig. 5). Section of an outgrowing spore in 1-hr preparation showed appearance of a transparent area under a ridge site of the outer spore coat (Fig. 5-a), and this break down of the inner structure eventually spread to every prominent site as seen in a 2-hr outgrowing cell (Fig. 5-b). The core swelled and then moved toward the crack region during this period. The inner coat structure largely disappeared, whereas the outermost electron-dense layer remained unaltered and also laminated components were seen in a late stage of the outgrowing , after 4-hr incubation (Fig. 5-c). A young vegetative cell wall surrounding the swollen core was well defined. Section of an outgrowing cell in a 4-hr preparation is also shown in Fig . 5-d. Although fine structure is not well discernible, the figure reveals that the spore coat is ruptured at an equatorial site and a
Changes of Ultrastructure during Outgrowth Morphological changes occurring during germination and outgrowth by incubation with nutrient broth were then examined
Changes of Ultrastructure during Germination To understand morphological changes which occur during L-alanine-induced ger mination, the surface feature and cross section of germinated spores were examined . As shown in Fig. 1, germination was complete in 20 min with an accompanying rapid reduction of optical density in the cell suspension. In this work germinated spores were prepared from cells incubated with L alanine for 45 min. Scanning electron micrograph of the germinated spores is shown in Fig. 2-b. There was no change in surface feature as compared with dormant spores. Section of a germinated spore fixed with KMnO4 revealed a coat structure consisting of three layers closely resembling that observed in the section of dormant spores, whereas a marked change observed
Fig.
4.
Ultrathin
section disappeared
of
a but
spore no
after change
45 in
young vegetative cell is now escaping through crevice, leaving an exuviae of the spore coat . Figure 5-e shows a remaining spore coat , in which the multilayered coat structure was well preserved and there is little difference in
min the
germination coat
structure
in was
1.-alanine seen
. •~
(10 74
000.
rim).
The cortex
ULTRASTRUCTURE
morphology
between
exuviae
of
the
OF BACTERIAL
out
specimen
growing spore and the spore coat of dormant cells. No crack was found around the outer
above.
layer,
panying
because
direction
of sectioning
of the
tion
SPORES
was These
and
different results
outgrowth morphological
a
× 45000
b
×40000
c
× 40000
d
× 25000
e
× 45000
191
from
that
described
suggest
that
germina
proceed changes
with
accom
which
Fig. 5. Ultrathin sections of outgrowing cells in nutrient broth in 1-hr (a), 2-hr (b), and 4-hr preparations (c, d, e), showing morphorogical changes in the spore coat during the developmental progress.
can
192
K. WATABE
AND M. KONDO
be classified into three steps ; the first is a rapid breakdown of the cortex and swelling of the core, the second degradation of the inner structure at a prominent region, and the last rupture of the spore coat and em ergence of a young vegetative cell.
rapid swelling and toward the breakage spore coat is ruptured cell emerges.
REFERENCES [ 1 ]
Fitz-James, Cytological
DISCUSSION
The ultrastructural components of B. thiaminolyticusspore are not markedly different from those found in various other bacterial endospores. Morphology of the spore coat closely resembles that of Bacillus popilliae [7] and Bacillus polymyxa[9, 10], and the spore coat consists of at least four layers; an electron-dense outer spore coat layer, a middle spore coat layer including two com ponents of an electron-dense and a trans parent layers, and an inner spore coat layer composed of six to seven laminated layers. It can be assumed that the different features in the middle spore coat layer between spores fixed with KMnO4 and those fixed with 0s04 is attributable to the different fixing agents. Kondo and Foster [5] demon strated three characteristic components of the spore coat of B. megaterium; an alkali soluble component, a paracrystal (keratin like substance) component, and a resistant fraction (phosphomuramyl polymer). They suggested that the paracrystal component was sandwiched between alkali-soluble and resistant fractions. Further study [3] sug gested that the outer coat was composed mainly of the resistant layer, and the alkali soluble and paracrystal fractions came from the inner layer. L-Alanine was incorporated into the spore coat, mainly into the para crystal fraction during an early stage of germination of B. thiaminolyticus[15]. Al though the relationship between morphology of the spore coat and the coat components remains unexplained, the incorporation site of L-alanine can be presumed to be the middle and/or the inner spore coat layer in view of the results reported by Kondo et al [3, 5]. Successive observation of thin sections during outgrowth in nutrient broth has indicated that breakdown of the spore coat first occurs at an inner part of a prominent site of the coat layer. This is followed by a
removal of the core region, and finally the and a young vegetative
strains
P. C., comparison
of
Bacillus
and
Young, spores
of megaterium.
I. E. 1959. of different
J.
Bacteriol.
78:
755-757. [ 2 ]
Holt,
S. C.,
parative forming
bacteria
Rev. [ 3 ]
and
Leadbetter,
ultrastructure
of
tions
[ 4 ]
: a freeze-etching
isolated
in
spore
II.
Bacteriol.
T., and relation
coat
Kondo, to the
of Bacillus
different
A., study
Vegetative
as compared
chem.
with
M. frac
megaterium.
and
mature
phage
bacterial
states.
J.
J.
nucleoids
Biophys.
Bio
4 : 671-678.
Kondo. M., electron
and Foster, microscope
from
Microbiol.
and Sechaud, of DNA-contain
normal
physiological
Cytol.
prepared
[ 6 ]
study.
its
E., Ryter, microscope
plasms.
DNA
and
Com spore
97 : 944-946.
Kellenberger, 1958. Electron ing
Nishihara, and
from
Bacteriol.
[ 5 ]
1969.
aerobic
33 : 346-378.
Kawasaki, C., 1969. Ultrastructure
J.
E. R. selected
coats
J. W. studies
of
1967. on
bacillus
Chemical fractions
spores.
J.
Gen.
47•@: 257-271.
Luft, embedding
J.•@ H.
1961. methods.
Improvements J. Biophys.
in epoxy Biochem.
resin Cytol.
9 : 409-414. [ 7 ]
Mitruka, and
B. M.,
Pepper,
refractile J. [ 8 ]
bodies,
Bacteriol.
and
Black,
S. H.,
Comparisons
spores
N. 1959. bacteria.
: 301-344.
(in
Murphy, features of scanning
R. N.,
1967.
of
of
Bacillus
cells,
popilliae.
94 : 759-765.
Mochizuki, of thiaminase 66
[ 9 ]
Costilow,
R. E.
Electron J. Kyoto
microscope Pref. Univ.
study Med.
Japanese)
J. A., and Campbell, Bacillus polymyxa electron
L. L. 1969. Surface spores as revealed by
microscopy.
J.
Bacteriol.
98 :
737-743. [ 10 ]
Murray, 1970. The
R. G. E., maturation
germination J. [ 11 ]
of
Microbiol. Ohye,
tion J. [ 12 ]
Cell
Biol. pH
and [ 14 ]
an J.
J.
Cell
into nation.
Biol.
1970. J.
Cell
Biol.
K.,
Canad.
1962.
Forma
of Bacillus
coagulans.
Japan.
of
of
17:
J.
of spore
Bacteriol.
formation
33
: 1-12.
579-592. T.,
and of
Incorporation
Bacillus
at
electron
D. F., and Murrell, W. G. and structure of bacterial
16:
I.
citrate in
208-212.
Cytology Appl.
lead
stain
studies
spores.
spores
use
Ichikawa,
Biochemical
bacterial
W. G.
spore
The
A. D., Ohye, composition
Watabe, 1974.
Murrell,
1963.
germination.
spores. [ 15 ]
spores.
electron-opaque
P. D.
Warth, 1963. The
polymyxa
of the
E.S. as
Walker,
and Marak, J. structure during
15 : 111-123.
microscopy. [ 13 ]
and
structure
Reynolds, high
Bacillus
M. M., cell wall
16 : 883-887. D. E.,
and
Hall, of
M. of
of 14C-e-alanine
thiaminolyticus
Microbiol.
Kondo,
germination
during
18 : 173-180.
germi
Changes of Ultrastructure in Spore thiaminolyticus during Germination Kazuhito
WATABE
and
Masaomi
Coat of Bacillus and Outgrowth KONDO
Faculty of PharmaceuticalSciences,Kobe-Gakuin University,Kobe, and Faculty of PharmaceuticalSciences, Osaka University, Toyonaka (Received
for publication,
October
31, 1974)
ABSTRACT
least coat
Electron microscopic observation showed that the spore coat of Bacillus thiaminolyticus consisted four layers; a high electron dense outer spore coat layer with five prominent ridges, a middle layer including two layers of a high and a low electron density, and an inner spore coat layer
of at spore com
posing six to seven laminated layers. Rapid breakdown of the cortex and swelling of the core occurred in spores which were allowed to germinate by L-alanine for 45 min, whereas no change of surface feature was observed by scanning electron microscopy. Germination and outgrowth of spores in nutrient broth proceeded, being accompanied by morphological of the cortex and swelling of the core, the second the spore coat, and the last rupture of the spore
changes, degradation coat and
paper,
changes
of ultrastructure
of
the spore coat of B. thiaminolyticusduring germination and outgrowth are described. MATERIALSAND METHODS
The morphology of the spore coat of bacilli and clostridia has been studied ex tensively by many workers [1, 2, 9, 13, 14]. When germination has been completed and a new cell emerges, the spore coat is generally left behind as a collapsed membranous structure. The spore coat consists of two or three layers ; the coat of Bacillus megaterium spores consists of at least three characteristic components [5], and that of Bacillus cereus, Bacillus subtilis, Bacillus coagulans and Bacillus stearothermophilus spore is composed of two layers, an outer and an underlying laminated coat [11, 14]. In the previous paper [15] it was demon strated that L-alanine was incorporated mainly into a paracrystal fraction of the spore coat during an early stage of germina tion of Bacillus thiaminolyticus, suggesting that the coat layer of spore plays an important role in the initiation of germination. In the present
in three steps ; the first is a rapid breakdown of the inner layer at a prominent region emergence of a young vegetative cell.
Organism. B. thiaminolyticusMatsukawa et Misawa spores were produced on nutrient agar medium at 37 C for about 1 week as previously described [15]. Germination and outgrowth medium. For germination and outgrowth of spores, the following media were used. (i) Lyophilized spores (20 mg dry wt/10 ml) were heat activated at 65 C for 15 min and incubated in 100 ml of germination medium containing 10 mM L-alanine and 5 mM tris (hydroxymethyl)aminomethane-hydrochloride buffer(pH 7.8) at 37 C for 45 min. Germinated spores were harvested and washed by repeated cen trifugations. (ii) Heat-activated spores (40 mg dry wt/20 ml) were incubated in 200 ml of nutrient broth with shakings (120 rpm) at 37 C. Cells were harvested after incuba tion for 1, 2, and 4 hr and washed by repeated centrifugations. Figure 1 shows the time course of germination and outgrowth of spores, in which arrows indicate the times
in
Requests for reprints should be addressed to Dr. Kazuhito Watabe, Faculty of Pharmaceutical Sciences, Kobe-Gakuin University, Ikawadani-ma chi Arise, Tarumi-ku, Kobe 673, Japan. 187
K. WATABE
188
AND M. KONDO
when samples were removed for electron microscopic observation. Electron microscopy. Samples (2.0 to 5.0 ml) of spores, germinated spores, and outgrowing cells were fixed by addition of KMnO4 and phosphate buffer (pH 7.2) to a final concentration of 0.6% and 50 mM, respectively, or by adding a 4% solution of °sal to a final concentration of 1%. The KMnO4-fixed samples were kept at 2 C for 6 to 8 hr, whereas the 0s04-fixed samples
Fig.
1. of
Time
B.
were (•›) times
course
of germination
thiaminolyticus incubated or
spores. in
an
and
L-alanine
(10
in nutrient broth (•œ). Arrows when samples were removed
microscopic
outgrowth
Heat-activated
spores mM)
solution
indicate the for electron
were kept at 2 C for 3 days and then washed four times by centrifugation. Both types of cells were suspended in melted, warm agar (2%) and allowed to solidify. The resulting agar-embedded pellets were cut into little cubes (2 to 5 mm), and placed in 30% ethanol. The specimens were dehydrated through an ethanol series of 50, 70, 80, 95, and 100% and absolute acetone. Em bedding in Epon resin (Shell Oil Co., New York, N. Y.) was achieved by Luft's method [6]. Sections cut with glass knives on an LKB ultratome (L. K. B., Anerley Road, London S. E. 20) were mounted on carbon coated grids and stained with uranyl acetate [4] and lead citrate [12]. The various specimens were examined and photographed in a JEM type 120 electron microscope (JEOL, Japan) at an accelerating voltage of 120 kV. Observation of surface feature of spores or germinated spores was carried out as follows; samples were pipetted onto a circular polished-aluminum specimen stub in small droplets and allowed to air dry. The specimen was placed in a vacuum evaporator and a thick coating (40 to 50 nm) of goldpalladium (60/40 alloy) was evaporated onto a specimen at angles ranging from 35 to 40. The samples were examined in a JSM type U 3 scanning electron microscope (JEOL, Japan) at an accelerating voltage of 10 kV.
observation.
b
a
Fig.
2.
Scanning
germination
electron in
L-alanine
micrographs (10
mm),
of dormant showing
ridges
spores in
(a)
parallel
and to
germinated the
long
spores axis
of the
(b)
after
spores. •~20
45
min 000.
ULTRASTRUCTURE
OF BACTERIAL
RESULTS
Morphology of Spore Surface feature of spores envisaged by scanning electron microscopy is shown in Fig. 2-a. Ridges are seen to run parallel to the long axis of the spores, coalescing at its ends into a polygonal pore-like structure. Scanning observation also indicates an average size of 1.4 nm by 0.6 nm, with a length to width ratio of 2.3 : 1. Results observed by an electron micro scopic technique of thin sectioning of B. thiaminolylicus have previously been reported
SPORES
189
[8], but fine structure has remained obscure because of technical limitation inherent to the electron microscope. Figure 3-a and b shows cross sections of spores. A set of five ridges forms an outer most part of the spore coat. The coat of a KMnO4-fixed dormant spore (Fig. 3-a) consists of three layers ; a high electron-dense outer spore coat layer, an electron-transparent middle spore coat layer in which an electron-dense thick layer was sandwiched, and an inner spore coat layer composed of six to seven laminated components. An OsO4-fixed
cross scction
Fig.
3.
a,
fixed
ofspore
Ultrathin
with
layered
section
KMnal, spore
is illustrated
coat
of
showing
a spore a multi
structure'. •~ 75
Fig. 3. b, Ultrathin fixed with Os04, layered
spore
coat
section showing
of
000.
a spore multi
a
structure. •~ 85 000
1 AbbrCViations MC, middle GCW. germ
used
in
the
following
spore coat ; OMC, outer-middle cell wall. In all figures. markers
eleCtron spore indicate
micrograms coat ; IMC. 0.5 nm.
: C , COre ; CO, inner-middle
cortex ; OC, spore
coat
; IC,
outer
spore
coat ;
inner
spore
coat ;
190
K.
WATABE
AND M.
KONDO
in Fig. 3-b. Compared with the k MnO4fixed one, the only difference lays in the fine structure of the middle spore coat layer which is further separated by two components of a low electron-dense outer-middle and an inner-middle spore coat layer . The fine structure observed in a specimen prepared by
was disappearance of the cortex with swelling of the core (Fig. 4).
prefixation with glutaraldehyde and successive fixation with ()sal closely resembled that of the OsO4-fixed specimen described above (unpublished data) , the spore coat appearing to consist of at least four layers.
(Fig. 5). Section of an outgrowing spore in 1-hr preparation showed appearance of a transparent area under a ridge site of the outer spore coat (Fig. 5-a), and this break down of the inner structure eventually spread to every prominent site as seen in a 2-hr outgrowing cell (Fig. 5-b). The core swelled and then moved toward the crack region during this period. The inner coat structure largely disappeared, whereas the outermost electron-dense layer remained unaltered and also laminated components were seen in a late stage of the outgrowing , after 4-hr incubation (Fig. 5-c). A young vegetative cell wall surrounding the swollen core was well defined. Section of an outgrowing cell in a 4-hr preparation is also shown in Fig . 5-d. Although fine structure is not well discernible, the figure reveals that the spore coat is ruptured at an equatorial site and a
Changes of Ultrastructure during Outgrowth Morphological changes occurring during germination and outgrowth by incubation with nutrient broth were then examined
Changes of Ultrastructure during Germination To understand morphological changes which occur during L-alanine-induced ger mination, the surface feature and cross section of germinated spores were examined . As shown in Fig. 1, germination was complete in 20 min with an accompanying rapid reduction of optical density in the cell suspension. In this work germinated spores were prepared from cells incubated with L alanine for 45 min. Scanning electron micrograph of the germinated spores is shown in Fig. 2-b. There was no change in surface feature as compared with dormant spores. Section of a germinated spore fixed with KMnO4 revealed a coat structure consisting of three layers closely resembling that observed in the section of dormant spores, whereas a marked change observed
Fig.
4.
Ultrathin
section disappeared
of
a but
spore no
after change
45 in
young vegetative cell is now escaping through crevice, leaving an exuviae of the spore coat . Figure 5-e shows a remaining spore coat , in which the multilayered coat structure was well preserved and there is little difference in
min the
germination coat
structure
in was
1.-alanine seen
. •~
(10 74
000.
rim).
The cortex
ULTRASTRUCTURE
morphology
between
exuviae
of
the
OF BACTERIAL
out
specimen
growing spore and the spore coat of dormant cells. No crack was found around the outer
above.
layer,
panying
because
direction
of sectioning
of the
tion
SPORES
was These
and
different results
outgrowth morphological
a
× 45000
b
×40000
c
× 40000
d
× 25000
e
× 45000
191
from
that
described
suggest
that
germina
proceed changes
with
accom
which
Fig. 5. Ultrathin sections of outgrowing cells in nutrient broth in 1-hr (a), 2-hr (b), and 4-hr preparations (c, d, e), showing morphorogical changes in the spore coat during the developmental progress.
can
192
K. WATABE
AND M. KONDO
be classified into three steps ; the first is a rapid breakdown of the cortex and swelling of the core, the second degradation of the inner structure at a prominent region, and the last rupture of the spore coat and em ergence of a young vegetative cell.
rapid swelling and toward the breakage spore coat is ruptured cell emerges.
REFERENCES [ 1 ]
Fitz-James, Cytological
DISCUSSION
The ultrastructural components of B. thiaminolyticusspore are not markedly different from those found in various other bacterial endospores. Morphology of the spore coat closely resembles that of Bacillus popilliae [7] and Bacillus polymyxa[9, 10], and the spore coat consists of at least four layers; an electron-dense outer spore coat layer, a middle spore coat layer including two com ponents of an electron-dense and a trans parent layers, and an inner spore coat layer composed of six to seven laminated layers. It can be assumed that the different features in the middle spore coat layer between spores fixed with KMnO4 and those fixed with 0s04 is attributable to the different fixing agents. Kondo and Foster [5] demon strated three characteristic components of the spore coat of B. megaterium; an alkali soluble component, a paracrystal (keratin like substance) component, and a resistant fraction (phosphomuramyl polymer). They suggested that the paracrystal component was sandwiched between alkali-soluble and resistant fractions. Further study [3] sug gested that the outer coat was composed mainly of the resistant layer, and the alkali soluble and paracrystal fractions came from the inner layer. L-Alanine was incorporated into the spore coat, mainly into the para crystal fraction during an early stage of germination of B. thiaminolyticus[15]. Al though the relationship between morphology of the spore coat and the coat components remains unexplained, the incorporation site of L-alanine can be presumed to be the middle and/or the inner spore coat layer in view of the results reported by Kondo et al [3, 5]. Successive observation of thin sections during outgrowth in nutrient broth has indicated that breakdown of the spore coat first occurs at an inner part of a prominent site of the coat layer. This is followed by a
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