JOURNAL OF BACTERIOLOGY, Oct. 2008, p. 6741–6748
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 190, No. 20
Characterization of Spores of Bacillus subtilis That Lack Most Coat Layers?
Sonali Ghosh,1Barbara Setlow,1Paul G. Wahome,1† Ann E. Cowan,1,2Marco Plomp,3
Alexander J. Malkin,3and Peter Setlow1*
Department of Molecular, Microbial and Structural Biology1and Center for Cell Analysis and Modeling,2University of
Connecticut Health Center, Farmington, Connecticut 06030-3305, and Chemistry, Materials, Earth and Life Sciences Directorate,
Lawrence Livermore National Laboratory, Livermore, California 945513
Received 30 June 2008/Accepted 18 August 2008
Spores of Bacillus subtilis have a thick outer layer of relatively insoluble protein called the coat, which
protects spores against a number of treatments and may also play roles in spore germination. However,
elucidation of precise roles of the coat in spore properties has been hampered by the inability to prepare spores
lacking all or most coat material. In this work, we show that spores of a strain with mutations in both the cotE
and gerE genes, which encode proteins involved in coat assembly and expression of genes encoding coat
proteins, respectively, lack most extractable coat protein as seen by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, as well as the great majority of the coat as seen by atomic force microscopy. However, the cotE
gerE spores did retain a thin layer of insoluble coat material that was most easily seen by microscopy following
digestion of these spores with lysozyme. These severely coat-deficient spores germinated relatively normally
with nutrients and even better with dodecylamine but not with a 1:1 chelate of Ca2?and dipicolinic acid. These
spores were also quite resistant to wet heat, to mechanical disruption, and to treatment with detergents at an
elevated temperature and pH but were exquisitely sensitive to killing by sodium hypochlorite. These results
provide new insight into the role of the coat layer in spore properties.
Spores of various Bacillus species are metabolically dormant
and extremely resistant to a variety of harsh treatments, in-
cluding heat, radiation, and many toxic chemicals (37). This
extreme resistance is the main reason that spores are major
causative agents of food spoilage and food-borne disease and
why spores of Bacillus anthracis are a potential biological war-
fare agent. Spore resistance is due to a variety of factors, but a
significant one is the spore coat. The coat is the outer layer of
spores of a number of Bacillus species and consists primarily of
protein, with ?70 different individual proteins in the coat
of Bacillus subtilis spores, many of which are cross-linked (7, 8,
10). Most of the latter proteins are components of the coats
only, although a few coat proteins also have significant roles in
coat assembly (7, 8, 10, 12). The coat layer is only semiperme-
able, generally allowing passage of molecules of ?5 kDa to
interior layers, in particular the spore’s inner membrane,
where receptors that sense the presence of nutrient molecules
that trigger spore germination are located (7, 10, 36). Because
of its permeability properties, the spore coat is very important
in preventing access of exogenous lytic enzymes such as ly-
sozyme from gaining access to the spore’s peptidoglycan (PG)
cortex, located beneath the coat layer, and the germ cell wall
beneath the cortex (7, 10, 13). This is important in spore
survival, since hydrolysis of cortex and germ cell wall PG by
lysozyme can cause spore lysis. The coat is also important in
the resistance of spores to many, albeit not all, reactive chem-
icals (37). The precise mechanism whereby the coat protects
against reactive chemicals is not known, but coat proteins may
react with and detoxify such chemicals before they can gain
access to and damage more sensitive targets further in the
spore’s interior. The spore coat also appears to contain en-
zymes such as superoxide dismutase and perhaps catalase that
may assist in detoxification of some reactive toxic chemicals, as
well as the CotA laccase, which can contribute to spore hydro-
gen peroxide resistance (7, 10).
In addition to a role in spore resistance, the spore coat has
been suggested to play roles in spore germination. Thus, some
proteins suggested to be important in allowing permeation of
nutrient germinant molecules into the interior regions of the
spore are coat proteins (36). At least one enzyme, CwlJ, im-
portant in lysis of the B. subtilis spore’s PG cortex during
germination is also in the spore coat, perhaps at the cortex-coat
boundary (2, 4). Indeed, proper assembly of CwlJ in B. subtilis
spores requires the coat protein GerQ (30). Some of the sec-
ond redundant B. subtilis spore cortex-lytic enzyme, SleB, also
appears to be located at the coat-cortex boundary (4).
Most studies probing the importance of the coat in various
spore properties have used B. subtilis spores with coats that are
defective because of either chemical removal of much coat
protein by extraction with detergents at high pH or the lack of
CotE, a protein essential for assembly of many coat proteins as
well as the outer coat layer (7, 10). However, both cotE and
chemically decoated spores retain much coat protein, most
notably as an insoluble “rind” that is also extremely resistant to
a variety of digestive enzymes (7, 10, 13). Therefore, it would
be most helpful if spores lacking all or almost all coat proteins
were available to more rigorously test the role of the coat in
spore properties. GerE is a DNA binding protein acting in the
mother cell compartment late in sporulation which, in addition
to having other activities, positively regulates the expression of
genes coding for a number of proteins in the spore coat that
* Corresponding author. Mailing address: Department of Molecular,
Microbial and Structural Biology, University of Connecticut Health Cen-
ter, Farmington, CT 06030-3305. Phone: (860) 679-2607. Fax: (860) 679-
3408. E-mail: firstname.lastname@example.org.
† Present address: Wadsworth Center, 120 New Scotland Avenue,
Albany, NY 12208.
?Published ahead of print on 22 August 2008.
are resistant to extraction at a high pH with detergent plus a
reducing agent; these proteins constitute the insoluble fraction
of the spore coat (8, 10). gerE mutants also make spores in
which much of the coat can be readily lost, and it has been
reported that cotE gerE spores appear to lack visible coats (6,
21, 38). Consequently, in this communication we describe the
generation of a cotE gerE strain and the properties of the cotE
gerE spores that appear to lack the majority of coat proteins.
MATERIALS AND METHODS
Strains used and spore preparation. The strains used in this work are isogenic
derivatives of strain PS832, a prototrophic laboratory derivative of strain 168.
Strain PS533 (35) (wild type) carries plasmid pUB110, which encodes resistance
to kanamycin (Kmr; 10 ?g/ml). PS3328 (24) carries a tetracycline resistance (Tcr;
10 ?g/ml) cassette replacing the majority of the cotE coding sequence. Strain
PS4149 carries a spectinomycin resistance (Spr; 100 ?g/ml) cassette replacing
much of the gerE coding sequence (see below), and strain PS4150 carries the cotE
and gerE deletion mutations from strains PS3328 and PS4149.
For construction of strain PS4149, the DNA region from bp ?1 to ?282
relative to the translation start site of gerE was PCR amplified from chromosomal
DNA of strain PS832 using primers with additional base pairs that included an
EcoRI site in the forward primer and a PstI site in the reverse primer (all primer
sequences are available upon request). The PCR product was purified, digested
with EcoRI and PstI, and cloned between the EcoRI and PstI sites in plasmid
pJL74 (18) to generate plasmid pJL74gerE1. A second DNA fragment encom-
passing bp ?181 to ?469 relative to the gerE translation start site was PCR
amplified from chromosomal DNA of strain PS832 using primers with additional
base pairs, including a NotI site in the forward primer and a BamHI site in the
reverse primer. This PCR product was purified and cloned between the NotI and
BamHI sites in plasmid pJL74gerE1, giving plasmid pJL74gerE2 with fragments
upstream and downstream of gerE flanking a Sprresistance cassette. Cloning of
the correct fragments and the proper orientation of these fragments in
pJL74gerE2 were confirmed by PCR. Plasmid pJL74gerE2 was linearized with
XhoI and used to transform strain PS832 to Spr. The replacement of much of the
gerE coding sequence with a Sprcassette in one Sprtransformant, termed strain
PS4149, was confirmed by PCR. Chromosomal DNA from strain PS4149 was
then used to transform strain PS3328 to a SprTcrphenotype, and the expected
loss of much of the gerE coding sequence in one transformant (termed strain
PS4150) was again confirmed by PCR.
Spores of all strains were prepared at 37°C on 2? SG medium agar plates
without antibiotics as described previously (23). After 2 to 3 days at 37°C, plates
were held at 23°C for 2 to 4 days to allow further lysis of growing or sporulating
cells, and then spores were scraped from plates and purified as described previ-
ously (23). All spores used in this work were free (?98%) of growing or sporu-
lating cells or cell debris. In some cases, spores were purified even further by
centrifugation through a solution of 50% Nycodenz, as described previously, to
remove cell debris (5).
Analysis of spore coat proteins. Coat proteins were extracted from spores at an
optical density at 600 nm (OD600) of ?25 for 2 h at 70°C in 1% sodium dodecyl
sulfate (SDS)–0.1 M NaOH–0.1 M NaCl–0.1 M dithiothreitol (1). The decoated
spores were washed as described previously (1), the material extracted was run
on SDS-polyacrylamide gel electrophoresis (PAGE) using a 10% acrylamide gel
(14), and the gel was stained with Coomassie brilliant blue.
Chemically decoated wild-type spores and intact gerE, cotE, and cotE gerE
spores at an OD600of ?10 in 1 ml of 20 mM Tris-HCl (pH 8) were digested with
hen egg white lysozyme (20 ?g/ml) for 1 h at 37°C, followed by addition of
pancreatic DNase to 10 ?g/ml and further incubation for 20 min to reduce the
viscosity of the extract. The extracts were then centrifuged in a microcentrifuge
for 5 min, the pellet fraction was washed twice with 20 mM Tris-HCl (pH 8), and
the final pellets were prepared for either differential interference contrast (DIC)
microscopy or electron microscopy (EM) as described below. In other experi-
ments, this final pellet was suspended in 0.5 ml of 25 mM Tris-HCl buffer (pH 8)
and incubated for 6 h at 37°C with 50 ?g/ml pronase or boiled for 20 min with
Analysis of spore properties. Atomic force microscopy (AFM) on intact spores
was carried out and images were collected using a Nanoscope IV AFM (Veeco
Instruments, Santa Barbara, CA) operated in tapping mode as described previ-
ously (26–28). For DIC microscopy, pellets from lysozyme-digested spores ob-
tained as described above were suspended in 25 to 50 ?l of 25 mM KPO4(pH
7.4)–0.1 M NaCl, 5-?l aliquots were applied to agarose-coated slides, and the
slides were imaged using DIC optics on a Zeiss LSM510 laser scanning micro-
scope with a 63-by-1.4 numerical aperture planapochromat lens. For EM, pellets
from lysozyme-digested spores obtained as described above were fixed, pro-
cessed, sectioned, and photographed as described previously (9).
The core wet densities of spores with or without prior decoating were deter-
mined as described previously (19, 29) by banding in equilibrium density gradi-
ents of Nycodenz with intact wild-type spores labeled with ruthenium red added
to all density gradients as an internal standard to correct for slight differences
between different gradients (39; K. Griffiths and P. Setlow, unpublished results).
Spore resistance to wet heat at ?90°C was determined using spores in water at
an OD600of 1. At various times, the heated spores were diluted 1/100 in 23°C
water and then diluted further, aliquots were spotted on LB medium (25) agar
plates containing an appropriate antibiotic, plates were incubated for ?24 h at
37°C, and colonies were counted. Further incubation gave no increase in colony
Spore resistance to sodium hypochlorite was determined essentially as de-
scribed previously (40) by incubation of spores at an OD600of 1 at 23°C in 50 mM
KPO4buffer (pH 7.0) plus a 1/105dilution of commercial sodium hypochlorite
(Sigma; 10 to 13% available chlorine). At various times, aliquots were diluted
1/10 in 1% sodium thiosulfate; this dilution was incubated for at least 10 min at
23°C, samples were diluted further in water, and numbers of survivors were
determined as described above. Spore resistance to mechanical abrasion in liquid
was determined as described previously (11).
Spore germination. Spores were germinated following heat activation (30 min;
75°C) of spores at an OD600of 10 in water. Spores were germinated at an OD600
of ?1 and at 37°C in 25 mM Tris-HCl (pH 8)–1 mM L-alanine. The progress of
germination was assessed by measuring the OD600of the cultures and was also
checked by phase-contrast microscopy. Analysis of spore germination with do-
decylamine did not use heat-shocked spores and was carried out with spores at
an OD600of ?2 at 40°C in 20 mM KPO4buffer (pH 7.5)–1 mM dodecylamine
(33). Germination of spores with dodecylamine was assessed by measuring the
OD270of the supernatant fluid from 1-ml aliquots of the germinating culture,
which monitors the release of dipicolinic acid (DPA) from the spores, an early
event in spore germination. Analysis of spore germination with the 1:1 chelate of
Ca2?and DPA (Ca-DPA) again used spores that were not heat shocked and was
carried out in 60 mM Ca-DPA–25 mM Tris-HCl (pH 8) at 23°C (24). The
progress of spore germination was assessed by phase-contrast microscopy. Spore
germination was also assessed by the ability of the spores of the various strains
to form colonies on nutrient plates with appropriate antibiotics as described
Generation of severely coat-defective spores. While spores
of strains with mutations in either cotE or gerE have defective
coats, as evidenced most notably by the sensitivity of these
spores to lysozyme, these spores do retain significant amounts
of coat protein (7, 10). However, these two mutations likely
result in defective spore coats by different mechanisms: a cotE
mutation, which eliminates a protein essential for the assembly
of many coat proteins as well as the outer coat layer, and a gerE
mutation, which eliminates the expression of a number of coat
protein genes, including some coding for proteins in both the
inner and outer coat layers, as well as derepressing the expres-
sion of other proteins (7, 10, 43). Thus, it seemed worthwhile
to examine spores of a cotE gerE strain for the presence of any
residual coat, especially since spores of a ?cotE strain that also
carry a point mutation in gerE are reported to lack a visible
coat (6, 32, 38). Consequently, we constructed a strain with
deletions of both cotE and gerE and examined the spores of
this strain for coat material. Strikingly, the spores of such a
strain had very little coat protein extractable at a high pH with
SDS plus a reducing agent, as determined by SDS-PAGE of
extracts from intact spores, in contrast to the large amount of
protein extracted from gerE and from cotE spores (Fig. 1).
However, there was some protein extracted from the cotE gerE
spores (Fig. 1).
6742 GHOSH ET AL.J. BACTERIOL.
Analysis by AFM also showed the presence of significant
coat material in cotE and gerE spores, as expected (Fig. 2a to
d). However, the great majority of cotE gerE spores appeared
to lack the obvious coat material present on wild-type, cotE,
and most gerE spores, most notably the prominent outer
ridges, outermost amorphous and rodlet layers, and inner coat
crystalline layers seen previously (3, 27; M. Plomp, A. M.
Carroll, P. Setlow, and A. J. Malkin, unpublished results), as
the outer surfaces of the great majority of the cotE gerE spores
appeared to be quite smooth (Fig. 2e and f). Pellets of purified
cotE gerE spores were also almost white, in contrast to the
brown to salmon color of pellets of wild-type, cotE, and gerE
spores (data not shown). In addition, there was a significant
amount of darkly pigmented material in the top layer of initial
pellets from sporulating cotE gerE cells harvested by centrifu-
gation that was not seen with sporulating cells of the other
three strains (data not shown). This pigmented material may
be in part coat material that misassembled in the cytoplasm of
the sporulating cotE gerE cells and was easily removed during
spore purification. Alternatively, this material may be an ag-
gregate that was generated by the action of a misassembled
coat protein, perhaps by CotA, which can act as a laccase (10).
As a further check on the presence of coat material, in
particular the presence of the insoluble material that has been
termed a rind and is seen with cotE or decoated wild-type
spores treated with lysozyme (12), the spores of these two
strains as well as gerE spores were treated with lysozyme, and
the insoluble material was purified and examined by EM and
DIC microscopy, the latter a light microscopic technique sim-
ilar in many respects to phase-contrast microscopy (Fig. 3 and
4). As expected (7, 10, 12), DIC microscopy of material re-
maining after centrifugation and washing of lysozyme digests
FIG. 1. SDS-PAGE of coat extracts from spores of various strains.
Purified spores were extracted with decoating solution, an aliquot (10
?l) of the extract was run on SDS-PAGE, and the gel was stained as
described in Materials and Methods. The samples were from spores of
strains PS533 (wild-type), PS3328 (cotE), PS4149 (gerE), and PS4150
(cotE gerE). The numbers to the left of lane 1 give the migration
position of molecular mass standards, in kDa.
FIG. 2. AFM analyses of spores of various strains. Purified spores
of various strains were analyzed by AFM and photographed as de-
scribed in Materials and Methods. The spores analyzed were from
strains PS533 (wild-type) (a), PS3328 (cotE) (b), PS4149 (gerE) (c and
d), and PS4150 (cotE gerE) (e and f). Bars, 500 nm. The asterisks (e)
indicate a few spores that retained obvious coat material.
FIG. 3. DIC micrographs of spores with or without coat defects
after digestion with lysozyme and DNase. Spores of strains PS533
(wild-type) (a), PS3328 (cotE) (b), PS4149 (gerE) (c), and PS4150
(cotE gerE) (d) were digested, and the material remaining after diges-
tion was examined by DIC microscopy as described in Materials and
Methods. The PS533 spores had been chemically decoated prior to
lysozyme treatment, and the arrowheads (b) point to a few spores that
were not digested with lysozyme. Bar, 20 ?m (all images are at the
VOL. 190, 2008PROPERTIES OF SEVERELY COAT-DEFICIENT SPORES 6743
revealed that the lysozyme digestion left behind an insoluble
rind that is almost certainly derived largely from the insoluble
proteins in the spore coat (Fig. 3a to c). Surprisingly, there was
also what appeared to be rind material left behind after ly-
sozyme digestion of cotE gerE spores (Fig. 3d), and this mate-
rial was not destroyed by digestion with pronase or boiling with
SDS (data not shown). The sizes of the rinds from wild-type
and cotE spores appeared to be similar in DIC microscopy
(Fig. 3a and b), but the gerE spore rinds appeared to be
smaller, with the cotE gerE spore rinds appearing even smaller
(Fig. 3c and d).
EM of the rind material from the cotE and decoated wild-
FIG. 4. Electron micrographs of rind material generated by lysozyme and DNase digestion of spores with or without coat defects. Spores of
strains PS533 (wild-type) (a), PS3328 (cotE) (b), PS4149 (gerE) (c), and PS4150 (cotE gerE) (d) were digested, and rind material was isolated, fixed,
and examined by EM as described in Materials and Methods. Bars, 1 ?m (all figures are at the same magnification). Arrows indicate thin rind
material in gerE spores (c) and cotE gerE rinds that collapsed upon themselves (d).
6744GHOSH ET AL.J. BACTERIOL.
type spores revealed relatively thick rinds that largely retained
the shape of the intact spores (Fig. 4a and b). Most rinds
generated from gerE spores were similar in thickness to the
rinds from the cotE and decoated wild-type spores (Fig. 4c).
However, the gerE spore rinds appeared less rigid, as many
were deformed (Fig. 4c); this deformation is most likely the
reason that the gerE spore rinds appeared to be smaller than
wild-type and cotE spore rinds by DIC microscopy. Surpris-
ingly, EM of insoluble material remaining after lysozyme di-
gestion of cotE gerE spores indicated that these spores did have
rinds (Fig. 4d). However, these rinds were significantly thinner
than those from cotE, gerE, or decoated wild-type spores, and
again, almost all of the cotE gerE rinds did not retain the spore
shape, with many having collapsed almost completely (Fig. 4d).
Again, this is most likely the reason that the cotE gerE spore
rinds appeared to be smaller than rinds from other types of
spores in DIC microscopy. In any event, it appears that while
cotE gerE spores have much less coat material than wild-type,
cotE, or gerE spores, they do retain some coat protein.
Viability and germination of coat-deficient spores. An obvi-
ous question about spores that lack most coat material is
whether these spores are viable. Analysis of the viability of the
cotE gerE spores on nutrient plates indicated that these spores
had essentially the same viability as wild-type spores and that
both gerE and cotE spores also had similar viability (Table 1).
The nearly identical viability of spores of these four strains
indicated that these spores could germinate with comparable
efficiencies. However, the viability data did not indicate how
fast these various spores germinated. Consequently, we mea-
sured the germination of these spores with the nutrient germi-
nant L-alanine by monitoring the OD600of germinating cul-
tures (Fig. 5A). As reported previously, the germination of
both cotE and gerE spores was slower than that of wild-type
spores (7, 21, 43). Germination of the cotE gerE spores was
also slower than that of wild-type spores but was actually faster
than that of cotE spores. The observation that cotE gerE spores
germinated only about twofold more slowly than wild-type
spores with L-alanine was also obtained by monitoring spore
germination by measuring the release of the dormant spore’s
large depot of Ca-DPA, an early event in spore germination
(data not shown).
In addition to specific nutrients, spores can also be triggered
to germinate by a number of nonnutrient agents; two of these
are the cationic surfactant dodecylamine and exogenous Ca-
DPA. When endogenous Ca-DPA release was monitored to
assess spore germination, cotE gerE spores germinated signif-
icantly faster with dodecylamine than did wild-type spores, as
did cotE and gerE spores (Fig. 5B). In contrast, while wild-type
spores germinated ?90% in 2 h with exogenous Ca-DPA, as
determined by phase-contrast microscopy, ?5% of cotE gerE
spores germinated in 2 h with Ca-DPA (data not shown).
Similarly, cotE and gerE spores also did not germinate with
exogenous Ca-DPA (data not shown). The lack of germination
of cotE spores with Ca-DPA was not unexpected, since CwlJ,
the enzyme whose action on the spore cortex is triggered by
Ca-DPA, does not assemble into the spore coat in cotE strains
(2, 4, 30). Presumably this is also the case in gerE strains.
Resistance properties of cotE gerE spores. While the cotE
gerE spores germinated reasonably well with several germi-
nants, it was possible that loss of most of the coat might
drastically alter the resistance properties of these spores. How-
ever, there was only a relatively small effect on spore resistance
to wet heat due to loss of most of the spore coat, as the D88°C
value (time for inactivation of 90% of spores) decreased only
about threefold in cotE gerE spores compared to the value for
wild-type spores (Fig. 6A). The heat resistances of cotE and
TABLE 1. Viability of spores of various strainsa
CFU/ml at OD600of 1.0
PS533 (wild type)
PS4150 (cotE gerE)
1.2 ? 108
1.4 ? 108
1.1 ? 108
1.3 ? 108
1.2 ? 108
1.3 ? 108
1.0 ? 108
9 ? 107
aAliquots of purified intact or chemically decoated spores were spotted on
plates with appropriate antibiotics, plates were incubated, and colonies were
counted as described in Materials and Methods.
FIG. 5. L-Alanine (A) and dodecylamine (B) germination of spores with or without coat proteins. (A) Spores of various strains were heat
shocked and germinated with L-alanine, and the OD600of cultures was monitored to assess spore germination, as described in Materials and
Methods. The OD600falls ?60% upon completion of wild-type spore germination, and this value was used to calculate the degree of spore
germination at various time points. (B) Spores of various strains were incubated with dodecylamine, and DPA release was monitored to assess
spore germination, as described in Materials and Methods. E, PS533 (wild-type); F, PS3328 (cotE); ‚, PS4149 (gerE); Œ PS4150 (cotE gerE).
VOL. 190, 2008 PROPERTIES OF SEVERELY COAT-DEFICIENT SPORES6745
gerE spores were identical to that of wild-type spores and
slightly less, respectively (Fig. 6A).
A major factor determining the resistance of spores of B.
subtilis and other Bacillus species to moist heat is the water
content of the spore core, with a higher core water content
giving spores with lower moist-heat resistance (37). Determi-
nation of the wet density of the core of the spores of these four
strains by equilibrium density gradient centrifugation of de-
coated spores indicated that spores of all four strains had very
similar core wet densities, and thus essentially identical core
water contents, although the core water content of the cotE
gerE spores was the highest of the spores of the four strains
examined, consistent with their lower wet-heat resistance
While there were no large differences in the resistance of
wild-type and coatless spores to moist heat, this was expected
not to be the case for resistance to many toxic chemicals.
Previous work has shown that coat proteins are important in
protecting spores against a number of chemicals, including
sodium hypochlorite, chlorine dioxide, and ozone, and cotE or
chemically decoated wild-type spores are much more sensitive
to these agents than are intact wild-type spores (37, 40, 41).
This was also seen in the current work when extremely dilute
sodium hypochlorite was used, as cotE spores were more sen-
sitive than wild-type spores, and gerE spores were even more
sensitive (Fig. 6B). However, the cotE gerE spores were even
more sensitive than the gerE spores, with ?0.01% survival after
1 min of treatment. The killing of the cotE gerE spores by this
sodium hypochlorite concentration was actually slightly faster
than the killing of vegetative cells of strain PS533 (data not
shown). In contrast to the greater sensitivity to sodium hypo-
chlorite of the spores with various coat defects, these coat-
defective spores were resistant to treatment with SDS at a high
pH and temperature, as this treatment, which is used to re-
move much spore coat protein, had only a minimal effect on
the viability of even cotE gerE spores (Table 1).
In addition to having resistance to moist heat and chemicals,
dormant spores are also more resistant to mechanical disrup-
tion than are germinated spores or growing cells (11). The
factors providing spore resistance to mechanical disruption are
not known, but one such factor has been suggested to be the
highly insoluble rind of coat protein, which is retained even in
cotE spores. However, examination of spore killing by mechan-
ical disruption, a process that has been shown to be due almost
certainly to actual disruption of the spores (11), indicated that
spores of the wild-type, cotE, gerE, and cotE gerE strains ex-
hibited almost identical rates of killing (data not shown).
The cotE gerE spores described in this report appear to be
the most severely coat-defective yet stable B. subtilis spores
reported to date. The spore coat’s normal outer layers as well
as the more inner rodlet layer are absent from these cotE gerE
spores, and they retain little detergent-extractable coat pro-
tein, although they do contain some. This observation is con-
sistent with previous work that reported the extraction of sev-
eral coat proteins from cotE gerE spores, although the gerE
mutation used in the previous work is most likely a point
mutation (22, 32, 38). It is, however, notable that the amount
of detergent-extractable coat protein in our cotE gerE spores is
significantly less than that reported by others. Whether this is
due to differences in the gerE mutations used or to differences
in the B. subtilis strains, sporulation conditions, or spore puri-
fication regimens is not clear. Indeed, B. subtilis spores pre-
pared on plates, as was done in this work, are reported to have
less extractable coat protein than spores prepared in liquid, the
method used for spore preparation in much other work (31). In
any event, the most notable finding about our cotE gerE spores
is that they appear to retain a very thin layer of detergent-
FIG. 6. Resistance of spores with and without coat defects to moist heat (A) and sodium hypochlorite (B). Spores of various strains were
incubated in water at 88°C or sodium hypochlorite, and spore viability was determined as described in Materials and Methods. E, PS533
(wild-type); F, PS3328 (cotE); ‚, PS4149 (gerE); Œ, PS4150 (cotE gerE).
TABLE 2. Core wet density of spores of strains with coat defectsa
Core wet density,
PS533 (wild type)...................................................................1.382 (34)
PS3328 (cotE).........................................................................1.380 (34)
PS4149 (gerE).........................................................................1.379 (35)
PS4150 (cotE gerE)................................................................1.376 (36)
aPurified spores of various strains were decoated, and their core wet densities
were determined as described in Materials and Methods.
bValues in parentheses are percentages of core wet weight that were water,
calculated as described previously (20).
6746 GHOSH ET AL.J. BACTERIOL.
insoluble protein that is resistant to protease digestion, al-
though this structure does not appear to be as rigid as the
wild-type spore coat. Presumably, much of the thin coat rem-
nant in these cotE gerE spores is composed of cross-linked
proteins, although the identity of these proteins is not clear.
The combination of a cotE and a gerE mutation almost cer-
tainly does not block synthesis of a number of coat proteins,
although the lack of GerE abolishes synthesis of some (8, 10,
42, 43). Indeed, we observed that pigmented material likely
derived from or generated by misassembled coat protein ac-
cumulated in cultures of fully sporulated cotE gerE cells, but
this material was not associated with the mature spores. Pre-
sumably the combination of the cotE and gerE mutations pre-
vented the adherence of the normal outer layers of the coat to
the spore, either because the coat misassembles or because it
lacks some essential protein(s) or an essential coat modifica-
tion crucial for adherence. This is clearly a topic for future
investigation, as is the precise composition of the insoluble
coat material remaining in cotE gerE spores.
It was notable that the cotE gerE spores germinated reason-
ably well with the nutrient germinant L-alanine, suggesting that
most coat protein is not essential for nutrient germination.
Previous work has indicated that a gerE mutation has major
effects on spore germination with nutrients (6, 7, 10, 21), but
we did not see this with either our gerE or cotE gerE spores.
The reason(s) for the better germination of our gerE spores is
not clear, although we note that (i) the genetic background for
our strains is different from that used previously, and (ii) the
gerE mutation used in our work is a deletion mutation, in
contrast to the gerE point mutation used in other work. How-
ever, the cotE gerE spores did not germinate with Ca-DPA.
The explanation for this result is most likely that the cortex
lytic enzyme CwlJ is absent from cotE gerE spores, as it is from
cotE spores. CwlJ is essential for B. subtilis spore germination
with Ca-DPA (2, 24). Presumably the second redundant cortex
lytic enzyme, SleB, remains in the cotE gerE spores and is
sufficient for the completion of their germination (4), although
the absence of CwlJ from these spores may be at least in part
the reason for their slower germination with L-alanine. Since
gerE spores also did not germinate with Ca-DPA, we expect
that these spores also lack CwlJ.
In contrast to their slower germination with L-alanine, the
cotE gerE spores actually released DPA faster than wild-type
spores when germination was triggered by the cationic surfac-
tant dodecylamine. The reason for this is not completely clear,
but it may be simply that the hydrophobic dodecylamine has an
easier time accessing the spore’s inner membrane, the likely
site where this chemical triggers spore germination, in cotE
gerE spores, which have only a very thin layer of coat material,
than in wild-type, cotE, and gerE spores, which have a much
thicker coat layer (33).
The relatively normal resistance of the cotE gerE spores to
moist heat was not unexpected, as the spore coats are not
thought to play a major role in spore resistance to moist heat.
However, the combination of the absence of most of the coat
from these spores and their relatively normal core water con-
tent is consistent with the idea that any restraining action of the
spore coat is not essential for the reduction in core water
content late in spore development, which is the major cause of
spore resistance to wet heat. The absence of any effect of the
much thinner and evidently less rigid coat in cotE gerE spores
on spore resistance to mechanical disruption further indicates
that the coat is not of major important in this resistance prop-
erty either, and by the process of elimination, this must be due
at least in part to the spore’s thick PG cortex. The extremely
high sensitivity of the cotE gerE spores to hypochlorite, how-
ever, indicates that the coat is extremely important in the
resistance to this and probably other chemicals. Indeed, the
cotE gerE spores were at least as sensitive to this agent as were
growing cells. The precise coat component responsible for
spore resistance to hypochlorite and other reactive chemicals is
not known, and it may be due simply to the normally large
amount of protein that can detoxify reactive chemicals by non-
specific reactions with them.
One additional outcome of the identification of these stable
and severely coat-defective cotE gerE B. subtilis spores is that
such spores provided an excellent reagent for determining the
reason that spores often give faint peripheral staining with
stains that are thought to be relatively specific for nucleic acids
or membranes (15, 20, 34), as this could well be due to adsorp-
tion of these stains to coat protein. In addition, the relative
lack of coat proteins from cotE gerE spores may allow the
identification of the spore components responsible for the
spore’s significant autofluorescence, a property that has been
suggested may be useful for spore detection (16, 17). Recent
work has indicated that the cotE gerE spores described in this
work do indeed exhibit decreased peripheral staining with a
number of reagents and, perhaps more importantly, exhibit
greatly decreased autofluorescence (19a). The latter property
may make these coat-defective spores useful in studies of the
location of fluorescent reporter proteins fused to extremely
low-abundance proteins in spores, such as the germinant re-
ceptors in the spore’s inner membrane (36).
This work was supported by grants from the National Institutes of
Health (GM-19698) and the Army Research Office to P.S. The Center
for Cell Analysis and Modeling at the University of Connecticut
Health Center is supported by NIH RR022232. Part of this work was
performed under the auspices of the U.S. Department of Energy by
Lawrence Livermore National Laboratory under contract number DE-
We are grateful to Art Hand for assistance with EM, to Keren
Griffiths for advice and reagents for the analysis of spore core water
content, to Michael Mallozzi and Adam Driks for helpful advice, and
to one reviewer for helpful suggestions.
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