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Journal of Bacteriology, October 2000, p. 5556-5562, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Analysis of Nucleoid Morphology during Germination
and Outgrowth of Spores of Bacillus Species
Katerina
Ragkousi,1
Ann E.
Cowan,1,2
Margery
A.
Ross,1 and
Peter
Setlow1,*
Department of
Biochemistry1 and Center for Biomedical
Imaging Technology,2 University of
Connecticut Health Center, Farmington, Connecticut 06032
Received 17 April 2000/Accepted 5 July 2000
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ABSTRACT |
After a few minutes of germination, nucleoids in the great majority
of spores of Bacillus subtilis and Bacillus
megaterium were ring shaped. The major spore DNA binding
proteins, the 
/
-type small, acid-soluble proteins (SASP),
colocalized to these nucleoid rings early in spore germination, as did
the B. megaterium homolog of the major B. subtilis chromosomal protein HBsu. The percentage of ring-shaped
nucleoids was decreased in germinated spores with lower levels of
/-type SASP. As spore outgrowth proceeded, the ring-shaped
nucleoids disappeared and the nucleoid became more compact. This change
took place after degradation of most of the spores' pool of major
/-type SASP and was delayed when /-type SASP degradation
was delayed. Later in spore outgrowth, the shape of the nucleoid
reverted to the diffuse lobular shape seen in growing cells.
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INTRODUCTION |
During the process of sporulation in
Bacillus subtilis the sporulating cell is divided into two
unequally sized compartments, the larger mother cell and the smaller
forespore. Each compartment of the sporulating cell ultimately contains
a complete chromosome, but the patterns of transcription from the
genomes in these two compartments are quite different (8).
The gross structures of the nucleoids in the two compartments are also
very different. After formation of the septum separating the mother
cell and forespore, the forespore nucleoid appears extremely compact,
while the nucleoid in the mother cell retains the diffuse lobular
appearance of the nucleoid in growing cells (19). The cause
of the forespore nucleoid condensation is not clear but may be in part
a reflection of the small size of the forespore compartment. Several
hours later, the forespore nucleoid decondenses slightly and takes on
the appearance of a ring-shaped or doughnut-like structure. This change
is due to the binding of forespore DNA by a group of small,
acid-soluble proteins (SASP) of the / type, which have been
localized on the ring-shaped nucleoid (14). These proteins
are the products of a multigene family that is expressed only in the
forespore just prior to the conversion of the forespore nucleoid to a
ring-shaped structure. There are two major /-type SASP in
B. subtilis, termed SASP- and -, as well as two minor
proteins of this type (21, 22). Deletion of the genes
encoding SASP- and - (termed sspA and -B,
respectively) results in spores (termed 
) lacking ~85% of total /-type SASP. The
spores are much more sensitive than
are wild-type spores to a variety of treatments, including heat and UV
radiation, and extensive work has shown that /-type SASP saturate
the spore chromosome and protect spore DNA from many types of damage
(21, 22). During sporulation of the
strain, the forespore nucleoid condenses normally but
does not assume the ring-shaped structure seen in wild-type forespores (14). These findings indicate that /-type SASP are
essential for formation of the ring-shaped nucleoid structure. However, the precise function of this nucleoid structure is not clear, since
sporulation of strains appears
relatively normal, although not completely so (21,
23; B. Setlow, K. A. McGinnis, and P. Setlow,
unpublished data). In addition to /-type SASP, the major
chromosomal protein in growing B. subtilis cells, HBsu, has
also been localized to the ring-shaped forespore nucleoid
(17).
Although the structure of the forespore nucleoid has been studied to
some degree, much less is known about the structure of the nucleoid
early in spore germination. One study presented a compelling electron
micrograph of a germinating spore, suggesting that the nucleoid is ring
shaped in this stage of development as well (15, 16).
However, this structure has not been studied in detail and there has
not been any assessment of the contribution of /-type SASP to the
nucleoid structure in the germinated spore. Their contribution is of
special interest since /-type SASP are degraded early in spore
germination, and thus the nucleoid structure should revert to that
found in growing cells. In addition, analysis of the nucleoid structure
early in spore germination and comparison with that in the forespore
may give further insight into the nucleoid structure in the dormant
spore, since it has been impossible to directly assess the structure of
the nucleoid in the dormant spore, which is relatively impermeable to
the fixatives and stains used in microscopy. In this work we report the
analysis of the structures of the nucleoids of germinating spores, with or without various /-type SASP, and analyze the locations of /-type SASP and the Bacillus megaterium homolog of
HBsu in the germinating spore. These analyses were carried out using
both B. subtilis, for which strains lacking gpr
as well as strains containing and lacking a variety of /-type
SASP genes are available, and B. megaterium, for which a
gpr mutant is available (9, 10, 18, 24). The
major reason for the use of B. megaterium was that the
larger size of its spores relative to those of B. subtilis
greatly simplifies microscopy and, in particular, localization of
proteins by immunofluorescence microscopy.
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MATERIALS AND METHODS |
Bacterial strains used and preparation of spores.
The
bacterial strains used in this work are listed in Table
1. B. megaterium strains are
derivatives of strain QMB1551; B. subtilis strains are
derivatives of strain PS832. B. megaterium strains were
sporulated in supplemented nutrient broth (5) at 30°C, and
B. subtilis strains were sporulated in 2× SG medium (12) at 37°C. Spores were purified and stored as described
previously (5, 12). Antibiotics were added to media at the
following concentrations: 3 µg/ml for chloramphenicol and 10 µg/ml
for kanamycin.
Spore germination and SASP extraction and analysis.
Spores
(1 to 5 mg [dry weight]/ml) in water were heat shocked for 15 min at
60°C (B. megaterium) or 30 min at 70°C (B. subtilis). After being cooled in ice, spores were germinated at
0.13 mg [dry weight]/ml at either 30°C (B. megaterium)
or 37°C (B. subtilis) in Tris-Spizizen's minimal medium
(3) supplemented with 10 mM L-alanine. In one
experiment this medium was supplemented with either 10 mM KCN or 50 µg of chloramphenicol per ml.
For measurements of SASP levels, samples (65 ml) were harvested by
centrifugation at various times during spore germination
and outgrowth
and the pellet was lyophilized. The dry spores were
disrupted in a
dental amalgamator (Wig-L-Bug) with glass beads
(100 mg) as the
abrasive, SASP were extracted with cold 3% acetic
acid, and the
supernatant fluid was dialyzed and lyophilized as
described previously
(
12). The dry residue was dissolved in
a small volume of 8 M
urea and subjected to polyacrylamide gel
electrophoresis at low pH, and
the gel was stained with Coomassie
blue as described previously
(
12).
Fixation, staining, and microscopy of germinated and outgrowing
spores.
For fixation and DNA staining, samples (250 µl) of
germinating spores were collected at various times and mixed with 250 µl of fixative solution (3% [wt/vol] paraformaldehyde and 0.4%
[vol/vol] glutaraldehyde in HEPES-buffered saline [pH 7.05] [per
liter, 16 g of NaCl, 0.74 g of KCl, 0.27 g of
Na2 HPO4 · 2H2O, 2 g of dextrose, 10 g of HEPES]). After 15 min at room temperature,
fixation was continued on ice for 50 min. The samples were then washed twice by centrifugation with phosphate-buffered saline (PBS) (10 mM
NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM
KH2PO4 [pH 7.4]) and resuspended in 100 µl
of GTE (5 mM glucose, 25 mM Tris-HCl, 10 mM EDTA [pH 8.0]). Aliquots
(10 µl) of this suspension were mixed with 10 µl of 2-µg/ml
4',6'-diamino-2-phenylindole (DAPI) and held at room temperature for 15 min before being loaded on poly-L-lysine-coated multiwell
slides (ICN Biomedicals). Slides were washed twice with PBS, mounted
using a Slow Fade Anti Fade kit (Molecular Probes), and stored at
4°C.
For fixation and immunostaining of SASP and the HBsu homolog in
B. megaterium, samples of germinating spores were collected
and fixed as described previously (
6,
14,
17) but freshly
prepared lysozyme (3 mg/ml) in GTE was added and incubation was
for 15 min at room temperature before cells were distributed in
the wells of
multiwell slides. Some of these cells were stained
with guinea pig
antiserum against HBsu and rabbit antiserum against
/
-type SASP,
the guinea pig immunoglobulin G (IgG) was detected
with fluorescein
isothiocyanate (FITC)-labeled secondary antibody,
the rabbit IgG was
detected with biotinylated goat anti-rabbit
IgG and then with
indocyanine (Cy3)-conjugated streptavidin, and
the cells were mounted
as described previously (
18). Other cells
were stained with
10 µl of 2-µg/ml DAPI or 2.4 nM quinolinium,
1,1'-[1-3-propanediylbis [(dimethylimino) - 3,1 - propanediyl]
]bis [ 4 - [ ( 3 - methyl - 2 (3H) - benzoxazolylidene)methyl]]-tetraiodide
(YOYO) (Molecular Probes, Eugene, Oreg.), to visualize DNA. After
15 min at room temperature, slides were washed twice with PBS
and mounted
as described
above.
Slides were viewed on a Zeiss (Thornwood, N.Y.) Axiovert 100 microscope
using either a 63×, 1.4 numerical aperture oil immersion
Plan
apochromat lens or a 63×, 1.25 numerical aperture oil immersion
Plan
neofluar lens and a 1.6× optivar lens and appropriate filter
blocks.
Images were collected using a Roper Scientific (Tuscon,
Ariz.)
PXL-cooled charge-coupled device camera and processed using
Adobe
Photoshop version 5.0. Measurements of spore, nucleoid,
or SASP ring
dimensions were by pixel counting using SCION software
downloaded from
the National Institutes of Health. Confocal microscopy
was done using a
Zeiss LSM410 confocal
microscope.
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RESULTS |
Appearance of nucleoids in germinated spores.
Examination of
spores of either B. megaterium or B. subtilis in
the first minute of germination after staining with DAPI revealed that
the majority of nucleoids in the germinated spores were ring shaped
(Fig. 1; Table
2; and data not shown). While all the
spores had not yet initiated germination by 2 min, only the nucleoids of germinated spores were stained by DAPI. Analysis of DAPI-stained germinated spores by confocal fluorescence microscopy further revealed
that the nucleoid is indeed ring shaped and not a hollow sphere (data
not shown). It was somewhat surprising that we observed such a high
percentage of ring-shaped nucleoids in germinated spores, as depending
on the precise orientation of the nucleoid in the microscope field, not
only rings but also sharp lines of DAPI staining should be observed.
While some of the latter structures were seen (Fig. 1), the percentage
was very low. Possibly the spores, which are not perfect spheres, affix
to microscope slides in nonrandom orientations, which increases the
percentage of spores which show ring-shaped nucleoids in a microscope
field. Analysis of the nucleoid rings in germinated spores indicated
that these were generally spherical (Fig. 1) and that the average
diameters of the nucleoids were similar in both B. megaterium and B. subtilis spores, even though the
germinated B. megaterium spores themselves had almost twice
the diameter of the germinated B. subtilis spores (Table
3). Note that the treatment conditions of
the samples in Fig. 1 and the upper half of Table 3 are the same but
different from those used in the lower half of Table 3.
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FIG. 1.
Analysis of nucleoid shape during germination and
outgrowth of wild-type spores. Wild-type spores of B. megaterium were germinated; samples were taken at the times
indicated, fixed, and stained with DAPI; and nucleoids were visualized
by fluorescence microscopy as described in Materials and Methods. The
arrow points to a bar-shaped nucleoid. The scale bar is 5 µm.
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Previous work has shown that formation of the ring-shaped nucleoid in
the developing forespore is dependent on the synthesis
of
/
-type
SASP (
14), so it was of obvious interest to analyze
the
nucleoid structure in germinated spores of
B. subtilis
strains
with various
/
-type SASP contents. As expected, the
frequency
of ring-shaped nucleoids was greatly reduced in germinated
spores, which lack the two major
/
-type SASP, although a few
such ring-shaped nucleoids were
observed (Table
2). Germinated
spores lacking either SASP-
or
SASP-
(
and
spores, respectively)
exhibited an intermediate level of ring-shaped
nucleoids, while
germinated
spores with a plasmid
overexpressing a normally minor
/
-type
SASP (termed
SspC
wt) to the level of SASP-
plus -
in wild-type
spores (
21,
22,
24) had levels of ring-shaped nucleoids
close to that in wild-type
spores. However, when the
/
-type SASP
overexpressed in
spores to levels
similar to those of SASP-
plus -
in wild-type
spores was
SspC
Ala, a variant of SspC
wt that binds both
poorly and relatively nonproductively to DNA
(
24;
C. S. Hayes and P. Setlow, unpublished data), the level
of
ring-shaped nucleoids in germinated spores was only about one-third
of
that in wild-type spores (Table
2), although this value was
significantly higher than that in
spores.
Changes in nucleoid structure during spore germination and
outgrowth.
Analysis of the nucleoid appearance throughout spore
germination and outgrowth revealed that with both B. megaterium and B. subtilis the nucleoid rings
disappeared relatively rapidly, with very few ring-shaped nucleoids
remaining 20 to 60 min after the initiation of spore germination (Fig.
1 and 2). For these two species, >95%
of the spores had initiated spore germination after ~30 min (data not
shown). When the ring-shaped nucleoids present in the first minute of
germination disappeared, the nucleoids became slightly smaller and more
condensed (Fig. 1; Table 3). The size and appearance of these more
condensed nucleoids were essentially identical to those of the
nucleoids in the great majority of
spores immediately after the initiation of spore germination (Table 3
and data not shown). There should of course be intermediates in the
conversion of the ring-shaped nucleoids to the more compact forms, but
we did not observe any such intermediates, possibly because the
identification of such structures is beyond the resolution of our
microscopy. The condensed nucleoids present in germinated spores were
also similar to those in developing forespores prior to the synthesis
of /-type SASP (19). Eventually as outgrowth of
wild-type spores proceeded, the nucleoid shape changed once again to
the more diffuse lobular appearance of the vegetative cell nucleoid
(Fig. 1). In the medium used in this experiment, wild-type B. megaterium spores underwent their first cell division at ~250 to
~300 min, as determined by septal staining with wheat germ agglutinin
coupled to Oregon Green (data not shown). Analyses using B. megaterium further showed that in spores germinating as described
in Materials and Methods but with KCN to block ATP production or
chloramphenicol to block protein synthesis, the nucleoid rings
disappeared at the same rate as in spores germinating without these
inhibitors (data not shown).
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FIG. 2.
Percentage of ring-shaped nucleoids in germinating
spores of B. megaterium (A) and B. subtilis (B).
Spores were germinated, fixed, stained, and examined by microscopy for
ring-shaped nucleoids as described in Materials and Methods. From 220 to 1,000 stained nucleoids were examined at each time point. The
symbols used and the spores analyzed were as follows: in panel A,  ,
B. megaterium QMB1551 (wild type), and  , B. megaterium PS1551 (gpr); and in panel B, , B. subtilis PS832 (wild-type), and , B. subtilis PS1029
(gpr).
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Analysis of levels of major
/
-type SASP showed that in
B. megaterium and
B. subtilis spores, these proteins were
gone (
95%)
by 15 min and 40 min after the initiation of germination,
respectively
(data not shown), similar to what has been found
previously (
17).
Thus, the nucleoid rings can persist for at
least some time without
high levels of major
/
-type SASP. When
nucleoids were examined
in germinated
gpr spores which lack
the major protease initiating
SASP degradation during spore germination
(
18), the nucleoid
rings persisted longer after the
initiation of spore germination
than in wild-type spores (Fig.
1 and
2A); this was particularly
striking with the
B. megaterium
gpr spores. Note that
B. megaterium gpr spores do not
undergo their first cell division until >500
min after the start of
spore germination (data not shown). Analysis
of the levels of major
/
-type SASP in germinated
gpr spores
showed that these
proteins persisted at significant levels at
least 150 min after the
initiation of germination of
B. megaterium gpr spores but
that they were largely, if not completely, gone
from
B. subtilis
gpr spores after 60 to 80 min (reference
18 and
data not shown). Presumably
B. subtilis spores have higher
levels than
B. megaterium spores of proteases other than the
gpr gene product that can degrade
/
-type SASP during
spore
germination.
Analysis of possible nucleoid-associated proteins in germinated
spores.
Analyses using a number of techniques have shown that
/-type SASP are associated with the nucleoid in developing
forespores, dormant spores, and spores early in germination (4,
14, 18, 20). Thus, it was of obvious interest to examine the
distribution of /-type SASP in germinated spores. Not
surprisingly, immunostaining of /-type SASP early in spore
germination gave ring-shaped structures with both B. megaterium and B. subtilis gpr and wild-type spores (Fig. 3 and data not shown). These
ring-shaped structures were absent in
B. subtilis spores early in germination
(data not shown). Previous work has shown that the /-type SASP
rings colocalize with DNA rings in the developing forespore
(14). However, we were unable to effectively stain
germinated spore DNA when the samples had been immunostained for
/-type SASP, even when a number of fluorescent DNA stains were
used. The reason for this failure is not clear; possibly the antibody
reaction with /-type SASP alters the DNA sufficiently to preclude
DNA staining. However, the size of the /-type SASP rings in
germinated spores was identical to that of the nucleoid rings (Table
3), consistent with the colocalization of /-type SASP and DNA in
the germinated spore, as occurs in the developing forespore and the
dormant spore (4, 14, 20).
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FIG. 3.
Localization of /-type SASP in germinated
gpr spores by immunofluorescence microscopy. Spores of
B. megaterium (PS1551 gpr) or B. subtilis (PS1029 gpr) were germinated for 10 min,
fixed, treated, immunostained for /-type SASP, and visualized by
fluorescence microscopy as described in Materials and Methods. The
scale bar is 1 µm.
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To provide further evidence for the colocalization of
/
-type SASP
and DNA in germinated spores, we also examined the location
of the HBsu
homolog in germinated spores of
B. megaterium. HBsu
is the
major protein on the nucleoid in vegetative cells of
B. subtilis, where it covers ~5% of the DNA (
8,
11,
17). HBsu
also colocalizes with
/
-type SASP in the
developing forespore
and is almost certainly on dormant spore DNA,
where again it is
present in sufficient amounts to cover ~5% of the
genome and likely
modulates some of the effects of
/
-type SASP on
DNA properties
(
17). Immunostaining of either wild-type or
gpr germinated spores
of
B. megaterium for its
HBsu homolog again revealed ring-shaped
structures (Fig.
4). As expected, these rings largely
colocalized
with
/
-type SASP rings (Fig.
4) and the HBsu homolog
rings were
also the same size as the
/
-type SASP rings (Table
3),
suggesting
that
/
-type SASP and the HBsu homolog are together on
the ring-shaped
nucleoid early in the germination of wild-type
B. megaterium spores.
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FIG. 4.
Localization of /-type SASP and the HBsu homolog
in germinated B. megaterium spores by immunofluorescence
microscopy. Wild-type (WT) and gpr (PS1551) spores of
B. megaterium were germinated for 2 and 10 min,
respectively, fixed, treated, immunostained for both /-type SASP
and HBsu, and examined by fluorescence microscopy as described in
Materials and Methods. The panels labeled "/-type SASP" show
the Cy3 images, the panels labeled "HBsu" show the FITC images, and
the panels labeled "overlay" show the merged images from the Cy3
and FITC images. The scale bar is 5 µm.
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DISCUSSION |
The work reported in this communication allows a number of
conclusions. First, the nucleoid has a ring-shaped structure early in
spore germination. The nucleoid structure in the dormant spore has not
been determined because of the impermeability of the dormant spore core
to fixatives and stains. Consequently, the demonstration that
germinating spores contain a ring-shaped nucleoid, as well as previous
work indicating that developing forespores contain ring-shaped
nucleoids (14), strongly suggests that dormant spores also
contain ring-shaped nucleoids. Second, both /-type SASP and HBsu
homologs are on the ring-shaped nucleoid of the germinating spore.
Again, this has not been demonstrated for the dormant spore, whose core
is refractory to immunolocalization techniques. Since /-type SASP
and HBsu and its homologs are on the ring-shaped nucleoid in developing
forespores (4, 14, 17), our new findings strongly suggest
that both types of protein are also on the nucleoid in the dormant
spore. Third, the size of the ring-shaped nucleoids is the same in
germinated spores of both B. megaterium and B. subtilis. Given the significantly larger diameter of germinated B. megaterium spores, this observation indicates that the
size of the ring-shaped nucleoid is not determined directly by the cell's diameter. However, the precise mechanisms determining the size
as well as the shape of the ring-shaped nucleoid in germinated spores
and developing forespores are not clear. Fourth, high levels of major
/-type SASP are required for maximal levels of ring-shaped nucleoids, as was suggested previously based on analyses of ring-shaped nucleoids in developing forespores (14). However, we also
observed a low level of ring-shaped nucleoids in germinated
spores and ring-shaped nucleoids
persisted in germinated wild-type spores well after the great majority
of /-type SASP had been degraded. These observations suggest that
high levels of major /-type SASP are not essential for the
formation and maintenance of at least some ring-shaped nucleoids. There
are a number of minor /-type SASP in spores (21, 22),
and some of these bind more tightly to DNA than do the major
/-type SASP (C. S. Hayes and P. Setlow, unpublished data).
It is possible that in some cells
there are enough of these minor /-type SASP to trigger formation
of ring-shaped nucleoids in developing forespores, and presumably this
process persists in germinated spores. Interestingly, a small
percentage of Escherichia coli cells have also been reported to contain ring-shaped nucleoids under some conditions (13).
Some minor /-type SASP, especially ones that bind tightly to DNA,
may also be degraded much more slowly by GPR during spore germination
than the major /-type SASP (C. S. Hayes and P. Setlow, unpublished data), and again it may be these minor proteins which cause
the retention of the ring-shaped nucleoid structure after the great
majority of the major /-type SASP have been degraded. Alternatively, it may be that degradation of a small amount of major
/-type SASP is extremely slow, perhaps because a fraction of
these proteins is bound very tightly to particular regions of the
chromosome; again, this small amount of residual /-type SASP may
be sufficient to preserve the structure of the ring-shaped nucleoid.
Any of these scenarios or even some combination of them would then
explain the long lag between the degradation of most of the major
/-type SASP during spore germination and the disappearance of
ring-shaped nucleoids. This lag is seen most dramatically with spores
of B. megaterium, as >50% of germinated spores retained ring-shaped nucleoids 25 min after the start of spore germination, when
95% of all /-type SASP are gone. However, /-type SASP degradation does appear to be essential for the ultimate loss of
ring-shaped nucleoids during spore germination, as this loss was
greatly slowed during germination of gpr spores but was not affected by inhibition of ATP production or protein synthesis. Thus,
neither transcription nor translation is needed for loss of ring-shaped
nucleoids during spore germination.
Previous work has shown that levels of total major /-type SASP
are decreased somewhat in spores lacking the gene for either SASP-
or - (10), and presumably this is the reason for the decrease in the percentages of germinated spores of
and strains with ring-shaped nucleoids.
Overproduction of SspCwt is sufficient to saturate the
spore chromosome with this protein, and this largely reverses the
phenotypic effects of the deletion of genes coding for SASP- and
- (24). Since SspCwt binds to spore DNA and
has the same effects on DNA properties in vivo as major /-type
SASP (21, 22, 24), it is not surprising that germinated
spores with pSspCwt
have near wild-type levels of ring-shaped nucleoids. However, it was
somewhat surprising that overexpression of SspCAla resulted
in a significant amount of ring-shaped nucleoids in germinated
spores. This SspC variant has an
Ala-for-Gly substitution in a residue that is conserved in /-type
SASP, and SspCAla does not restore UV and heat resistance
to spores and does not cause the
change in DNA structure and properties caused by binding of
SspCwt or other wild-type /-type SASP (21, 22,
24). However, SspCAla does bind to DNA (C. S. Hayes and P. Setlow, unpublished data), and possibly this is sufficient
to promote formation of some ring-shaped nucleoids.
While high levels of /-type SASP are clearly essential for
maximal formation of ring-shaped nucleoids in spores, if not for their
maintenance, it seems likely that other proteins may also be involved
in the formation of these structures. Two such proteins are HBsu and
its B. megaterium homolog, which are associated with the
ring-shaped nucleoids in developing forespores (17) and
germinated spores as shown in this work. Unfortunately, HBsu is an
essential protein in B. subtilis (11), so its
role in the formation of ring-shaped nucleoids in spores cannot be
easily tested. Additional candidates for a role in formation of
ring-shaped nucleoids in spores are minor SASP which are not related to
/-type SASP (1). These proteins are present in spores
at levels lower than those of /-type SASP but still at quite
substantial levels. While these minor SASP have not been shown to be
DNA binding proteins, many are basic proteins and at least two have
some as yet unknown role in spore outgrowth (1, 2).
Interestingly, loss of either of these two proteins slows outgrowth of
wild-type but not spores, as might
be expected if these minor proteins altered the spore's nucleoid
structure. It will be of interest to analyze spore nucleoid structures
in strains lacking some of these minor SASP.
While knowing the constituents in and the structure of the ring-shaped
spore nucleoid is clearly of interest, the function of this unusual
nucleoid structure is of even more interest. At present no specific
function can be ascribed to this structure, although it is not
absolutely essential for either sporulation or spore germination and
outgrowth, as strains sporulate and
spores germinate and grow out.
However, the time for return of
spores to vegetative growth is significantly longer than that for
wild-type spores (9). Some of this time difference may be
due to the lack of production of amino acids through degradation of
/-type SASP in spores, but
even in very rich media, spores
take longer to grow out than wild-type spores (9). Possibly
the ring-shaped nucleoid structure somehow facilitates optimal
transcription during spore germination and outgrowth. If this is the
case, one might also expect some defect in sporulation efficiency or
kinetics in strains. While
sporulation of strains is
qualitatively similar to that of wild-type strains (9),
recent work has indeed suggested that there may be some differences
between the sporulation of wild-type and that of
strains (23; B. Setlow,
K. A. McGinnis, and P. Setlow, unpublished data). Consequently, it
might be worthwhile to analyze sporulation of
strains quantitatively, as it is difficult to imagine
that such a structure as the ring-shaped nucleoids of spores has no
effect on gene expression. This work is in progress.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from the National Institutes
of Health (GM19698).
Microscopy was performed at the Center for Biomedical Imaging
Technology at the University of Connecticut Health Center, except for
analysis of the colocalization of the /-type SASP and the HBsu
homolog, which required the microscope of Timothy Hla. We are grateful
to Lotte Pedersen and Jason Kirk for assistance and advice and to
members of the Setlow lab for suggestions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, University of Connecticut Health Center, Farmington, CT 06032. Phone: (860) 679-2607. Fax: (860) 679-3408. E-mail:
setlow{at}sun.uchc.edu.
| |
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Journal of Bacteriology, October 2000, p. 5556-5562, Vol. 182, No. 19
0021-9193/00/$04.00+0
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Top
Abstract
Introduction
