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Microbiology and Molecular Biology Reviews, June 2004, p. 234-262, Vol. 68, No. 2
1092-2172/04/$08.00+0 DOI: 10.1128/MMBR.68.2.234-262.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Compartmentalization of Gene Expression during Bacillus subtilis Spore Formation
David W. Hilbert
and
Patrick J. Piggot*
Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
Gene expression in members of the family Bacillaceae becomes
compartmentalized after the distinctive, asymmetrically located
sporulation division. It involves complete compartmentalization of the
activities of sporulation-specific sigma factors, 
F
in the prespore and then E in the mother
cell, and then later, following engulfment, G in
the prespore and then K in the mother
cell. The coupling of the activation of F to
septation and G to engulfment is clear; the
mechanisms are not. The factors provide the bare framework of
compartment-specific gene expression. Within each regulon are
several temporal classes of genes, and for key regulators, timing is
critical. There are also complex intercompartmental regulatory signals.
The determinants for F regulation are assembled
before septation, but activation follows septation. Reversal of the
anti-F activity of SpoIIAB is critical.
Only the origin-proximal 30% of a chromosome is present in the
prespore when first formed; it takes 
15 min for the
rest to be transferred. This transient genetic asymmetry is important
for prespore-specific F activation.
Activation of E requires F
activity and occurs by cleavage of a prosequence. It must occur rapidly
to prevent the formation of a second septum. G is
formed only in the prespore. SpoIIAB can block
G activity, but SpoIIAB control does not
explain why G is activated only after engulfment.
There is mother cell-specific excision of an insertion element in
sigK and E-directed transcription of
sigK, which encodes pro-K. Activation
requires removal of the prosequence following a
G-directed signal from the
prespore.
Cell differentiation is a fundamental biological process. Central to
it are the coordination of gene expression with morphological change
and the establishment of distinct programs of gene expression in the
different cell types involved. Formation of spores by Bacillus
subtilis is a primitive system of cell differentiation (Fig.
1), which has become a paradigm for the study of cell differentiation in
prokaryotes (59,
183,
228,
231,
281). The spores formed
are dormant and show greatly increased resistance to stresses such as
heat and noxious chemicals compared to what is seen with vegetative
cells. It was shown 25 years ago through a study of genetic mosaics
that gene expression is compartmentalized during sporulation of B.
subtilis, with different genes being expressed in the
prespore and the mother cell, the two cell types involved
(43,
226). In the years
since, the completeness of compartmentalization has been demonstrated
first by immunoelectron microscopy
(48,
83,
189), then by
fluorescence microscopy with immunofluorescence and green fluorescent
protein (105,
170,
233,
299,
314), and most recently
through the use of a two-part transcriptional probe
(173).
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FIG. 1. Schematic
representation of the stages of spore formation. A vegetatively growing
cell is defined as stage 0. It is shown as having completed DNA
replication and containing two complete chromosomes (represented as
disordered lines within the cells), although replication is not
completed at the start of spore formation. Formation of an axial
filament of chromatin, where both chromosomes (or a partially
replicated chromosome) form a continuous structure that stretches
across the long axis of the cell, is defined as stage I. Asymmetric
division occurs at stage II, dividing the cell into the larger mother
cell and smaller prespore; for clarity, the septum is
indicated as a single line. At the time of division, only approximately
30% of a chromosome is trapped in the prespore, but the
DNA translocase SpoIIIE will rapidly pump in the remaining
70%. Stage III is defined as completion of engulfment, and the
prespore now exists as a free-floating protoplast within the
mother cell enveloped by two membranes, represented by a single
ellipse. Synthesis of the primordial germ cell wall and cortex, a
distinctive form of peptidoglycan, between the membranes surrounding
the prespore is defined as stage IV and is represented as
thickening and graying of the ellipse. Deposition of the spore coat,
protective layers of proteins around the prespore, is defined
as stage V. The coat is represented as the black layer surrounding the
engulfed prespore. Coincident with coat and cortex formation,
the engulfed prespore is dehydrated, giving it a phase-bright
appearance, represented here as a light grey shading. Stage VI is
maturation, when the spore acquires its full resistance properties,
although no obvious morphological changes occur. Stage VII represents
lysis of the mother cell, which releases the mature spore into the
environment.
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The cell
type-specific, compartmentalized programs of gene expression result
from the cell type-specific activity of RNA polymerase sigma factors:
F and then G in the
prespore and E and then
K in the mother cell (Fig.
2) (231).
Compartmentalization of gene expression in each cell type is coupled to
morphogenesis, with F and E
becoming active after asymmetric division and G and
K becoming active after engulfment of the
prespore by the mother cell. Thus, the activation of
G and K also represents
compartmentalization in the sense of expression after but not before
completion of engulfment. The key regulators of sporulation discussed
below have been identified in all species of endospore former whose
genomes have been sequenced, including the pathogens Bacillus
anthracis and Clostridium difficile
(277). Thus, conclusions
from the study of B. subtilis are generally valid for members
of the family Bacillaceae and illustrate general features of
cell differentiation.
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FIG. 2. Intercompartmental
communication during sporulation. The parallel vertical
lines represent the two membranes separating the prespore
(right) from the mother cell (left). Diagonal red lines represent
pathways of intercompartmental posttranslational activation, and
vertical black arrows represent intracompartmental transcriptional
activation. Fluorescent micrographs represent cells stained with FM4-64
(red) to visualize the cell membranes and expressing
compartment-specific gfp fusions to spoIIQ,
spoIID, sspA, and gerE for
F, E, G,
and K, respectively. The prespore
membranes are not stained in the G and
K images because engulfment is complete and the
prespore membranes are now inaccessible to the lipophilic
FM4-64 stain. F, active in the prespore,
is the first compartmentalized factor during sporulation. It
triggers expression of SpoIIR, which activates the inferred
receptor protease SpoIIGA, located in the asymmetric septum.
Upon receipt of the signal, SpoIIGA processes the inactive
precursor pro-E into active E
in the mother cell. RNA polymerase with E
transcribes the spoIIIA operon, whose products then signal
across the prespore membrane to activate
G, expressed in the prespore under the
control of F but held inactive by SpoIIAB
(and probably other factors) until this signal is received.
SpoIIIJ is also required for this signaling; although only
required (and therefore only represented) in the prespore, it
is expressed vegetatively and is presumably present in both
compartments. Although not represented here, transcription of
spoIIIG (encoding G) requires an unknown
signal from the mother cell as well as the SpoIIQ protein,
expressed in the prespore under the control of
F. Once G becomes active, it
causes expression of SpoIVB, which is inserted into the inner
prespore membrane. SpoIVB triggers processing of
pro-K, which is synthesized in the mother cell from
the E-directed sigK gene. The processing
enzyme is thought to be SpoIVFB, which also expressed in the mother
cell under the control of E but does not act upon
pro-K until it receives the SpoIVB signal from the
prespore.
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In this article we review the process of
spore formation. We focus primarily on B. subtilis but include
discussion of other species where we think it appropriate. We discuss
in detail the genetic and biochemical experiments that have led to the
discovery and characterization of cell-specific programs of gene
expression. Since sporulation follows a distinct series of
morphological and genetic stages, we detail steps in the order that
they naturally occur, as though following a single cell through the
entire developmental process. The cell-specific changes in gene
expression that occur during sporulation are coupled to morphogenesis.
The two major phases of compartmentalization are associated with two
major morphological events, completion of septation and completion of
engulfment. Consequently, we start with a brief description of the
morphological changes during sporulation. We discuss in depth the
events leading to the asymmetrically located sporulation division,
which primes the organism for the compartmentalization of gene
expression that follows the division. Compartmentalized gene expression
is associated with the activation of the four sporulation-specific
factors. We discuss the activation of each. We pay particular
attention to regulation of F because it is the
first factor whose activity is compartmentalized during
sporulation and because in vitro and in vivo analysis of its activation
has progressed furthest.
Many of the loci discussed in this
review were identified almost three decades ago as spo loci,
because mutations in them blocked spore formation. Since then, our
understanding of the roles of those loci has increased enormously. In
general, spo loci encode proteins with unique roles in spore
formation and, in some cases, in regulation of compartmentalization.
They have provided the underpinning of much of our knowledge of
compartmentalization. However, in the last decade it has become clear
that there are also regulators, or regulatory mechanisms, which have
overlapping rather than unique roles. The corresponding genes were
generally missed in earlier studies because mutation in them had a
comparatively mild effect on spore formation. Nevertheless, such
overlapping regulatory mechanisms play an important part in the
compartmentalization of gene expression, and they are an active area of
research. For the main sections in the review, we use a historical
approach in describing the development of our knowledge of
compartmentalization. We think that this historical approach is
important for appreciating much of the present and past thinking about
compartmentalization.
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MORPHPOLOGICAL STAGES OF SPORULATION
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In order to discuss compartmentalization
of gene expression, we first review the morphological changes in the
developmental process, with which changes in gene expression are
associated. Formation of heat-resistant spores from vegetative cells of
B. subtilis takes about 7 h at 37°C. The
morphological changes during sporulation were initially characterized
by electron microscopy
(145,
251). The basic sequence
of changes is similar for all species of Bacillus and
Clostridium that have been studied
(79) and is illustrated
in Fig. 1. Identification
of successive stages by Roman numerals follows the convention
introduced by Ryter
(251) and now generally
used. The vegetative cell is designated stage 0. Formation of an axial
filament of chromatin, where two copies of the chromosome condense and
elongate to form a filament that stretches across the long axis of the
cell, is defined as stage I
(21,
29,
308). Subsequently, the
cell divides at a subpolar site, resulting in the formation of two
unequally sized daughter cells.
Completion of septation is
designated stage II. At the time of asymmetric division, only
approximately one-third of a chromosome is present in the smaller
prespore (also called the forespore), but the
remaining two-thirds is rapidly pumped in by the DNA translocase
SpoIIIE (17,
303), resulting in two
cells with unequal volumes but identical genomes. Next, the polar
septum undergoes septal thinning, followed by bulging of the
prespore into the mother cell and migration of the septal
membrane around both sides of the prespore. When the migration
is complete, the membranes fuse at the cell pole, pinching off the
prespore and releasing it within the mother cell as a
free-floating protoplast that is surrounded by two membranes, one
derived from each of the two cells; completion of engulfment is
designated stage III
(227). Engulfment is
followed by the deposition of two peptidoglycan layers, the primordial
germ cell wall and the cortex, in the space between the membranes
surrounding the prespore (stage IV)
(81). Following this
deposition, a complex structure of proteins on the outside surface of
the prespore, known as the coat, is constructed (stage V)
(47,
109). This stage is
followed by maturation of the spore (stage VI), when it gains
resistance to UV radiation and high temperature
(208). Lastly, the
mother cell lyses (stage VII), releasing the mature spore into the
environment. Germination and outgrowth, followed by a resumption of the
vegetative growth cycle, occur when the spore finds itself in a
nutrient-rich environment
(213). Sporulation
mutants are denoted by the stage in the process at which they are
blocked (e.g., spoII mutants complete asymmetric septation but
fail to complete engulfment). The names for sporulation loci include
the stage of blockage caused by mutation and a distinguishing letter
designation (e.g., spoIIA)
(228,
231).
The profound
morphological changes that occur during sporulation are coupled to
global changes in gene expression, which are effected by activation of
alternative RNA polymerase factors (Fig.
2)
(228,
231). Activation of
H (and the response regulator Spo0A) in the
predivisional cell leads to expression of factors important for axial
filament formation, asymmetric division, and compartmentalization of
gene expression. Immediately after asymmetric division,
F becomes active in the prespore, rapidly
followed by activation of E in the mother cell. The
separate lines of gene expression drive engulfment of the
prespore by the mother cell and result in synthesis of the
late-compartment-specific factors. Upon completion of
engulfment, G becomes active in the
prespore and K becomes active in the
mother cell. Coat and cortex synthesis, spore maturation, and mother
cell lysis are driven by these late stages of cell-specific gene
expression. Each step is dependent upon completion of all of the
previous steps except axial filament formation (see
below).
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INITIATION OF SPORULATION
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Gene expression becomes compartmentalized immediately after the
spore septum has formed. To understand how this compartmentalization
happens, it is important to explore the events leading to it. In this
section we briefly discuss the activation of the master sporulation
response regulator Spo0A and the alternative factor
H. For more focused reviews on the initiation of
sporulation, we refer the reader to references
27 and
223.
The Phosphorelay
Spo0A is the master regulator for entry into spore
formation. It is activated by the phosphorelay, a more complex version
of the classic two-component system
(26). In turn,
Spo0A-PO4 activates transcription of genes required for
axial filament formation and for asymmetric division. It also activates
transcription of the genes encoding the early compartmentalized
factors, F and E, as
well as their regulators. Sporulation is initiated in response to a
number of external and internal signals that are integrated into the
phosphorelay, including signals for nutrient starvation, cell density,
and cell cycle progression
(27,
223,
291).
At least
five kinases are involved in the phosphorelay: KinA, KinB
(290), KinC
(160), KinD
(134), and KinE
(67), of which KinA and
KinB are the primary kinases for initiation of sporulation. In response
to unidentified stimuli, they autophosphorylate and then donate their
phosphate groups to the response regulator Spo0F. Spo0F lacks an output
domain and is incapable of activating transcription; it serves only as
an intermediary in the phosphorelay. The phosphotransferase
Spo0B transfers the phosphate from Spo0F-PO4 to Spo0A
(26). The phosphorelay is
also subject to negative regulation: for example, the phosphatases
Spo0E, YisI, and YnzD dephosphorylate Spo0A, thereby preventing its
activation (210,
221). In order to ensure
that sporulation occurs only under the appropriate conditions, the
phosphorelay must integrate different intracellular and extracellular
signals. The mechanisms responsible for this integration are described
below.
Cell density.
Efficient sporulation requires high cell density
(101). When cell density
is low, the Rap (response regulator aspartyl phosphatase) proteins
RapA, RapB (222), and
RapE (133)
dephosphorylate Spo0F-PO4, preventing Spo0A activation. The
rapA and rapE genes are cotranscribed with a
downstream open reading frame encoding the signaling peptide precursors
PhrA and PhrE, respectively, which are processed and exported out of
the cell. As cell density increases, the processed peptides are
imported by the oligopeptide permease (Opp) and inhibit the activity of
RapA and RapE; similarly, the processed product of PhrC (CSF
[competence- and sporulation-stimulating factor])inhibits RapB. Inhibition of the Rap proteins prevents
dephosphorylation of
Spo0F-PO4 and allows
phosphorylation of Spo0A and the
initiation of sporulation when cell density is high
(133,
220).
Nutrient starvation.
In addition to high cell
density, nutrient starvation is also required for the initiation of
sporulation. A dramatic drop in the concentration of GTP and GDP
correlates with the onset of sporulation, and inhibition of GMP
synthesis by decoyinine treatment induces sporulation in the absence of
nutrient starvation
(200). CodY has recently
been identified as the key sensor of guanine nucleotide levels.
Disruption of the codY gene allows sporulation to occur in the
presence of excess nutrients, and the ability of the CodY repressor to
bind DNA correlates with the GTP concentration. As a consequence, when
GTP levels drop upon entry into stationary phase, CodY-regulated genes
are derepressed (239).
Microchip array analysis has identified phrA, phrE,
and kinB, all positive regulators of the phosphorelay, as
targets of CodY repression
(204). Therefore, one
way that nutrient starvation is integrated into the decision to
sporulate is transcriptional regulation of phosphorelay components via
CodY. In addition, sporulation is also subject to catabolite repression
(117) and requires a
functioning Krebs cycle
(131), although the
molecular basis of these dependencies is
unknown.
Cell cycle.
In addition to factoring extracellular conditions
such as cell density and nutrient availability into the decision to
sporulate, the intracellular environment is monitored as well. Damage
to DNA and blocking of either the initiation or progression of DNA
replication prevent the initiation of sporulation
(126,
128,
129,
132,
165). These conditions
lead to the expression of Sda (suppressor of dnaA) because of
the presence of binding sites in the sda promoter region for
the repressors DnaA and LexA, which no longer repress when DNA
replication is blocked or DNA is damaged, respectively. Sda impairs
KinA autophosphorylation, blocking
the phosphorelay (28). As
a result, a developmental checkpoint is established that only allows
cells with undamaged, replicating chromosomes to proceed into
development.
An additional mechanism is thought to link
chromosome partitioning status to sporulation. Spo0J and Soj
(suppressor of spo0J) (also known as Spo0JB and
Spo0JA, respectively) are members of the
plasmid-partitioning families of proteins ParB and ParA, respectively
(130). Spo0J colocalizes
to cell poles with the chromosomal origin of replication
(175), binds to sites
near the chromosomal origin
(174), and is required
for optimal efficiency of chromosome segregation
(130). In the absence of
Spo0J, Soj binds to the promoter regions of at least four
Spo0A-responsive genes (spo0A, spoIIA,
spoIIE, and spoIIG) and represses their transcription
(33,
191,
237,
238). This effect
appears to be mediated by dynamic protein localization; when Spo0J is
present, Soj oscillates between sites near the poles of the cell,
presumably preventing stable DNA-protein interaction and
transcriptional repression. However, in the absence of Spo0J, Soj
remains static and represses developmental transcription
(191,
238). Although it is
tempting to speculate that Spo0J and Soj sense chromosome partitioning
status and regulate the initiation of sporulation accordingly, direct
evidence for this model is lacking. However, consistent with a role in
monitoring the cell cycle, recent studies have linked these proteins to
cell division, to initiation of DNA replication, and to axial filament
formation (11,
163,
209,
308).
Spo0A Regulon
The sum of all of these interactions determines if
enough Spo0A-PO4 has been generated to initiate sporulation.
Spo0A-PO4 can either activate or repress transcription by
binding to a 7-bp sequence, TGNCGAA, where N is any nucleotide, in or
near promoters recognized by the vegetative factor
A and the alternative factor
H
(223). This binding
results in global changes in gene expression, altering the expression
profile of over 500 genes, which represent approximately one-eighth of
the total genes in B. subtilis
(71). Additional genomic
analysis has revealed that 121 of these genes are under the direct
control of Spo0A, with approximately one-third being positively
regulated and the remainder being negatively regulated; 25 of the
regulated genes are themselves transcription factors, indicating that
many of the transcriptional changes caused by Spo0A are indirect
(203).
A number of
key spo loci are directly positively regulated by Spo0A: the
spoIIA and spoIIG operons, encoding the
prespore- and mother cell-specific transcription factors
F and E, respectively; and
spoIIE, encoding a bifunctional protein phosphatase that is
required for asymmetric division and F activation
(231). In addition, the
gene encoding the effector of axial filament formation, racA,
is under the control of Spo0A
(21,
308). Thus, Spo0A
activates the synthesis of factors required for chromosome remodeling,
asymmetric division, and the compartmentalized gene expression that
immediately follows. The racA gene was identified by
functional analysis of the Spo0A regulon
(21), and such analyses
will most likely reveal additional genes involved in
sporulation.
Role of H
In addition to the phosphorelay and the
main vegetative factor A, the
transition-state regulator H is also required for
the initiation of sporulation. Regulation of H
synthesis and activity is not well understood but involves both
posttranscriptional and posttranslational mechanisms
(106,
177).
H regulates a number of phosphorelay genes:
H-dependent transcription of spo0A is
essential for sporulation
(270), and
kinA, kinE, and spo0F also have
H-dependent promoters
(23,
236). In addition,
transcription of several phr genes encoding peptide
precursors, some of which function to reverse phosphorelay inhibition
by Rap phosphatases, is also dependent upon H
(197). Other
H-regulated genes are important for later events in
sporulation: H-dependent transcription of the
essential cell division operon ftsAZ is required for efficient
asymmetric septation (19,
94,
98), and the
spoIIA operon, which encodes the prespore-specific
transcription factor F and its regulators, is under
the control of H
(302). A substantial
portion of the H and Spo0A regulons overlap: for
example, spoIIA and racA are regulated by both
transcription factors. The H regulon has recently
been characterized by microchip array analysis
(23), and functional
analysis of this regulon has resulted in the independent identification
of racA
(308).
There are several substantial differences in
the events associated with the sporulation division compared with those
associated with the vegetative division
(114,
227). Any or all may be
important for priming the compartmentalization of gene expression which
follows that division. The first is the formation of an axial filament
of chromatin, in which both chromosomes in the predivisional cell
elongate into a filament that stretches the length of the long axis of
the cell. This structure was characterized by electron microscopy
(78,
188,
252) and later by
fluorescence microscopy
(29,
300).
Genetic Control
Standard genetic analysis failed to identify effectors
of axial filament formation, and only recent genomic and cell
biological studies have allowed identification of the components
involved. Despite their name, none of the classic spo0
mutations clearly prevented axial filament formation
(228): spo0H
mutants formed axial filaments
(29,
94), whereas
spo0A, spo0B, and spo0F mutants underwent an
additional symmetric division during sporulation, and it was not clear
if that was preceded by axial filament formation
(29,
53). Some insight into
the process was derived from analysis of the SMC (structural
maintenance of chromosomes) protein, required for chromosome compaction
and partitioning (25,
176). Mutants lacking
this protein are able to activate Spo0A but are unable to form axial
filaments (99),
suggesting that an effector of axial filament formation is either
missing or nonfunctional in this background. It was also found that
asymmetric division did not occur in the absence of axial filament
formation, suggesting the existence of a checkpoint that functioned to
couple the two events
(99). Such a checkpoint
would ensure that asymmetric division would trap the origin-proximal
region of a chromosome in the prespore, providing a template
for transcription by E-F (RNA
polymerase core enzyme, E, associated with F) and
an anchor for chromosome translocation into this
compartment
DivIVA.
Several lines of investigation provided insight into
the genetic control of axial filament formation. The first was
a study of the DivIVA protein of B. subtilis,
considered the functional homologue of Escherichia coli MinE
in that it restricts the division-inhibition proteins MinCD to the cell
poles and so ensures mid-cell division during vegetative growth
(34,
56). divIVA
mutants have severe growth and sporulation defects, but the
isolation of mutants specifically defective in sporulation suggested
that DivIVA played a dedicated role in development (Fig.
3). These mutants frequently formed anucleate prespores
(287), indicating that
the proposed checkpoint coupling axial filament formation and
asymmetric division (99)
had been disrupted.
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FIG. 3. Chromosome
partitioning and genetic asymmetry. A single cell progressing through
sporulation is represented on the right. Disordered internal lines
represent the nucleoids. The earliest (topmost) cell is drawn as having
two complete chromosomes, although it may contain one partially
replicated chromosome. Through the action of DivIVA, RacA, and Soj, the
two complete chromosomes (or the partially replicated chromosome) are
remodeled into an axial filament that extends across the long axis of
the cell, represented in the second cell. After asymmetric division
occurs, the prespore contains only the origin-proximal
one-third of a chromosome, whereas the mother cell contains one
complete chromosome and two-thirds of another; this partitioning
results in transient genetic asymmetry between the mother cell and the
prespore. For simplicity, the septum is represented as a
single line. A portion of the third cell has been expanded in order to
represent the asymmetry more clearly; the hatched ovals represent the
DNA translocase SpoIIIE; the locations of several genetic loci
are noted; and F is depicted as being active in the
prespore and E is depicted as being active
in the mother cell. Within about 15 min of the asymmetric
division, SpoIIIE pumps the remaining two-thirds of
the prespore chromosome into this
compartment, restoring genetic
symmetry.
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RacA.
Genomic approaches made it possible to elucidate the
role that DivIVA played in axial filament formation. One approach
identified Spo0A-regulated genes by microchip array
(71), systematically
disrupted them, and screened the mutants for changes in chromosome
segregation (21). In a
separate study, H-regulated genes were identified
by microchip array, and candidate genes for partitioning function,
including those predicted to have DNA-binding domains, were disrupted
and characterized (308).
Both approaches resulted in identification of a gene that, when
disrupted, caused an anucleate prespore phenotype. The encoded
protein, RacA (remodeling and anchoring of the chromosome A), was found
to bind nonspecifically to the chromosome and also to the pole of the
cell, acting as a bridge connecting the two. Localization of RacA to
the cell pole is dependent upon DivIVA, explaining why mutations in the
corresponding genes cause similar phenotypes, although those caused by
mutations in divIVA are more severe
(21,
287,
308). From their
observations, Ben-Yehuda and colleagues
(21) proposed that once
the origin-proximal region of the chromosome reaches the pole of the
cell, RacA binds DivIVA and displaces the division inhibitor MinCD
(192,
295), triggering
asymmetric division
(21).
Although the
prospect of a checkpoint linking axial filament formation to asymmetric
division is exciting, it still requires direct testing. The original
observation that smc mutants fail to form axial filaments
remains unexplained (99);
it may be that RacA cannot bind to the uncondensed and disorganized
chromosomes of smc mutants
(25,
176). racA and
divIVA mutants also undergo asymmetric division even though
axial filament formation is impaired
(21,
287,
308), whereas the
smc mutant is deficient in both processes
(99). Two plausible
explanations are that RacA and DivIVA prevent asymmetric division in
the smc mutant, enforcing the checkpoint proposed by Graumann
and Losick (99) (see
above) or that defects in asymmetric division and axial filament
formation are independent in this background, placing the existence of
this checkpoint in doubt. Epistasis tests could help distinguish
between these possibilities. For example, in the former case,
racA and divIVA mutations should restore asymmetric
division to an smc mutant, whereas in the latter case they
should not. More work is needed to explore the relationship between
axial filament formation and asymmetric
division.
Soj.
The fact that racA mutations cause a less
severe defect than divIVA mutations
(21,
287,
308) suggested that at
least one additional factor was involved in axial filament formation.
Indeed, it was found that a mutant lacking RacA and the transcriptional
repressor Soj (33,
191,
238) displayed a
phenotype approximating the more severe phenotype caused by a
divIVA mutation
(308), a surprising
result because no partitioning function had previously been attributed
to Soj. However, such a phenotype could be mediated by its interaction
partner, Spo0J, which is required for optimal efficiency of vegetative
chromosome partitioning
(130) and localizes to
the origin region of the chromosome
(174,
175). The RacA-Soj
system is an example of redundancy in sporulation controls and is
especially noteworthy because it led to the attribution of a novel
function to Soj, one that was unlikely to be observed in a
racA+ genetic
background.
Polar localization region.
In a separate study of axial
filament formation, the use of systematic chromosomal inversions
identified a polar localization region, located 150 to 300 kbp
from the origin of replication, that is required for efficient trapping
of DNA in the prespore
(307). Although an
attractive hypothesis is that this region is the principal binding site
for RacA, chromatin immunoprecipitation experiments suggest that a
different region (60 to 80 kbp from the origin) is preferentially bound
(21). As a result, the
relationship of the polar localization region to the RacA-Soj-DivIVA
system remains
unclear.
Once a cell has formed the axial filament, the next
major event in sporulation is asymmetric division. Since the cell
normally divides at mid-cell with remarkable accuracy
(198), this switch
requires a dramatic relocalization of the cell division apparatus
(61). By dividing at a
polar site, the cell becomes genetically and morphologically
asymmetric, and the asymmetry leads to different cell fates. The
asymmetric division is critical to the establishment of
compartmentalized gene expression. The division has much in common with
vegetative division (61,
114), but the
distinguishing features are presumptively ones that might lead to
compartmentalization of gene expression. It is the distinguishing
features that are considered here.
FtsZ Ring Switching
Dynamic repositioning.
During vegetative growth, the essential prokaryotic
tubulin homologue FtsZ forms a ring (the Z ring) at mid-cell, where
division subsequently occurs
(298). However, during
sporulation, activation of Spo0A triggers the formation of Z rings near
both poles of the cell
(167). The switch has
recently been characterized by deconvolution microscopy. The use of
this technique revealed that, during sporulation, a Z ring initially
forms at mid-cell but the FtsZ then redeploys to sites near both poles
through the formation of a dynamic helical intermediate
(19). This intermediate
resembles the helical structures formed by two bacterial actin
homologues, Mbl and MreB
(137); one possibility
is that FtsZ relocates by tracking along these
structures.
Genetic control.
Early studies showed that the ftsAZ operon
contained three promoters, one of which was activated during
sporulation by H
(94,
98). However, deletion of
this promoter had only a moderate effect on asymmetric division
(98). Similarly,
disruption of the spoIIE locus, encoding a critical activator
of F in the prespore
(8,
49), also had a moderate
effect on polar Z ring formation
(19,
149) and asymmetric
division (16,
228). However,
simultaneous ablation of spoIIE and the
H-dependent promoter of ftsAZ resulted in
severe impairment of polar Z ring formation and asymmetric division.
Conversely, if ftsAZ overexpression was combined with
expression of spoIIE, asymmetric division could be triggered
during vegetative growth
(19). Therefore,
spoIIE induction and increased ftsAZ expression play
overlapping roles during sporulation in ensuring polar Z ring formation
and asymmetric
division.
SpoIIE.
The discovery of a role for SpoIIE in polar
Z ring formation (19,
149) reinforced previous
ultrastructural studies that had implicated this protein in asymmetric
division. These studies found that null mutations in spoIIE
resulted in rare asymmetric septa that were aberrantly thick, whereas
several point mutations blocked sporulation but allowed the formation
of typical sporulation septa at normal frequency
(16,
228). Consistent with
this role, it was found that SpoIIE localizes to asymmetric
division sites (10,
14) in an FtsZ-dependent
manner (168). FtsZ and
SpoIIE also interact in vitro and in yeast two-hybrid assays
(186). However, how this
interaction assists polar Z ring formation and asymmetric division is
unknown. Some possibilities include anchoring an end of the FtsZ helix
to the cell membrane, antagonizing MinCD near the pole of the cell, and
recognizing a polar marker
(19). In addition to
SpoIIE and increased ftsAZ expression, there is
evidence that MinCD and SpoVG may play minor roles in selecting the
asymmetric division site
(15,
195).
Abortively Disporic Phenotype
In spo+ strains,
surface annular structures (cloisons) and Z rings appear at both polar
sites (19,
167,
251), indicating that
the cell has two potential polar division sites. However, a septum is
normally formed at only one of these sites
(251). In certain
mutants, both potential polar division sites are utilized, resulting in
a three-chambered organism consisting of two smaller prespores
separated by a larger central compartment. This is the abortively
disporic phenotype, which is associated with mutations in some
spoII loci
(228,
252). Mutants that
demonstrate the abortively disporic phenotype can initiate sporulation
but have defects in activating the mother cell-specific transcription
factor E
(124). Three proteins
expressed in the mother cell under E control,
SpoIID, SpoIIM, and SpoIIP, are required to
prevent the second asymmetric division
(57,
232). Although it was
puzzling why B. subtilis would generate two potential division
sites during sporulation, recent studies have provided some insight.
Cells with anucleate prespores are frequently observed in
racA mutant cells, which are deficient in axial filament
formation. However, a substantial proportion of these cells undergo a
second asymmetric division at the other end of the cell, and if they
successfully capture DNA in the second prespore, they form
spores (21,
308). This result
suggests that the ability to divide at both asymmetric division sites
is a failsafe mechanism to ensure successful sporulation even in the
absence of axial filament
formation.
Which End of the Cell?
An unanswered question is how the cell determines at which end of
the cell it will divide. Although FtsZ rings form at both potential
polar division sites, the next known protein to assemble, FtsA,
localizes to only one of them
(76), indicating that
FtsA may play some role in selecting which of the two potential
division sites is utilized. Chromosome segregation into the
prespore or mother cell appears to be essentially random with
respect to time of replication, and so chromosome age is presumably not
a factor in determining which end becomes the prespore
(43,
65). In most
circumstances and in a range of species, spores are formed almost
exclusively at the older pole of the cell, clearly suggesting that the
pole is a determinant of asymmetry and compartmentalization
(52,
112,
113). However, when the
sporulation procedure involves centrifugation (with a force of perhaps
5,000 x g) and 25 mM Mg2+, spore
position is essentially random with respect to pole age
(52). Thus, polar
determinism can be lost without losing asymmetric division or
compartmentalization.
Differences between Sporulation Septum and Vegetative Division Septum
The
asymmetrically located sporulation division is often considered the
defining early morphological event in sporulation. The machinery for
asymmetric division is similar to that used for vegetative division
(61). However, there are
several distinctive features of the sporulation division
(114,
227) in addition to
those described above. Since the division is critical to the
compartmentalization of gene expression that follows, it is useful to
summarize those distinctive features. (i) The sporulation division
septum is much thinner than the vegetative division septum. (ii) The
two cells that result from the sporulation division do not separate
from each other, as occurs following vegetative division. Rather, the
mother cell engulfs the prespore. (iii) Autolysis of the wall
material (peptidoglycan) within the sporulation septum begins in the
center of the septum, and ultimately there is apparently complete loss
of wall material. In contrast, autolysis of the wall material of the
vegetative septum begins at the periphery of the septum and proceeds
inwards. Moreover, there is little loss of wall material—the
split septum provides the wall for the poles of the nascent cells
(227). (iv) Prior to the
sporulation division, the two chromosome origins and associated
proteins move to the extreme poles of the cell rather than to a
subpolar location, as in vegetative division
(21,
175,
300,
308). (v) The septum is
asymmetrically located, with respect to the cell poles, during
sporulation but not during vegetative growth
(251) (vi) Several
proteins become associated with the sporulation septum that are not
associated with the vegetative division septum
(10,
14,
72). (vii) Complete
partitioning of a chromosome into the prespore occurs after
septation (303,
310), so that there is
genetic asymmetry between the prespore and the mother cell
when they are first formed
(54,
84,
305). (viii) After the
sporulation division, different programs of gene expression are
initiated in the two daughter cells
(231). These programs
are driven by activation of cell-specific factors that direct
RNA polymerase to transcribe different genes, which encode factors
responsible for establishing the very different fates of the
prespore and the mother
cell.
|
TRANSFER OF DNA INTO THE PRESPORE
|
|---|
At this stage of sporulation, the two chromosomes have
extended into a filament stretching along the long axis of the cell,
with their origin regions near the poles
(21,
300,
308). The cell divides
near one of the poles, trapping the origin-proximal one third of a
chromosome in the smaller prespore and leaving one chromosome
and two thirds of another in the larger mother cell (Fig.
3)
(305). As a consequence,
the two cells are now genetically asymmetric. This asymmetry has
important implications for compartmentalization of gene expression
(54,
84), as discussed below.
The developing organism also has a major challenge in that it must
ensure that the prespore compartment receives a complete
chromosome The transfer of the origin-distal two-thirds of a chromosome
from the mother cell into the prespore is concomitant with the
activation of different transcription factors in the prespore
and mother cell. Although both of these events are thought to occur
simultaneously, for the sake of clarity we will first discuss the
problem of DNA transfer and then turn to the compartmentalization of
gene expression.
The critical locus for chromosome translocation
is spoIIIE. In spoIIIE mutants, the origin-distal
two-thirds of a chromosome remains trapped in the mother cell
(305). In
spo+ strains, the remaining DNA is actively
pumped across the asymmetric septum from the mother cell into the
prespore (303,
310). The DNA
translocase SpoIIIE localizes to the center of the sporulation
septum (264,
304), apparently by
anchoring to the chromosome
(20). SpoIIIE
then uses ATP to transport the chromosome into the prespore
(Fig. 3)
(17).
The question
remains why DNA is transported only from the mother cell into the
prespore. Does the location of the chromosome origin in the
prespore determine the direction of transfer? Or is it the
orientation of SpoIIIE in the center of the spore septum,
toward or away from the prespore? When discussing this
question, it is important to note that SpoIIIE is widely
conserved in nonsporeformers
(267), is expressed
vegetatively in B. subtilis
(82), and is involved in
chromosome partitioning in circumstances other than sporulation. For
example, SpoIIIE removes trapped nucleoids from minicells,
prevents chromosome bisection when DNA replication is transiently
inhibited (267) and when
cells lack SMC (24), and
is required for efficient partitioning in mutants defective in
terminating DNA replication or resolving chromosome dimers
(164). These situations
require either bidirectional movement or transfer out of the smaller of
two cells (the minicell), suggesting that SpoIIIE lacks an
inherent polarity.
Two recent studies have attempted to address
the question of the polarity of SpoIIIE during spore formation
by synthesizing SpoIIIE exclusively in either the
prespore or mother cell
(36,
265). Both laboratories
agree that expression of spoIIIE only in the mother cell is
sufficient to obtain DNA translocation into the prespore and
substantially to restore spore formation. However, they disagree about
the effect of expression only in the prespore. There were a
number of technical differences between the two studies that could
account for their different conclusions. As a consequence, more work
may be necessary to fully understand how polarity of DNA transfer by
SpoIIIE is
regulated.
|
COMPARTMENTALIZATION OF GENE EXPRESSION
|
|---|
After axial filament formation and asymmetric
division and concomitant with DNA transfer by SpoIIIE,
different programs of gene expression are established in the
prespore and the mother cell. These programs are directed by
cell-specific factors whose activation is coupled to landmark
morphological events. Asymmetric division triggers the activation of
F in the prespore, followed by activation
of E in the mother cell. Later in sporulation, the
completion of engulfment of the prespore by the mother cell
leads to the activation of G in the
prespore and K in the mother cell.
Therefore, in addition to complete spatial compartmentalization between
prespore and mother cell, sporulation gene expression is also
divided into temporal (pre- and postengulfment) phases. Throughout the
intermediate and late stages of sporulation, the mother cell and
prespore communicate with each other, sending and interpreting
biochemical signals to ensure that their genetic programs are
coordinated (Fig.
2).
In the sections
that follow, we briefly review the historical development of evidence
that gene expression is indeed compartmentalized. We then focus on the
activation of the particular factors that have been shown to
direct the compartmentalized gene expression. It is presumed that those
activation mechanisms hold the key to why activation is
compartmentalized. Whereas we now consider the evidence that gene
expression directed by the different factors is
compartmentalized to be compelling, our understanding of the mechanisms
of compartmentalization is still incomplete. A variety of regulators of
activation have been identified. Some of the regulators have
an essential function in spore formation, so that their mutational
inactivation eliminates spore formation. However, other regulators
appear to have partially or completely overlapping functions, so that
mutational inactivation of only one regulator may have little or no
effect on spore formation. Regulators of both types may be critical for
compartmentalization of the activity of the different
factors.
Developing Evidence that Gene Expression Is Compartmentalized
The different fates of
the prespore and the mother cell suggested that different
genes are expressed in the two compartments. This suggestion was
supported by biochemical characterization of extracts enriched for the
contents of the prespore or the mother cell
(5,
55,
88,
269). The first clear
evidence that expression of spo loci was compartmentalized
came from the study of genetically mosaic bacteria
(43,
226). The mosaics were
obtained by transforming spo mutants at the start of
sporulation so that only one of the two copies of the chromosome became
spo+. After division, mutant and wild-type
alleles were distributed randomly into the prespore and the
mother cell. Since the mother cell is destroyed and only the chromosome
in the prespore is inherited upon spore germination, it was
possible to infer the location of spo locus expression. For
several loci, the spores obtained gave rise to spo
mutant progeny, indicating that only the mother cell
chromosome required a spo+ allele for
sporulation to occur. Other loci that were tested yielded only
spo+ spores, indicating that, for
sporulation to occur, the allele on the prespore chromosome
had to be transformed to spo+
(43,
226,
230). Subsequently,
determination of ß-galactosidase activity in prespore-
and mother cell-enriched extracts from strains expressing
spo-lacZ transcriptional fusions provided strong support for
differential gene expression between mother cell and prespore
(reviewed in reference
59).
Direct
evidence that the expression of particular genes was completely
compartmentalized was obtained by the use of immunoelectron microscopy
with antibodies to small acid-soluble proteins (SASPs), which revealed
that these proteins are found exclusively within the prespore
(83). The utility of this
technique was expanded by using antibodies to ß-galactosidase
on samples from strains expressing spo-lacZ transcriptional
fusions (48,
189). The experiments
demonstrated that the activities of F and
G were confined to the prespore and those
of E and K were confined to the
mother cell (48,
83,
189).
Although
informative, immunoelectron microscopy is time-consuming and difficult
and suffers from low sensitivity. The study of compartmentalization
took a leap forward through the use of immunofluorescence microscopy
and then fluorescence microscopy of cells expressing transcriptional
fusions to gfp (encoding green fluorescent protein
[GFP]). These techniques provided greater sensitivity and
ease of use than electron microscopy, and GFP studies have the added
advantage that living cells can be analyzed. The use of these
techniques demonstrated that F and
E became active very soon after completion of
septum formation and that the activities were completely
compartmentalized into the prespore and mother cell,
respectively, within the limits of detection. Likewise, they
demonstrated that G and K
became active very soon after completion of engulfment, also completely
compartmentalized into the prespore and the mother cell,
respectively (105,
169,
233,
299,
314). A two-part
transcription probe provided a very different type of evidence for the
completeness of compartmentalization and also provided evidence that
the vegetative factor A continues to be
active in both prespore and mother cell throughout sporulation
(173).
|
ACTIVATION OF F
|
|---|
spoIIA Operon
Studies of the spoIIA locus have been critical
to our understanding of compartmentalization of gene expression. The
locus was found to be a tricistronic operon
(80,
229) that was
transcribed prior to asymmetric septation
(93,
218) in a Spo0A- and
H-dependent manner
(290,
301,
302). When the
spoIIA operon was sequenced, none of the open reading frames
bore obvious similarity to any genes in the limited database of the
time. However, shortly thereafter, it became clear that the third gene
in the operon, spoIIAC, encoded a product that was homologous
to an RNA polymerase factor
(62,
274), named
F
(183). The first targets
identified for E-F action were spoIIIG and
gpr (282,
285), and the site of
expression of spoIIIG was shown to be the prespore
(93,
142,
189), as it has been for
all E-F-directed genes analyzed subsequently
(231).
Posttranslational Regulation of F
Although the spoIIA
operon is expressed prior to asymmetric division
(93,
218),
F does not become active until after asymmetric
division (93,
142). It seemed likely
that F was subject to some form of
posttranslational regulation, and genetic analysis revealed that the
other two products of the spoIIA operon, SpoIIAA and
SpoIIAB, regulate its activity. Thus, overexpression of
SpoIIAB inhibited F activity, and mutation
of SpoIIAB increased F activity. Mutation
of SpoIIAA blocked F activation, but
activity could be restored to these strains by mutation of
SpoIIAB. Taken together, these results indicated that
SpoIIAB antagonized F, and that in turn,
SpoIIAA might antagonize SpoIIAB
(260). Consistent with
an inhibitory role for SpoIIAB, a separate study showed that
mutation of SpoIIAB caused hyperactivity of
F, blocked sporulation prior to asymmetric
septation, and caused extensive lysis
(38). Given that the
activity of F was confined to the prespore
during sporulation
(189), it was
hypothesized that its regulation by SpoIIAA and
SpoIIAB was responsible for
compartmentalization.
Biochemical analysis of the interactions
between members of the regulatory pathway began to shed light on the
mechanism of F regulation. It was shown that the
negative role of SpoIIAB was direct in that it bound
F, thus acting as an anti-sigma factor;
SpoIIAA antagonized the action of SpoIIAB and so is
an anti-anti-sigma factor
(51,
199). SpoIIAB
bore significant similarity to protein kinases, and it was found to
phosphorylate SpoIIAA on a serine residue at position 58
(199,
207). Mutation of this
serine residue to an alanine (mimicking
dephosphorylation)
resulted in constitutive F activity, and mutation
to an aspartic acid (mimicking
phosphorylation) blocked
F activity in vivo
(44), indicating that the
phosphorylation state of
SpoIIAA is critical for its ability to function as an
anti-anti-sigma factor. Therefore, SpoIIAB inhibits
F both directly, as an anti- factor, and
indirectly, by inactivating the anti-anti- factor
SpoIIAA. SpoIIE, a membrane-bound serine phosphatase,
dephosphorylates and thus activates SpoIIAA
(8,
49). Therefore, it
reverses the inactivation of SpoIIAA by SpoIIAB and
promotes activation of F. In addition,
SpoIIE localizes to asymmetric division sites
(10,
14), suggesting that it
may mediate a link between asymmetric division and
prespore-specific gene expression. The basic model of
F activation is illustrated in Fig.
4A.
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|
FIG. 4. Models
of F regulation. AA, AB, and E refer to
SpoIIAA, SpoIIAB, and SpoIIE, respectively.
The anti- factor SpoIIAB binds F
as a dimer but is represented here as a monomer for simplicity.
(A)Basic model of F regulation. The anti-
factor SpoIIAB binds F and holds it
inactive. This inhibition can be reversed by the anti-anti-
factor SpoIIAA. SpoIIAA is subject to regulation by
its phosphorylation state; it is
inactive when phosphorylated by
SpoIIAB (a serine kinase as well as an anti- factor)
and active when
dephosphorylated by
SpoIIE. Once
dephosphorylated,
SpoIIAA can bind SpoIIAB and liberate
F, activating prespore-specific
transcription. In this model, the
phosphorylation state of
SpoIIAA is directly correlated with F
activity, and the fate of SpoIIAB after F
liberation and the nucleotide binding status of SpoIIAB are
not considered. (B) Integrated model of F
regulation. In the predivisional cell, SpoIIAA and
SpoIIAB are present in two forms:
phosphorylation of SpoIIAA
by SpoIIAB results in free
phosphorylated (inactive)
SpoIIAA and a
SpoIIAA-SpoIIAB-ADP complex, while
unreacted SpoIIAB-ATP forms an inhibitory complex with
F. As long as the level of
dephosphorylated
SpoIIAA remains below a certain threshold, it will be absorbed
by the SpoIIAB-ADP sink. Asymmetric division triggers
activation of F in the prespore through
three possible mechanisms: generation of excess
dephosphorylated
SpoIIAA so that the sink can no longer absorb all of it,
sequestration of SpoIIAB in a long-lived complex with
SpoIIAA, and proteolysis of SpoIIAB. Asymmetric
division is thought to increase the level of
dephosphorylated
SpoIIAA either by activation of the phosphatase activity of
SpoIIE or equivalent distribution of SpoIIE into both
compartments, resulting in a much higher
SpoIIE/SpoIIAA-PO4 ratio
in the prespore. The complexes listed are not intended to
reflect a stoichiometric biochemical reaction; rather, they reflect the
different combinations thought to be formed by these factors and how
they correlate with asymmetric division and activation of
F. The mother cell (not shown) is presumed to
resemble the predivisional
cell.
|
|
Mechanisms of Compartmentalization
Although the known factors involved in F
regulation have been identified, it is still not clear why
F activity is confined to the prespore.
Models of compartmentalization have evolved over time; we will briefly
review them in roughly the order that they were proposed. Although some
of them are no longer widely accepted, we think that a discussion of
their supporting data and potential flaws is
useful.
ATP/ADP ratio.
Early in vitro experiments indicated that
SpoIIAB bound F in the presence of ATP,
whereas it bound SpoIIAA in the presence of ADP
(3,
44,
50). This result
suggested that different concentrations of these nucleotides in the two
compartments might be responsible for compartmentalization of
F activity to the prespore. However, it
was not clear how the proposed differential ATP/ADP ratio was
established in vivo, and direct evidence for the regulatory roles of
these nucleotides was lacking. Subsequent in vitro studies of the
interaction betwe
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