METHODS 20, 95–110 (2000)
Article ID meth.1999.0909, available online at http://www.idealibrary.com on
Structure and Assembly of the Bacterial
Endospore Coat
Adriano O. Henriques* ,† and Charles P. Moran, Jr.* ,1
*Department of Microbiology and Immunology, School of Medicine, Emory University, 3001 Rollins
Research Center, Atlanta, Georgia 30322; and †Instituto de Tecnologia Quı́mica e Biológica,
Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras, Portugal
Many biological processes are mediated through the action of
multiprotein complexes, often assembled at specific cellular locations. Bacterial endospores for example, are encased in a
proteinaceous coat, which confers resistance to lysozyme and
harsh chemicals and influences the spore response to germinants. In Bacillus subtilis, the coat is composed of more than 20
polypeptides, organized into three main layers: an amorphous
undercoat; a lamellar, lightly staining inner structure; and closely
apposed to it, a striated electron-dense outer coat. Synthesis of
the coat proteins is temporally and spatially governed by a cascade of four mother cell-specific transcription factors. However,
the order of assembly and final destination of the coat structural
components may rely mainly on specific protein–protein interactions, as well as on the action of accessory morphogenetic proteins. Proteolytic events, protein–protein crosslinking, and protein
glycosylation also play a role in the assembly process. These
modifications are carried out by enzymes that may themselves be
targeted to the coat layers. Coat genes have been identified by
reverse genetics or, more recently, by screens for mother cellspecific promoters or for peptide sequences able to interact with
certain bait proteins. A role for a given locus in coat assembly is
established by a combination of regulatory, functional, morphological, and topological criteria. Because of the amenability of B.
subtilis to genetic analysis (now facilitated by the knowledge of its
genome sequence), coat formation has become an attractive
model for the assembly of complex macromolecular structures
during development. © 2000 Academic Press
Bacterial endospores are complex structures whose
biogenesis culminates a developmental process initiated in response to nutritional deprivation (1–3). The
1
To whom correspondence should be addressed. Fax: (404) 707–
3637. E-mail: moran@microbio.emory.edu.
1046-2023/00 $35.00
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.
spore is a dormant cell type, extremely resistant to
environmental challenges, but that nevertheless retains the ability to monitor its environment and to
germinate and resume vegetative growth within minutes of exposure to specific germinants (1, 4). The basic
endospore architecture (Fig. 1) is probably conserved
across species. The core compartment (Cr), which contains the chromosome, is delimited by a membrane
(IFM), covered by a thin germ cell wall (PGCW). This
basic unit of viability is then enveloped by two protective layers of different properties, the cortex peptidoglycan (Cx), apposed to the forespore outer membrane (OFM), and the protein coat (Uc, IC, and OC).
The morphological changes that occur during sporulation are similar in a number of bacilli and clostridia (2,
5), as well as in the round-shaped Sporosarcina ureae
(but see below) (6). Other endospore formers have not
yet been examined in detail (5). At the onset of spore
formation, the sporangial cell is divided by a polar
septum into two dissimilar compartments (Fig. 2A), a
smaller prespore and a larger mother cell, each of
which accommodates a copy of the genome (the sporulation division in S. ureae is, however, symmetric) (6).
Soon after the asymmetric division (morphological
Stage II), the septal membranes migrate around the
prespore, a process that eventually converts it into a
free protoplast separated from the mother-cell cytoplasm by two membranes of opposing polarity, the
inner and outer forespore membranes (Stage III) (1–3).
During Stages IV and V, the engulfed prespore (or
forespore) is encircled first by the germ cell wall and
then by the cortex (Fig. 1). Finally, the spore is encased
within the coat, which consists of a 60- to 100-nm-thick
protein layer at the spore surface. Most of the morphological variations seen in spores from different species
occur at the level of the coat layers (see below). The
95
96
HENRIQUES AND MORAN
mature spore is then released into the environment, on
lysis of the terminal mother cell (1–3).
The process of coat assembly is best understood in
the bacterium B. subtilis, mainly because the advanced
genetic tools available for this organism have allowed
the cloning and analysis of a large number of coat
genes [see (3) for a review] (Table 1). The coat can be
viewed as a complex, multichambered organelle, to
which different proteins are targeted during its maturation. Formation of the coat spans a developmental
window of some 6 h (1, 3). During that period, four
mother cell-specific transcription regulators ensure
that the synthesis of proteins with specific functions in
coat assembly is kept in register with the course of
spore morphogenesis (1, 3, 7, 8). In addition to the
intricate transcriptional control, the assembly of individual proteins depends on a topological imprint established early by the expression of genes encoding a
unique class of morphogenetic proteins. These guide
the assembly of the coat components, but may not
themselves be part of the final structure (9 –13). The
process of assembly further depends on specific but as
yet poorly understood interactions (possibly sequential) among specific components (see below) and on
secondary modifications, both enzymatic and nonenzymatic (14 –18). The mature coat is required for the
spore’s ecological fitness (both its resistance to environmental challenges as well as its responsiveness to germinants). This discussion focuses on the approaches
and methodology that have been employed in the analysis of the composition, structure, and function of the
B. subtilis spore coat. The methods used and the con-
cepts derived from the study of coat assembly in this
organism should be generally applicable to other spore
formers. General methods for the manipulation and
genetic analysis of B. subtilis have been compiled recently (19, 20).
STRUCTURAL DIFFERENTIATION OF THE
ENDOSPORE COAT
In B. subtilis, sporulation triggered by exhaustion of
a key nutrient in rich liquid medium [Difco sporulation
medium, or DSM (21)] or by resuspension in a defined
medium [Sterlini Mandelstam, or SM (22)] is a rapid
and efficient process. Sporulation is essentially completed some 8 to 10 h after its onset, defined by the end
of exponential growth or by the resuspension moment,
accordingly. Routinely, more than 90% of the viable
cells in a liquid culture form spores that are resistant
to heat, organic solvents, hydrogen peroxide, or lysozyme, all of which are treatments that would
promptly kill vegetative cells (1–3, 20). When examined by electron microscopy [see (23) for a protocol],
spores purified from cultures 9, 24, or 48 h after the
onset of sporulation appear structurally equivalent
(20). The coat is clearly separated from the cortex by a
zone of stained amorphous material commonly referred
to as the undercoat (13) (Fig. 1). This layer appears to
form a continuum with the cortex peptidoglycan, but
may be separated from it by the forespore outer membrane (24, 25) (Fig. 1). The inner coat, which rests on
FIG. 1. Spore structure. Shown is an electron micrograph of a thin cross section of a B. subtilis endospore (A) and a schematic
representation (B) of a radial section of the spore in (A). (B) All the spore structures (as well as their presumptive locations) that are relevant
to the present discussion but are not easily recognizable on the electron microscopy examination of wild-type spores. Cr, spore core; IFM,
inner forespore membrane; PGCW, primordial germ cell wall; Cx, cortex; OFM, outer forespore membrane; UC, undercoat; IC, inner coat; OC,
outer coat; SL, surface layer. Bar 5 0.2 mm.
ENDOSPORE COAT ASSEMBLY
the undercoat, is some 20 – 40 nm wide, and is formed
by the juxtaposition of three to five thin lightly staining
laminae. The outer coat, closely apposed to the inner
layer, is wider (40 – 88 nm), and formed by thicker,
electron-dense striations (Fig. 1). Freeze-etching studies revealed that the outer coat is organized in a pattern of closely aligned rods or bars that tend to run
97
along the longitudinal axis of the spore, just underneath a thin surface layer (26, 27). This surface layer
assumes distinctive patterns from species to species
(26, 27). The surface layer revealed by the freezeetching studies in B. subtilis is not clearly distinguished on conventional electron microscopy examination of spores (Fig. 1). However, several lines of
FIG. 2. Stages of endospore formation and spore coat assembly. The relevant morphological stages of sporulation (as described in the text)
are represented (A), in parallel with the course of the mother-cell cascade of gene expression (B). Note that the cotE gene is initially
transcribed (from its P1 promoter) under the direction of s E-containing RNA polymerase, and in a second period (from its P2 promoter), by
s E in conjunction with SpoIIID. (C) magnification of a region of the forespore outer membrane and a view of the assembly process. The
putative final destination of the different coat proteins is represented, based on the evidence discussed in the text. Three stages in coat
assembly are represented (see text). Coat assembly may be initiated with the synthesis, under s H control, of proteins that accumulate in the
compartment defined by the two forespore membranes (e.g., TasA) and that later may in part associate with the cortex (at this stage cortex
synthesis has not been initiated). TasA may also associate with the inner coat layers. At this early stage CotE forms a ring in a
SpoIVA-dependent manner, at a distance from the forespore outer membrane. In a second period, coat structural proteins, most of which are
synthesized under s K control, are targeted to different coat layers. At this stage, when cortex synthesis commences, SpoVID is required to
keep the CotE ring in place. In a third period, deposition of the coat proteins continues. In addition, several gene products, some of which
are made under GerE control (e.g., Tgl, CgeD), are involved in the modification of certain coat components. Some may act from the outside
of the spores (e.g., CgeD, SpsC, SodA), without becoming associated with the structure. Others, e.g., Tgl, may associate with the outer coat
layers. The outermost coat layer is proposed to contain proteins (e.g., CotM, CotX), crosslinked in a Tgl-dependent manner, defining a surface
layer (SL) (see text). CotB and CotG may be subjected to crosslinking reactions, as discussed in the text.
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HENRIQUES AND MORAN
evidence suggest that the outermost coat layer may
have properties that are distinct from the rest of the
structure. For example, certain treatments result in
detachment of a diffuse layer from the surface of the
spores, without affecting the overall coat appearance
(28). Also, mutants in which the surface properties of
the spores are dramatically altered (cgeD or spsC; see
below) display a normal coat morphology, suggesting
that the mutations introduce subtle effects in an outermost layer that is not normally recognizable (29, 30).
In a cotH mutant, in which the structure of the coat
layers is profoundly altered, a diffuse surface layer
that appears to peel off the compromised coats is evident (31). It can be questioned whether in this case the
mutation reveals a normal preexisting structure or
results in an abnormal one. However, it should be
noted that in cotM and tgl mutants (see also below),
which display specific defects in the external layers of
the outer coat (29), this loosely attached layer is not
observed, suggesting that its synthesis or assembly
may be in part controlled by the two loci.
There is considerable diversity, within the genus
Bacillus, with respect to the structure, extent, and
composition of the coat layers (26, 27). In some species
the spore is further enveloped by a more or less conspicuous exosporium (of loose definition), normally separated from the coats (26, 27). The surface layer discussed above may correspond to an exosporium, in
which case in B. subtilis it would be atypically joined to
the outer coat (26, 32). Other spore appendages of even
more obscure nature and function have been described.
These include intriguing hair-like fibers projecting
from the surface of B. subtilis or B. anthracis spores
(26).
FUNCTIONS OF THE COAT AND THEIR
ASSESSMENT
Lysozyme Resistance
The spore’s resistance and germination properties
develop late in development, during Stage V (Fig. 2A),
TABLE 1
B. subtilis Genes Encoding Structural Components of the Coat
Gene
Location a
(kb)
Product(s)
(kDa)
cotA
cotB
cotC
cotD
cotE
684.7
3714.9
1904.6
2332.3
1774.4
58.5
42.9
8.8
8.8
20.9
cotF
cotG
cotH
cotJA
cotJC
cotM
cotS
cotT
cotV
cotW
cotX
cotF
cotZ
ynzH
ytaA
4166.3
3716.3
3716.2
755.9
755.9
1925.3
2845.1
1280.40
1251.5
1251.1
1250.6
1250.0
1249.4
1900.6
3161.2
13/8 1 5 e
23.9
42.8
9.73
21.69
15.2
40.29
12.98/7.76 e
14.36
12.32
18.58
17.87
16.52
11.6
41.2
Probable
localization
OC
OC
OC
IC
OC
OC
OC
IC/OC
IC
IC
—
IC
IC
OC f
OC f
OC f
OC f
OC f
?
?
Transcriptional
regulation
s K; GerE(2) c
s K 1 GerE
s K 1 GerE
s K 1 GerE
s E(P 1) d
s E 1 SpoIIID (P 2) d
sK
s K 1 GerE
sK
s E 1 SpoIIID
s E 1 SpoIIID
s K; GerE(2) c
s K 1 GerE
sK
s K (GerE) g
s K (GerE)
s K 1 GerE
s K (GerE)
s K (GerE)
?
?
Assembly
requirements b
CotE
CotE
CotE, CotH
—
—
—
—
CotE, CotH
CotE, GerE
CotJC
CotJA
—
CotE
GerE
—
—
—
—
—
—
—
a
The positions of the various loci are based on the coordinates listed in the B. subtilis genome database [http://www.bioweb.pasteur.fr/
Genolist/Subtilist; see also (73)].
b
Assembly of the protein in question (but not its production) requires expression of the indicated loci.
c
GerE represses transcription from the cotA and cotM promoters.
d
The cotE gene is expressed from two promoters: one (P1) is used by s E-containing RNA polymerase, whereas the other (P2) requires the
ancillary transcription factor SpoIIID.
e
Size of precursor and mature forms, respectively.
f
These genes encode components of the coat-insoluble fraction (16).
g
The cotV, cotW, and cotX genes are transcribed from the P VWX promoter, whereas cotX is also expressed from the P X promoter. The P YZ
promoter drives transcription of the cotY and cotZ genes. GerE is required for transcription from the P x promoter, as well as for enhanced
transcription from P VWX and P YZ (85).
ENDOSPORE COAT ASSEMBLY
concomitantly with the formation of the cortex and coat
layers (33, 34). The cortex is critical for maintaining
the spore’s dormancy and heat resistance. Its protection from the action of lytic enzymes such as lysozyme
is probably the most important coat function and certainly the easiest to assay in the laboratory (see below).
Purified spores are plated before and after treatment
with lysozyme (250 mg/ml) for 15 min at 37°C, and the
percentage of survivors is calculated (20, 34). The acquisition of lysozyme resistance, but not of other resistance properties, requires de novo protein synthesis
during Stage V of sporulation, when most of the coat
structural proteins are made and assembled (33, 34).
Lysozyme resistance is prevented by the addition of a
serine protease inhibitor to the medium, suggesting
that proteolytic events are involved in coat assembly
(33, 34). It should be noted that very little coat structure is required to confer nearly normal lysozyme resistance and that only spores with highly compromised
coats are sensitive to lysozyme. Examples are those
produced by cotE, gerE, or spoVID mutants (10, 13, 35).
These mutants also show a slight decrease in heat
resistance, probably as an indirect consequence of exposure of large parts of the cortex. When a culture at
Hour 8 of sporulation is examined under a microscope
equipped with phase contrast optics, the endospores
appear as ellipsoidal phase bright bodies, either free or
still partially or completely enclosed within the phasedark mother cell (2, 20). Refractility correlates with the
degree of spore protoplast dehydration, which in turn
depends on correct formation of the cortex [see (36) and
references therein]. Certain coat mutants, including
coatless mutants, or spores from which the coats have
been chemically stripped remain phase bright and heat
resistant. However, they become lysozyme and hydrogen peroxide sensitive and susceptible to organic solvents, e.g., chloroform and octanol (13, 35, 37– 40) (see
also below), indicating that the coat acts as a permeability barrier. Moreover, these spores become deficient in germination.
Germination Defects
Germination is thus another important spore property that requires accurate formation of the coat layers
(13, 35, 41– 43). However, germination can also be affected by defects in the structure of the cortical peptidoglycan (PG) or by specific defects in the germination
machinery (e.g., germination receptors) that do not
translate into obvious structural defects in the cortex
or coat layers (25, 35, 36, 44). A germination (Ger)
phenotype may reveal a coat deficiency if it correlates
with functional, compositional, or structural changes
in other specific coat attributes, while not affecting
further spore properties, or if additional changes can
be shown to be indirect consequences of a coat deficiency (e.g., the cotT or cotD mutant) (41, 45).
99
Spores of different species and strains germinate in
response to a variety of physical and chemical stimuli
[see, e.g., (44, 46, 47)]. For B. subtilis, the chemical
germinants normally used include L-alanine (which
can be used in a range of concentrations between 0.1
and 10 mM, sometimes in combination with 1 mM
inosine) and related amino acids, Penassay broth, and
a mixture known as AGFK (asparagine, glucose, fructose, KCl) (20, 41, 44). Germination can be scored on
DSM plates, containing 3- to 4-day-old sporulated colonies that have been heat activated (48). The colonies
are overlaid with a rich medium containing the 2,3,5triphenyltetrazolium indicator (Tzm). After a 2- to 3-h
incubation in the dark, Ger 1 colonies are red, because
the reactivation of dehydrogenase activity causes reduction of the dye (20). Ger 2 colonies remain uncolored, whereas intermediate phenotypes usually correlate with the severity and stage of the germination
defect (4, 20). Because the overlay medium permits
stimulation of germination by L-alanine, only mutants
unable to respond to L-alanine or to both L-alanine and
AGFK will produce a Ger 2 phenotype. In contrast, a
red colony may correspond to a mutant unable to germinate in AGFK. These can be identified by a colony
transfer assay, in which sporulated colonies are first
transferred to a filter paper that (after treatments to
kill sporulating cells and to prevent premature germination) is placed on top of a germination agar plate
containing AGFK and Tzm (20, 49). Both tests can also
be used to identify mutants that germinate faster than
the wild type, although in this case liquid assays provide a more reliable measure of germination rates. In
the latter case, a heat activated purified spore suspension is exposed to different germinants, and the rate of
germination is followed, normally by measuring the
loss in optical density of the suspension at 580 nm (13,
16, 20, 41). Other parameters that can be used as a
measure of germination include loss of heat resistance,
release of dipicolinic acid, or release of hexoxaminecontaining material from the cortex [see (20) for a
discussion]. The various germinants should in principle be tested, as a mutation may interfere with specific
germination pathways (4). Heat activation is normally
required to achieve a maximum germination rate, but
some cortex mutants do not require heat activation to
achieve maximum germination (36). A similar test has
also been occasionally used to probe subtle differences
in coat structure (41). The ultrastructural analysis of
germinating spores reveals that the coat is cracked at
discrete locations, which may reflect the site of assembly of specific lytic enzymes (50). Because germination
is sensitive to serine protease inhibitors, it has been
suggested that proteases built into the structure play
an active role in the process (51). At least one intracellular protease (of about 30 kDa) is known to be deposited into the coat outer layers, but appears to play no
100
HENRIQUES AND MORAN
role in germination (52). The extracellular metalloprotease Mpr is also known to associate with the coat
layers, but its function in coat assembly or spore germination, if any, is not yet known (29).
The available evidence suggests some degree of functional differentiation between the two coat layers. This
distinction is generally made on the basis of the phenotypes of two pleiotropic mutants. One, cotE, fails to
assemble the outer coat and is lysozyme sensitive (13).
The other, gerE, lacks any inner coat structure and is
germination deficient [in fact the gerE mutant was
found in a screen for a germination deficient phenotype
(35)]. Although reiterated in the literature, this functional distinction cannot be precisely made on the basis
of the phenotypes displayed by the cotE or gerE mutant. First, the cotE mutant exhibits reduced inner
layers and is germination deficient (13, 29). Furthermore, it may also be deficient in undercoat formation
(53). Second, in addition to missing the inner coat
structure, the gerE mutant also lacks most of the outer
coat [and what remains of the outer coat is altered (29,
35)]. A better illustration of the functional differentiation of the coat is perhaps demonstrated by mutations
in two other coat loci (neither of which is associated
with lysozyme sensitivity). A mutant (cotD) that presumably lacks a single inner-coat component (a supposition that nevertheless relies on its presence in the
coats of a cotE mutant) responds slowly to the germinant L-alanine (45). Another mutant that displays a
germination phenotype results from the inactivation of
the cotT locus (41). That the locus encodes inner coat
components is supported by the observation that overexpression of cotT results in spores with an enlarged
inner coat (and no other structural alterations). These
spores are also germination deficient (41), supporting
the view that the inner coat plays an important role in
germination. The ambiguity in the distinction of functions for the inner and outer coats probably results
because of fundamental features of coat assembly and
structure and because both coat structures are functionally linked.
Redox Functions
Differences in composition and structure of the coat
layers among different species may reflect specific ecological pressures, but it is noteworthy that all endospores appear to have two different coat layers, inner
and outer. Spores or purified coat material from a
marine Bacillus species (strain SG-1) can oxidize Mn 21
to MnO 2, which precipitates on the outermost spore
coat layers (54). Vegetative cells, in contrast, can reduce MnO 2, suggesting that the oxide accumulated on
spores can be used as a terminal electron acceptor in
the metabolism of vegetative cells (55). This, in turn,
could confer a selective advantage over organisms relying only on oxygen (55). The genes involved in Mn 21
oxidation are clustered in an operon transcribed during
sporulation under s K control (54) (see also below). One
of the genes in the cluster (mnxG) encodes a protein of
the blue copper family of oxidases (54). MnxG is related
to CotA of B. subtilis, which is required for the production of the brown pigment that characterizes wild-type
sporulating colonies [see (45) and references therein].
Disruption of the cotA locus results in the formation of
unpigmented colonies and spores. These lack the 63kDa CotA protein (Table 1), but are functionally indistinguishable from wild-type spores (45). Two other redox enzymes appear to associate with the coat layers.
One, encoded by the cotJC gene (Table 1), is similar to
a Mn-dependent catalase from Lactobacillus plantarum, and is part of the internal coat layers (56, 57). The
other is a Mn-dependent superoxide dismutase (the
product of the sodA gene), which was found associated
with the coats of a cotE mutant (23) (Fig. 2). There is no
evidence that any of these enzymes are required for the
protection of the spore against oxidative stress (23,
56 –58), but they could participate in crosslinking reactions involving coat proteins (23) (see also below). It
has been recently suggested that the coat protects the
spores in the digestive tract of ruminants (A. Driks,
personal communication). However, spores in the soil
(or the intestinal tract) are likely to face challenges
profoundly different from those encountered by spores
of certain pathogens, e.g., Clostridium or B. anthracis,
in the host’s body, and to respond to different germination stimuli. Reinforcing the suggestion that different coats are made to meet specific conditions, we note
that even within the same species the coat composition
can be adjusted to the contents of the sporulation medium (60). This response is regulated at the transcriptional level for the abundant outer coat component
CotC of B. subtilis (59, 60).
PURIFICATION OF SPORES AND ISOLATION
OF THE COAT FRACTION
Spores are collected by centrifugation of cultures
normally 24 h after the initiation of sporulation, resuspended in cold distilled water, and incubated overnight
at 49C to lyse remaining cells, and the cycle is repeated
two or three times. A lysozyme treatment can be employed to enforce cellular lysis, but this is not recommended with mutants with severe coat lesions or with
uncharacterized mutations (20). This simple method
provides spores of reasonable quality for many applications including electron microscopy or quantitative
germination tests. However, during the extraction and
analysis of coat proteins from spores, we occasionally
faced proteolytic degradation of the sample, a problem
that could be circumvented by sedimentation of the
washed spores through a step gradient of 50%
Renografin-74 (or the equivalent Renocal-74, both from
ENDOSPORE COAT ASSEMBLY
Squibb Diagnostics) (20, 61). Vegetative cells will be
found together with cell debris at the top of the gradient, whereas sporulating cells containing endospores
are found in a more or less wide band about 2 cm below.
The sedimented spores are immediately washed with
cold distilled water, as traces of Renografin have been
found to induce some germination (26), and finally
resuspended in a suitable volume of water. This procedure often gives greater then 99% highly clean spores,
as assessed by phase-contrast microscopy. A correspondence can be established between the optical density of
the suspension at 578 nm and the number of spores,
either by plating or by direct counting in a Haussler
chamber. The spore suspension can then be stored at
49C for a period no longer than 2–3 weeks, after which
some alterations in the status of the coat layers can be
detected (see below). Other methods of storage (e.g.,
freezing or lyophilization) are not advisable for any of
the tests to probe the structure, composition, or function of the coat layers discussed here.
The analysis of purified spores (prepared by extensive washing and lysozyme treatment of 48-h cultures)
has revealed that the coat layers constitute 50 to 78%
of the total spore protein, with small amounts of polysaccharides or lipids [see, e.g., (37, 62– 64)]. This was
estimated following isolation of the spore coat fraction
(see below), on a protein basis or by monitoring the
radioactivity incorporated into protein during growth
and sporulation in the presence of one or several labeled amino acids or sulfate [see, e.g., (37, 62, 63)]. In
these and other classic studies, the coat fraction was
prepared by mechanical disruption of the spores with
0.11- to 0.12-mm glass beads in a cell disintegrator, at
neutral pH in the presence of EDTA and phenylmethylsulfonyl fluoride (PMSF), until no intact spores were
detected by phase-contrast microscopy. The coat fragments were recovered by centrifugation, treated with
lysozyme to remove the cortex peptidoglycan, and
washed with different solutions including sodium dodecyl sulfate (SDS) to remove membrane components
(37, 62, 63). The final insoluble residue, defined as the
coat fraction, often consisted of large coat fragments in
which the usual coat morphological features as seen by
electron microscopy were preserved (62). For storage,
the coat fraction is resuspended in water or lyophilized.
ANALYSIS OF THE COAT POLYPEPTIDE
COMPOSITION
Coat Soluble Fraction
Coat proteins are usually extracted from either purified coats or spores by two families of methods (20).
Treatment with 0.1 N NaOH at 49C for 15 min results
in solubilization of less than 5% of the coat protein, but
101
preferentially solubilizes a group of alkali-soluble proteins (37, 62, 63). The group includes the outer-coat
component CotC, of about 12 kDa, and at least two
polypeptides of 5 and 8 kDa that are cotF dependent
(15, 45). About 68% of the total coat protein (including
the alkali-soluble proteins) can be solubilized by treatments with reducing agents and detergents at pH values between 6.8 and 9.8, and resolved by SDS–
polyacrylamide gel electrophoresis (PAGE) (37, 62, 63).
The exact solubilization conditions differ among laboratories, but in general the SDS–PAGE profile of released proteins is rather conserved, as in the following
examples. Goldman and Tipper (62) boiled the coat
fraction for 3 min in 60 mM Tris, 3% SDS, 5% (v/v)
2-mercaptoethanol (2-ME), at pH 6.8, and noted no
significant improvement in extraction on addition of
denaturing agents such as guanidine hydrochloride
plus 6 M urea, 8 M urea, or 6 M guanidine thiocyanate.
In a related protocol Pandey and Aronson incubated
purified coats for 2–3 h at 37°C in a buffer containing 5
mM CHES, 1% SDS, 8 M urea, and 50 mM dithiothreitol (DTT), at pH 9.8 (63). These authors found that
about 6% of the solubilized material was polysaccharide (63) (see also below). In another study, Jenkinson
et al. (37) treated purified coats for 30 min at 68°C in 5
mM CHES, 1% SDS, 50 mM DTT, 2 mM PMSF, at pH
9.8. Subjecting whole spores to the same treatment
released only about 25% of the coat protein, consistent
with the protective role of the coat (37). Nevertheless,
the collection of SDS–PAGE-resolved polypeptides was
similar to that released from isolated coats. During the
extraction the spores retained refractility and heat resistance, indicating that no major structural disruption
of the cortex had occurred (37).
In our laboratory, we routinely boil intact spores for
8 min in the presence of 125 mM Tris, 4% SDS, 10%
(v/v) 2-ME, 1 mM DTT, 0.05% bromphenol blue, 10%
glycerol at pH 6.8 (23, 57, 65). After brief centrifugation, the supernate is directly applied to an SDS–
polyacrylamide gel. This procedure results in profiles
very similar to those reported by Jenkinson et al. (37),
and is highly reproducible (23, 57, 65). The pattern of
SDS–PAGE-resolved proteins extracted from wild-type
purified spores or coats generally coincides with, and
defines the collection of, soluble (or extractable) coat
structural components.
Different criteria have been used to define proteins
as coat components. Proteins that associate loosely
with the coat’s inner or outer interfaces are likely to be
released during the purification of the coat fraction,
but may remain bound to whole spores. Whether or not
these proteins are relevant must be established by
independent criteria (e.g., morphology, resistance, or
germination tests of the corresponding mutant). Proteins that associate with the surface of the spores may
be washed off with a salt solution. For example, a
102
HENRIQUES AND MORAN
protease activity associated with the spores of B. cereus
was easily released with 1 M KCl (66).
No proteins in the coat soluble fraction appear to be
covalently linked to the cortical peptidoglycan (32, 37).
To determine this, cells were labeled with N-acetyl-D[ 14C]glucosamine, and the coat fraction was prepared
with or without lysozyme treatment. None of the solubilized proteins was labeled, or its migration affected
by incubation of the coat fraction with lysozyme prior
to extraction (37). However, components of the cortex–
coat interface that may loosely associate with the peptidoglycan may be highly relevant for coat assembly or
function. It is not known whether the outer forespore
membrane remains functional after coat deposition,
but in B. subtilis at least 11 proteins in the total purified coat fraction are antigenically related to proteins
present in the vegetative cell membrane (24). Their
role, if any, in coat assembly needs to be established by
independent criteria.
In B. subtilis the collection of coat-soluble polypeptides consists of more than 25 different species, ranging in size from 6 to 63 kDa, as determined by the
analysis of Coomassie-stained gels, and confirmed by
pulse labeling of sporulating cells (23, 37, 43, 57, 65,
67). The profile is, however, dominated by a group of 4
to 6 species in the range 6 to 12 kDa (including CotC
and CotD) and by one major protein of about 36 kDa
(CotG), which together constitute about 50% of the
total solubilized coat protein (37, 45, 67). At least one
component of the soluble fraction (possibly two) is a
glycoprotein (37, 63) (see below). At least 16 polypeptides of the soluble fraction have been identified by
N-terminal sequence analysis (Table 1) (see below).
Some of the bands recognized on an SDS–PAGE coat
protein profile, however, correspond to more than one
protein. Therefore, a strategy is to determine the sequence of internal peptide fragments obtained by proteolysis. Alternatively, and in an effort to render the
protein profile less complex, pleiotropic mutants that
lack several coat components (e.g., cotE or gerE) can be
used (13, 35). One advantage of this approach is that
species extracted in insignificant amounts from wildtype coats or spores may, as the result of misassembly
or increased accessibility, be more readily extracted
from the mutant. The TasA protein, for example (GenBank Accession No. P54507), which has a role in coat
assembly, is barely detected in wild-type spores, but is
the most prevalent polypeptide that can be extracted
from the coats of gerE mutant spores (68).
The coat polypeptide composition differs from species to species (14, 40, 69 –71). For example, B. cereus
coats may contain a single main protein (14, 69),
whereas five major proteins as well as a few other
minor species are detected in B. megaterium (70, 71).
The soluble proteins show some propensity to selfassemble. Addition of coat protein to spores of B. cereus
that were stripped of both the inner and outer coat
layers and were lysozyme sensitive resulted in extensive deposition of coat material around the spore and
partial reconstitution of the coat layers (38). Moreover,
lysozyme resistance was partially restored (38). In B.
subtilis, the low-molecular-weight protein extracted
from purified coats with SDS and reducing agents at
alkaline pH was found to reassociate under certain
conditions to form structures that resembled coat fragments (62).
Proteins in the soluble coat fraction come from all
coat layers, as suggested by electron microscopy observations of B. subtilis coat fragments after extraction of
the soluble proteins (62). Extraction appears to solubilize the inner coat and most of the outer coat, but
leaves behind an amorphous component, a diffuse
darkly staining matrix, presumably derived from the
outer coat, and a thin outer layer (62) (see also section
on protein localization). This material (about 30% of
the total coat protein), probably corresponds to the coat
insoluble fraction. On the other hand, treatment with
alkali may preferentially solubilize material from the
outer coat layers (40, 69) (see below).
Coat Insoluble Fraction
The coat residue that remains after extraction resists solubilization by a variety of treatments and does
not contribute significantly to the pattern of electrophoretically resolved proteins. It consists of about 30%
of the total coat protein and defines the coat-insoluble
fraction (37, 62, 63). Amino acid analysis indicates that
this coat fraction has a high content of cysteine, as well
as other modified residues, suggesting that it is highly
crosslinked (62, 63). The importance of the cysteinerich fraction in coat assembly and function is illustrated by the observation that treatment with reducing
agents renders spores of various species sensitive to
lysozyme and H 2O 2 (39, 40). The insoluble residue can
be rendered more soluble by proteolysis or by acid
hydrolysis (16, 37). The purification by highperformance liquid chromatography (HPLC) of a peptide derived from a formic acid hydrolysis of the insoluble fraction and its sequence analysis offered the
possibility to clone the cotX gene, which codes for a
18.6-kDa protein (16). cotX belongs to the cotVWXYZ
cluster of functionally related genes (16) (see also Table
1). Its analysis revealed that some proteins can partition between the insoluble and soluble coat fractions.
The last two genes in the cluster encode highly similar
cysteine-rich proteins. CotY (predicted molecular
weight of 16.5) contains 15 cysteine residues, or 10% of
the total number, whereas CotZ (16.5 kDa) contains 10
cysteines, or 7% of the total. Both CotY and CotZ are
detected in the soluble fraction, as minor components
with electrophoretic mobilities of 26 and 18 kDa, respectively. CotY also exists as 52- and 76-kDa dimeric
and trimeric forms (with either itself or possibly CotZ).
ENDOSPORE COAT ASSEMBLY
These species can be detected on a Coomassie-stained
gel when CotY is overexpressed from a plasmid (16).
Higher-molecular-weight MW forms of CotY can be
detected only by Western. The multimeric forms of
CotY probably result from disulfide crosslinks, since
they can be completely reduced in the presence of 200
mM DTT (16). Deletion of cotXYZ results in spores
with a reduced outer coat, altered surface properties,
and increased accessibility to germinants (16).
The first three genes in the cluster, cotVWX, differ
from cotY and cotZ in that they do not encode cysteinerich proteins: cysteines are absent from CotW (12.3
kDa), whereas both CotV (14.4 kDa) and CotX contain
a single C residue. Nevertheless, CotX antigen can be
immunologically detected in the soluble fraction as
bands of 24 and 48 kDa, albeit in very low levels, as
well as a high-molecular-weight cross-reacting material that does not enter the gel (16). The CotX multimers cannot be solubilized by excess reducing agents.
CotW, CotV, and CotX are rich in glutamine and lysine
residues. This led to the suggestion that CotX could be
crosslinked via a transglutaminase-dependent formation of e-(g-glutamyl)lysine crosslinks (16). Interestingly, multimerization of CotY and CotZ is at least in
part dependent on CotX, since deletion of cotX results
in increased representation of monomeric CotY and
CotZ in the soluble fraction.
CotE is an abundant soluble protein, easily detectable on a Coomassie-stained gel (13), but recent studies
have suggested that most of the CotE antigen is associated with the insoluble fraction (53). A region in CotE
shares sequence similarity with the rod domain of the
acidic (type I) bovine cytokeratin 19, involved in the
formation of intermediate-size filaments (IFs) (53).
This region in IF proteins is arranged in coiled-coil a
helices, and is important in the interactions leading to
filament formation (72). Analysis by electron microscopy has suggested that CotE could be involved in the
formation of a network of filaments, whose complexity
was in part dependent on another coat protein, CotT
(53) (see also below). Oligopeptide repeats rich in glycine and aromatic residues (such as GGYGGG), characteristic of the head domain of type I cytokeratins, are
also found in the heads or tails of type II (basic) IF
proteins (72) and in the carboxy terminus of CotT (14).
In addition, CotT also contains several repeats of a
GGGY motif (see also below). Multiple antigenic forms
of both CotE and CotT were detected under the same
conditions that promoted filament formation, suggesting that both CotE and CotT could be crosslinked (53)
(see also below).
cot Genes Identified by Reverse Genetics
A total of 17 cot genes have now been identified by
reverse genetics (Table 1). These, by definition, encode
coat structural components that, in some cases, may
partition to various degrees between both the insoluble
103
and soluble fractions (see preceding section). The
cloned cot genes include those encoding the most abundant species in the soluble fraction (see above). Other
genes that participate in coat assembly, but that may
not encode structural components [e.g., spoVID, tgl (10,
17)] are generically designated as coat genes. Most of
the genes listed in Table 1 have been insertionally
inactivated. Surprisingly, with the exception of cotE
(13), none of the mutants displayed a lysozymesensitive phenotype, indicating extensive redundancy,
or minor roles for individual components. The cotH
gene was identified by reverse genetics (43), and its
product confirmed as a coat component by use of a
polyclonal antibody raised against purified CotH (31).
The cotH mutant forms spores that are lysozyme resistant, despite having highly disorganized and incomplete coat layers, from which several prominent proteins (including CotH) are missing (43). However, the
cotH mutant is impaired in germination (43). This example illustrates a recurring theme in coat studies; a
role for a given locus in coat assembly has to be established by the analysis of the impact of several parameters, including germination, on the assembly of other
coat proteins, morphology of the coat layers, or localization of the protein to the coats (see below). Interestingly, the cotH mutation exacerbates the germination
phenotype of a cotE mutant (43), depicting the principle that the use of multiple mutants must always be
considered.
Posttranslational Modifications of Coat Proteins
Several types of posttranslational modifications are
found in coat proteins, including glycosylation, proteolytic processing, and crosslinking.
Glycosylation
Purified coat fractions contain some carbohydrate,
and two low-molecular-weight polypeptides (of about 8
and 9 kDa) appear to be glycosylated (37, 63). It is
unclear whether they correspond to any of the abundant low-molecular-weight species (37). The analysis of
two divergent GerE-dependent transcription units,
called cgeAB and cgeCDE (30) (see below) has revealed
strong similarity between CgeD and the product of a
paralogous gene, spsC, of the spsA–K cluster (73). CgeD
and SpsC (as well as other products in the spsA–K
cluster) share sequence similarity with nucleotide
sugar transferases involved in the synthesis of extracellular polysaccharides (30, 73). These observations
suggest that CgeD and SpsC could be involved in the
glycosylation of coat proteins. Deletion of cgeD results
in spores with altered surface properties (30), as does
deletion of the entire spsA–K cluster (29). A somewhat
similar phenotype is conferred by a deletion of cotXYZ
(16). Because deletion of cgeD or of spsA–K does not
change the pattern of extractable coat proteins, it is
104
HENRIQUES AND MORAN
tempting to suggest that proteins in the insoluble fraction are glycosylated in a CgeD- and SpsC-dependent
manner. CgeD has a highly hydrophobic stretch of
amino acids near its N terminus (30). In contrast, the
overall hydrophobic CotX has a highly charged N terminus that could serve to anchor the protein to the
coat. In one scenario, CotX and CgeD could interact via
their hydrophobic parts, which would result in the
glycosylation of CotX and normal spore surface properties. It should, however, be emphasized that to date
neither CgeD nor SpsC has been shown to promote the
glycosylation of any coat component.
Proteolytic Processing
Some coat polypeptides or proteins required for coat
assembly are derived from proteolytic processing of
larger precursors. In at least one case, that of TasA, a
signal sequence seems to be removed by a type I signal
peptidase, suggesting that protein secretion may be
important for coat assembly (68). In comparison, the
primary products of the cotF and cotT genes are subjected to endoproteolytic cleavage (14, 15). The two
processing products of CotF (of 5 and 8 kDa) are found
associated with the coat (15). In contrast, only an
8-kDa processed form of the 13-kDa CotT precursor is
normally found in the coats (14). Mature CotT has an
unusual primary structure. Only 7 amino acids are
represented among its 44 residues, and of these 11 are
G (17.5%), 19 are P (30.2%), and 22 are Y (34.9%).
Moreover, these residues are arranged in repetitive
units of the form PYYYP (or PYYP), PRPP (or PRP),
and GGGY (see section on the insoluble fraction). The
C-terminal oligopeptide motif GGGYG is also reminiscent of cytokeratin 19 and other intermediate filament
proteins (53, 72) (see above). The sequences at the
carboxy-terminal side of the cleaved bonds in the CotF
and CotT precursors are identical (ER), and suggest
that a trypsin-like enzyme may be involved. The CotT
precursor but not its mature form accumulated in
spores of a gerE mutant that fails to produce a 30 kDa
protease (14, 52, 74). This suggests that processing
occurs on the spore and that gerE controls both the
production and assembly of the protease. Disruption of
cotT or accumulation of the CotT precursor interferes
with germination (41), but no other phenotypes derive
from the inactivation of cotF or cotT (14, 15). Given the
involvement of serine proteases in lysozyme resistance
and germination (33, 34, 51, 52), it is possible that
additional coat proteins serve as substrates for serine
proteases.
Protein–Protein Crosslinking
In several biological systems, crosslinking of structural proteins is know to result in the insolubilization
of specific structural components and to confer a high
degree of chemical and mechanical resistance to the
modified structure [(23, 75) and references therein]. In
addition to the formation of disulfide bonds that may
sequester proteins such as CotY and CotZ in the insoluble fraction (see above), there is also evidence for
irreversible crosslinking of coat proteins. The cotB
gene, for example, encodes a polypeptide with an apparent mobility of some 45 kDa that apparently also
accumulates as a dimer (67 kDa), resistant to detergents and reducing agents (29, 45). CotB has a predicted size of 42.9 kDa and does not contain cysteines.
In its last third (130 residues), CotB contains 21 K (or
16%) and 55 S (or 42,3%) residues, arranged in sequences of the form SSKS, SSSKSK, or SSDYQSS,
each repeated three times. The nature of the crosslinks
that hold the putative CotB dimer together are unknown.
A coat transglutaminase, proposed on the basis of
the properties of CotX (see above), was also suspected
from the analysis of the cotM locus (65). cotM belongs
to the a-crystallin family of small heat-shock proteins,
whose members can serve as substrates for a transglutaminase. Inactivation of cotM results in spores that
are specifically deficient in the assembly of the outermost coat layers, but did not change the SDS–PAGE
profile of proteins extracted from purified spores, suggesting that the protein is either not very abundant or
insoluble (65). More recently, (g-glutamyl)lysine
crosslinks were identified in purified spores and coat
material, a transglutaminase extracted from the surface of the spores, and its gene (tgl) cloned by reverse
genetics (17, 18). The Tgl substrates are unknown.
They could include CotX and CotM. Suggestively, the
electron microscopic observation of a tgl insertional
mutant revealed a phenotype very similar to that presented by the cotM mutant (29, 65). The tgl mutant has
unchanged resistance properties and, like the cotM
mutant, displays a normal SDS–PAGE profile of soluble proteins. It is possible that the outermost coat layer
(perhaps the thin outer layer seen on microscopic observation of the coat-insoluble residue mentioned
above) is crosslinked in a transglutaminase-dependent
manner, as already suggested (65).
Purified coat material has a relatively high tyrosine
content (63). Several coat proteins are tyrosine rich
and have peculiar primary structures. For example,
only 10 amino acids are represented in the 66-residuelong alkali-soluble CotC (45). Moreover, 3 amino acids
make up 75% of the total number of residues in CotC
(D, 18.2%; K, 28.8%; Y, 30.3%). Another protein, CotG,
is also Y-rich (21 residues, or 11%), although the most
represented amino acid is K (55 residues, or 28%).
CotG has a predicted size of 22.5 kDa, but migrates as
a 36-kDa species, possibly because of its unusual primary structure (67). Alternatively, CotG is an SDS/
DTT-resistant dimer, formed by crosslinks involving
tyrosine or lysine residues (67, 75). The protein is or-
ENDOSPORE COAT ASSEMBLY
ganized in nine repeating units with the consensus
H/Y KKS Y R/C S/T H/Y KKSRS (where the residues
in bold mark the least conserved positions). Deletion of
cotG resulted in spores that also lack CotB and in the
formation of an expanded outer coat (67), which is
missing its normal pattern of electron-dense striations
(23). We recently found that the amount of extractable
CotG was increased by mutations in the sodA locus,
encoding a Mn-dependent superoxide dismutase (23).
We proposed that SodA was required for the activation
of a putative peroxidase involved in the oxidative
crosslinking (and insolubilization) of CotG. Polymerization of CotG could be an important determinant of
the structural organization of the outer coat layers
(23). In the absence of functional SodA (which is not
required to be coat associated), more CotG could partition in the soluble fraction (23). In a striking parallel,
highly repetitive proline-rich plant proteins are known
to be crosslinked by the reaction of H 2O 2 with a peroxidase (76). It should also be noted that the repetitive,
P-rich CotT may also be crosslinked to itself or to CotE,
and that the latter may also be a prominent component
of the insoluble fraction (53) (see section on the insoluble fraction). o,o-Dityrosine bonds have been found in
coat material from B. subtilis (63), and a peroxidase
activity has been localized to the forespore membranes
of B. cereus (77). Spores, at least those of B. subtilis,
may contain dityrosine residues (78), but in minute
amounts, and it is unclear whether this type of modification can significantly contribute to the assembly
and function of the coat.
Localization of Coat Proteins within the Coat Layers
The structural differentiation of the coat has
prompted several attempts to assign individual proteins to different layers. These attempts relied on differential extraction techniques, surface iodination
studies, dependency on either cotE or gerE for assembly, or, more recently, direct localization of coat antigens by immunomicroscopy or by the use of fusions to
the green fluorescent protein (GFP). For example, the
alkali extraction of 2-mercaptoethanol-treated spores
of B. coagulans solubilizes a tyrosine-rich component
and results in loss of electrodensity from the outer coat
layers (40). Moreover, the treatment destroys the characteristic pattern of parallel fibrils seen in the surface
layers of the spores by freeze-etching (40). The
tyrosine-rich component may be the equivalent of the
12-kDa CotC protein from B. subtilis, which is also
tyrosine-rich, alkali-soluble, and associated with the
outer coat (see below). Surface iodination studies have
revealed that proteins of 36 and 12 kDa (the latter
alkali-soluble) were prominent components of the outermost coat layers in B. subtilis spores prepared at
Hour 9 of sporulation (37). From their electrophoretic
mobilities and relative abundance, these proteins are
likely to be CotG and CotC (45, 67) (see Table 1 and
105
Fig. 2C). Different proteins are surface exposed at earlier times (e.g., around Hours 5 and 6 of sporulation),
and those appear to be progressively covered by other
components as the spore matures (37). In combination
with pulse labeling experiments, these studies have
suggested that the order of assembly may not parallel
the order of synthesis of the coat components (37).
The properties of the cotE mutant are often used to
provide a first indication of the localization of a given
protein within the coat layers. The rationale is that any
protein missing from the coats of a cotE mutant must
be associated mainly with the outer layers (13). This
type of analysis strongly supports the view that CotG
and CotC, as well as CotA and CotB, are outer-coat
proteins (13, 45, 67) (Table 1 and Fig. 2). Absence of
CotA may explain why cotE mutant spores are unpigmented (13, 45). However, in the absence of more direct
evidence, these results should be interpreted with caution, as the mutant also displays a diminished inner
coat and undercoat (29, 53). Thus, CotE may also control the assembly of internal components. CotS, for
example (79), is produced under the joint control of s K
and GerE (see below), and has been localized by immunoelectron microscopy to the inner coat layers in B.
subtilis (80) (Table 1). However, its assembly requires
CotE (80). In addition, inner-coat proteins that are in
close proximity or in association with CotE may be less
represented or absent in the mutant. This may be the
case with CotH, although this protein is also thought to
be present in the outer coat (31, 43). Less reliable is the
assumption that a protein absent from the coats of a
gerE mutant is associated with the inner coat layers
(see also section on functions of the coat). The GerE
protein is a transcriptional regulator that acts to activate or repress transcription of several coat genes (60,
81) (see Fig. 2 and below). The gerE mutant lacks the
inner-coat structure (35), but is also impaired in the
production of several major outer-coat components, including CotC and CotG (60, 67, 81) (see also Table 1).
However, a cotE gerE double mutant lacks both the
inner- and outer-coat structure, and thus a coat protein
that persists in the double mutant must be in part
associated with internal layers of the coat. CotJC, for
example, was easily detected in whole spores of a cotE
gerE double mutant by immunofluorescence microscopy, but was barely detectable in intact wild-type
spores (56).
The subcellular localization of two proteins important for coat assembly, SpoIVA and CotE, has been
studied by immunofluorescence or by the use of fusions
to GFP (82– 84). These studies have confirmed earlier
immunoelectron microscopy results according to which
in sporulating cells, SpoIVA localizes around the forespore, in or close to the outer forespore membrane,
determining the assembly of CotE in a ring-like structure at a distance from it (9) (see below). In these
106
HENRIQUES AND MORAN
studies, an epitope-tagged version of CotE was used
(9). Essentially the same pattern of subcellular localization has been determined for SpoIVA and a CotE–
b-galactosidase fusion protein in the same cells by immunofluorescence microscopy (83). The cells were,
however, somewhat impaired in CotE function (83). A
SpoIVA–GFP fusion protein appears to assume the
expected subcellular localization, completely surrounding the forespore at Stage III (82). However the GFP
moiety interferes with SpoIVA function, and the fusion
protein assumes its correct localization only at low
temperatures and when the wild type spoIVA allele is
coexpressed in the cells (82). At later developmental
stages the GFP fluorescence disappears, suggesting
that the protein is covered by the deposition of the coat
proteins or is eliminated (82). A CotE–GFP fusion protein was shown to localize around the forespore in a
spoIVA-dependent manner, but in those cells (as for
the CotE–b-galactosidase fusion), assembly of the coat
was found to be somewhat aberrant (84). Ultimately,
the localization of individual components to different
coat layers may require the resolution of immunoelectron microscopy, as recently exemplified by CotS (80)
(see above).
GENETIC CONTROL OF COAT GENE
EXPRESSION
A Hierarchical Cascade of Gene Expression
In B. subtilis, the developmental pathway is governed at the transcriptional level by a cascade of four
compartment-specific RNA polymerase sigma (s) factors, which come into play in the order s F, s E, s G, and
s K (3, 7, 8). Activation of each s factor is linked to
specific stages of sporulation, thus coupling gene expression to the course of morphogenesis (1, 3, 7, 8). The
cloning of the cotA–E genes (13, 45), followed by that of
several other cot genes, allowed their transcriptional
analysis (14, 15, 17, 43, 57, 60, 65, 67, 68, 80, 85) (see
Table 1 and Fig. 2). Coat gene expression is initiated
soon after the sporulation division, and depends thereafter on the flow of a hierarchical cascade involving s E
and s K and two auxiliary DNA-binding proteins,
SpoIIID and GerE (60) (see also Fig. 2A and B). The
mother-cell genetic program is initiated by the activation of pro-s E to its active form s E, specifically in this
sporangial compartment (1, 3, 7, 8). Among the genes
that s E controls are spoIVA, spoVID, and cotE (the
latter from its P 1 promoter) (10 –12, 60), which encode
proteins with important morphogenetic roles in coat
assembly (see also the following section). s E also drives
the expression of the spoIIID gene (3, 7, 8), encoding
SpoIIID. The next class in coat gene expression is
represented by SpoIIID-dependent transcription of the
cotJ operon, and of the cotE gene, from its P 2 promoter
(57, 60). Then, with the help of SpoIIID, s E transcribes
the rearranged sigK gene, encoding an inactive pro
form of s K (3, 7, 8, 86). The activation of s K is delayed
until the completion of the engulfment sequence, which
converts the prespore into a free protoplast within the
mother-cell cytoplasm (Stage III) and marks the activation of s G in the forespore. Soon after, pro-s K is
proteolytically converted to its mature form in the
mother cell (3, 7, 8), where it replaces s E during the
postengulfment stages of mother-cell development. It
is then (from Hour 4 of sporulation onward) that the
transcription of most cot genes commences and that
the assembly of the coat structural components around
the engulfed forespore is first noted by microscopy (2,
60). A first wave of s K-dependent gene expression results in the transcription of cotA, cotD, cotH, cotF, cotT,
cotV, cotW, cotY, cotZ, and cotM (14, 15, 43, 60, 65, 85,
87) (Fig. 2). s K also directs transcription of the regulatory gene gerE (81, 88), whose product acts together
with Es K to activate or enhance transcription of the
last class of coat gene expression. The gerE-dependent
regulon includes a minimum of 12 genes and operons.
Transcription of cotB, cotC, cotS, and cotG of the soluble fraction, and cotX in the insoluble fraction is GerE
dependent (60, 67, 80, 85) (see Fig. 2). Transcription of
cotD, cotV, cotW, cotY, and cotZ is enhanced by GerE
(60, 81, 85). GerE also represses the transcription of
genes in earlier temporal classes (e.g., sigK, cotA, and
cotM) (60, 65, 81, 87), thus reinforcing the course of the
regulatory cascade (Fig. 2).
Thus, with the sole exception of tasA (68) (see below),
all the genes involved in the assembly of the spore coat
are transcribed in the mother-cell compartment of the
postdivisional cell.
Genetic Screens for the Identification of Coat Genes
Genetic screens aimed at the identification of
mothercell-specific promoters have been employed to
identify additional coat genes. In one example, the
gene encoding pro-s E (or its mature form) was placed
under the control of the IPTG-inducible P spac promoter
in a strain carrying a deletion of the sigE gene. The
strain was then transduced with a lysate of a temperate phage (SPb), bearing a random library of chromosomal fragments fused to the lacZ gene, and individual
colonies screened for a conditional, IPTG-dependent
Lac 1 phenotype (10). This type of screening allowed the
identification and cloning of the s E-dependent gene
spoVID and the cotJ operon, as well as the s Kcontrolled cotM gene (10, 57, 65). This approach had
the advantage of revealing mother-cell genes independently of a selectable phenotype, but a potential role in
coat assembly had to be investigated by complementary screens. The spoVID mutant was lysozyme sensitive, and the ultrastructural analysis confirmed a severe coat defect (10) (see below). In another case, the
ENDOSPORE COAT ASSEMBLY
disruption of cotJ resulted in spores with an altered
composition of the coat layers, and assembly studies, as
well as direct immunofluorescence localization, have
indicated that it encoded at least two components
(CotJA and CotJC) of the inner coat layers (56, 57).
Finally, disruption of cotM resulted in spores with an
altered coat composition and a specific ultrastructural
deficiency in the outermost coat layers (65).
Two loci, spoVIA and spoVIB, were identified in
screens for mutants that were germination deficient
and lysozyme sensitive (42, 89). The spoVIA mutant
lacked a 36-kDa coat protein, but the mutation does
not map to the cotG locus which encodes a prominent
36-kDa component (42, 67). The spoVIB mutation
maps to the leuB region, near spoVID, but is not allelic
to spoVID (10, 42). A third locus, spoVIC, was defined
by a mutation near cysB causing slow sporulation and
germination and abnormal assembly of the abundant
12-kDa protein (possibly CotC) (90). However, none of
the loci has been further characterized. More recently,
two suppressors of the lysozyme-sensitive phenotype
conferred by a cotE deletion have been obtained (53).
The mutations caused the assembly of minor polypeptides that were not seen in wild-type coat extracts and
that may help restore the assembly of several coat
polypeptides missing from the cotE mutant. The suppressors are lysozyme resistant, but not germination
proficient, and thus do not compensate for all the functions of CotE (53). The suppressor mutations have been
mapped to the aroD region of the B. subtilis chromosome, but are not yet characterized (53). Other than
cotA (45), only one other locus important for coat assembly, spoIVA, found in screens for mutations affecting sporulation (2), has been cloned and characterized
(11, 12) (see below).
EARLY, MIDDLE, AND LATE EVENTS IN COAT
ASSEMBLY
Morphogenetic Proteins and Structural Components
Immunoelectron microscopy studies have shown
that following septation, the CotE protein starts assembling in a ring-like structure that at engulfment
completely encircles the forespore, at a distance of
about 75 nm from it [see (9) and Fig. 2C]. This pattern
of CotE localization is dependent on the spoIVA locus,
which encodes a 490-residue protein with a nucleotide
binding motif (11, 12). SpoIVA itself localizes in close
proximity to the forespore outer membrane soon after
formation of the sporulation septum, and its pattern of
subcellular localization then follows the movement of
the engulfment membranes (9). Mutations in spoIVA
do not prevent synthesis of cotE or any other coat
proteins, except for CotC in DSM (60). However,
107
spoIVA mutants accumulate long swirls of coat material in the mother-cell cytoplasm that retain some of
the ultrastructural features of normal coats (2, 11, 12).
Thus, SpoIVA, which is not detected in mature spores,
appears to guide the assembly of the coat to the forespore, by allowing formation of the CotE ring (9). CotE
is thought to be initially kept in place by a matrix that
fills the gap defined by the SpoIVA and CotE rings. The
components of the matrix are not known, but are likely
to be encoded by genes in the s E regulon, such as those
in the cotJ operon whose products localize to the internal layers of the coat (56, 57). The matrix is thought to
define the site of assembly of the inner coat, whereas
the CotE ring marks the site of deposition of the outer
coat (9).
Appearance of active s K in the mother cell initiates
cortex synthesis and triggers the expression of many
coat genes (see preceding section and Fig. 2). At this
point, a third protein, SpoVID, is required to maintain
the CotE ring around the forespore (9). In a spoVID
mutant, the ring of CotE forms normally. However, at
a later stage, when synthesis of the cortex and deposition of most coat proteins are initiated, it detaches from
the forespore (9). As a consequence, coat material is
deposited in the mother-cell cytoplasm, leaving spores
in which the cortex is exposed and that are lysozyme
sensitive (10). The 63-kDa SpoVID protein, which is
very rich in glutamic acid (118 residues, or 20.5%), may
be required for binding the nascent coats to the cortex.
Interestingly, a protein recently identified as possibly
interacting with SpoVID shares sequence similarity to
cell wall-binding proteins (91). Like SpoIVA, the SpoVID protein is not detected in mature spores. SpoIVA,
CotE, and SpoVID guide the assembly of many other
coat proteins, through a complex series of morphogenetic steps, and are often termed morphogenetic proteins, to emphasize this property. Morphogenetic proteins such as SpoIVA and SpoVID differ from CotE in
that CotE is also an abundant structural component of
the coat (13). The formation of ordered coat deposits in
the mother cell of spoIVA and spoVID mutants (which
are reminiscent of inner- and outer-coat fragments)
suggests that at least to some extent coat assembly
relies on predetermined (possibly sequence-specific) interactions among the individual coat polypeptides.
However, no such structures are seen in a cotE mutant
(9), suggesting that a cascade of interactions leading to
coat assembly may start with CotE (see also below).
A View of the Assembly Process
Production of SpoIVA and CotE define an early organizational period in coat assembly, which is controlled by s E. A single amino acid substitution within
the 235 recognition region of s E (position 217) produced a protein that could direct transcription from s Kbut not from s E-dependent promoters (92). No signs of
coat assembly were detected in this strain, which was
108
HENRIQUES AND MORAN
blocked at Stage II of sporulation, further emphasizing
the role of the s E-controlled phase in the assembly
process. However, the transcription of certain genes
required for proper coat assembly (e.g., TasA) may
even start before, in the predivisional cell under s H
direction (3, 8, 68). In a tasA mutant the undercoat is
abnormal, suggesting that this layer is in part made
from the inside, through the action of secreted proteins
that may first accumulate in the cellular compartment
delimited by the forespore inner and outer membranes
(68). However, formation of the undercoat has also
been suggested to depend on CotE and CotT, which
would then be a late event, since assembly of CotT
requires GerE (14, 53). The efficient transcription of
genes encoding components of the internal coat layers,
such as the cotJ operon, requires s E and SpoIIID, and
thus follows the initial organizational events (57). Coat
gene expression then proceeds with s K. Two genes that
encode inner coat components, cotD and cotT, can be
transcribed by s K alone (14, 60). However, processing
and assembly of CotT are delayed until the GerEdependent production of a serine protease (14). It is
possible that the synthesis of several other components
of the internal layers of the coat is controlled by s E
(with SpoIIID) or by s K. s K also directs the expression
of several outer-coat genes, such as cotA, cotH, cotV,
cotW, cotY, cotZ, and cotM (see Fig. 2B).
Finally, in a late period s K (with GerE) activates or
reinforces the expression of at least three gene classes
(Fig. 2B): (1) those required for the consolidation of the
undercoat and inner-coat structures, encoding additional structural components [e.g., CotS (80)], or modification enzymes, which will probably include the gene
for a CotT-processing enzyme (14) or other proteases
(52, 74), and crosslinking enzymes [it is known, for
example, that the formation of a crosslinking product
of CotJC requires GerE (56)]; (2) those encoding outer
coat components [e.g., CotB, CotC, CotG, CotV, CotW,
CotX, CotY, CotZ (60, 67, 81, 85)]; and lastly, (3) genes
involved in the maturation of the outer coat layers
(crosslinking, glycosylation), whose products (e.g., Tgl,
or CgeD), in some cases, may act from the outside after
deposition of their substrates has occurred (17, 30).
Sequential Interactions in Coat Assembly
Assembly of the outer spore coat relies in part on a
cascade of genetic dependencies in the order CotE,
CotH, CotG, and CotB. In addition to CotE, a number
of other proteins are missing from the coats of a cotE
mutant (13), including CotB, CotC, CotG, and CotH
(43, 45, 67). Disruption of cotH results in spores that, in
addition to CotH itself, are missing CotG and CotB and
have reduced levels of CotC (43). In contrast, CotG
mutants lack CotB (67), whereas disruption of either
the cotB or cotC loci only results in loss of the corresponding polypeptides from the coat (45). Interestingly
cotB, cotH, and cotG form a gene cluster at about
3692.9 kb (73), although the significance of this clustering is unknown. Thus, the earlier a given locus acts
on the assembly pathway, the more pleiotropic its effects on the assembly of other coat components, suggesting that the genetic dependencies correspond to
sequential protein–protein interactions of the type A 1
B 3 AB 1 C 3 ABC, where C is a component that
cannot be recruited for assembly by either A or B alone.
However, other mechanisms of assembly are possible
[see, for example, (93)]. In only one case have interactions between coat proteins been characterized (56).
The yeast two-hybrid system was used to detect specific interactions between CotJA and CotJC, of the
types CotJA–CotJA, CotJC–CotJC, and CotJA–CotJC
(56). Coimmunoprecipitation results showed that
CotJA and CotJC were present in complexes at the
time of coat assembly (56). CotJA or CotJC was never
detected in spore extracts in the absence of the other
protein, indicating that complex formation was a prerequisite for assembly (56).
FUTURE DIRECTIONS IN COAT BIOLOGY
The genome sequence of B. subtilis (73) is a powerful
tool that will have an enormous impact in coat biology.
The inspection of the sequence has already revealed
that several “classic” coat genes were found to have
homologs in the chromosome. One striking example is
ynzH (GenBank Accession No. BG13471), whose product shares more than 88% of its residues with CotC,
suggesting that the two genes may be functionally
redundant (73). Examples of other genes that appear to
have homologs are cotF, cotH, cotJC, and cotS (73).
CotJC, for example, is related to a second putative
Mn-dependent catalase of B. subtilis, the YdbD protein, whose gene is transcribed during sporulation at
about the same time as the cotJ operon (29, 57). CotS
shares sequence similarity with YtaA (GenBank Accession No. BG12071), which was shown by N-terminal
sequence analysis to be a coat protein (94). The 41-kDa
YtaA protein is encoded in an adjacent but divergent
transcription unit, which may be functionally related
to the cotS operon (73, 79, 94). The cotS gene is preceded by a gene (ytxN) encoding a product similar to a
lipopolysacharide
N-acetylglucosaminyltransferase
(GenBank Accession No. BG11379) (73). In light of the
suggestion that a role of SpoVID could be to link the
nascent cortex and coat structures (see above), it is
interesting to note that ytxN is followed by the ytxO
gene (BG11380), which encodes an acidic protein related (although weakly) to SpoVID (10). The cotF gene
is in a special class, as several loci encode products
related to the CotF precursor (73): yraD and yraF encode products similar to its C-terminal half, whereas
yraG and yraE code for products similar to the
ENDOSPORE COAT ASSEMBLY
N-terminal half of the CotF precursor (GenBank Accession Nos. BG13759, 12268, 12269, and 13760, respectively). A third locus, yhcQ (GenBank Accession
No. BG11573) encodes a protein that (except for a short
central region) shares sequence similarity with the
full-length CotF precursor (72). Interestingly, the B.
subtilis CotF protein also shares sequence similarity
with a similarly sized protein from C. pasteurianum
(GenBank Accession No. AF062550), suggesting that
the availability of the genome sequences of other spore
formers will be important in future coat studies. The
genome sequences will allow the rapid identification of
the complete collection of coat components encoded by
paralogous and orthologous genes and the genetic and
functional analysis of the corresponding loci. The genome sequences will also help in the identification of
those coat genes that are unique to specific organisms.
Other studies will follow, such as the localization of
individual proteins to the coat layers and the analysis
of the assembly requirements of each component, including the characterization of specific protein–protein
(including enzyme–substrate) interactions leading to
assembly. Immunoelectron microscopy, as well as immunofluorescence techniques, will likely play an important role in the analysis of protein localization,
whereas genetic and biochemical techniques will be
instrumental in the characterization of specific interactions or protein regions required for assembly. Two
important advantages of using the yeast two-hybrid
system to look for interactions among spore components are: (1) the system can be used for the identification of interacting domains or regions in different
proteins; (2) it allows the use of libraries to screen for
potential partners of a specific bait protein. Phage display methodology has recently been used in the identification of at least one previously unknown coat protein that is probably capable of interacting with
SpoVID (91) (see also above). These and other types of
screens aimed at the identification of possible interacting partners of selected coat proteins will likely become
more common in the near future.
ACKNOWLEDGMENTS
We thank E. Ricca, R. Zilhão, and A. J. Ozin for critically reading
the manuscript. A. O. Henriques was the recipient of a fellowship
from the Fundacao pava a Ciencia e Tecnológica (JNICT). This work
was supported by PHS Grant GM54393 to C.P.M. from the National
Institutes of Health.
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