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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
Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
SUMMARY INTRODUCTION MORPHPOLOGICAL STAGES OF SPORULATION INITIATION OF SPORULATION The Phosphorelay Cell density. Nutrient starvation. Cell cycle. Spo0A Regulon Role of {sigma}H AXIAL FILAMENT FORMATION Genetic Control DivIVA. RacA. Soj. Polar localization region. ASYMMETRIC DIVISION FtsZ Ring Switching Dynamic repositioning. Genetic control. SpoIIE. Abortively Disporic Phenotype Which End of the Cell? Differences between Sporulation Septum and Vegetative Division Septum TRANSFER OF DNA INTO THE PRESPORE COMPARTMENTALIZATION OF GENE EXPRESSION Developing Evidence that Gene Expression Is Compartmentalized ACTIVATION OF {sigma}F spoIIA Operon Posttranslational Regulation of {sigma}F Mechanisms of Compartmentalization ATP/ADP ratio. Preferential inheritance. Inhibitor. Transient genetic asymmetry. SpoIIAB degradation. Cell division. SpoIIAB sink. Biochemical studies. Summary. {sigma}F Regulon ACTIVATION OF {sigma}E Compartmentalization Intercompartmental signaling. Protein localization. Role of Spo0A. Timing of activation. Regulation by gene position. {sigma}E Regulon ENGULFMENT OF THE PRESPORE BY THE MOTHER CELL Initiation of Engulfment Completion of Engulfment LATE PRESPORE-SPECIFIC TRANSCRIPTION FACTOR {sigma}G Transcriptional Regulation of spoIIIG Activation of {sigma}G {sigma}G Regulon LATE MOTHER CELL-SPECIFIC TRANSCRIPTION FACTOR {sigma}K Developmental Chromosome Rearrangement Pro-{sigma}K Processing Mother cell processing components. Prespore signaling. {sigma}K Regulon TEMPORAL CONTROL AND COMPARTMENTALIZATION SPORULATION OF COCCI DISRUPTION OF COMPARTMENTALIZATION CONCLUSION AND FUTURE DIRECTIONS ACKNOWLEDGMENTS REFERENCES
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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. |
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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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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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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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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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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. 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).
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.
HIn 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). |
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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).
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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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.
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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.
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).
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. 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.
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. |
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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.
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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.
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).
F
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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). FAlthough 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.
 View larger version (18K): [in a new window] |
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.
|
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 between these factors suggested that the ATP/ADP ratio was
not a critical factor in determining the partner to which
SpoIIAB bound
(187).
Preferential inheritance.
As an alternative mode of
regulation, it was proposed that a factor critical to
F activation was preferentially segregated to the
prespore during sporulation. As evidence, it was reported that
in protoplasts derived from sporulating cells expressing a
SpoIIE-GFP fusion protein, the fluorescent signal was found in
protoplasts derived from prespores and not from mother cells;
it was inferred that SpoIIE became located predominantly in
the prespore
(309). However, a
different study in which similar experiments were performed along with
careful computerized quantitation determined that the total
fluorescent signal was very similar in the prespore and in the
mother cell. Therefore, it was concluded that the previous result was
the consequence of similar amounts of protein being present in two
compartments of dramatically different sizes. In addition, time-lapse
microscopy of living cells clearly demonstrated that
SpoIIE-GFP is present in both the mother cell and the
prespore when first formed
(150), indicating that
preferential inheritance of SpoIIE into the prespore
is not a primary determinant ofcompartmentalization.
Inhibitor.
One assumption that had been made in models of
F regulation was that localization of
SpoIIE to the asymmetric division site was critical for its
role in F activation. When the N-terminal
transmembrane domains of SpoIIE were removed, the protein
failed to target to the asymmetric division site, yet sporulation was
only reduced by about 50% and about half of the bacteria
displaying F activity showed
prespore-specific expression
(9). This result contrasts
with the effect of inactivating spoIIE, where sporulation is
reduced at least 107-fold
(228) and
F activity is abolished
(9) and indicated that
when the location of SpoIIE in the cell was disturbed, some
other mechanism functioned to compartmentalize F
activity. It was suggested that a cytoplasmic inhibitor of
SpoIIE was able to regulate the soluble SpoIIE
protein and prevent it from becoming active in the mother cell.
However, the putative inhibitor has yet to be identified. It is
important to note that a separate study, using a different mutant of
SpoIIE that became solubilized during sporulation, found
similar results (73) but
interpreted them as supporting the preferential inheritance model
(309).
Transient genetic asymmetry.
Studies of chromosome
partitioning during sporulation helped generate a new concept
for compartmentalization studies. Analysis of the effects of
spoIIIE mutations revealed that at the time of asymmetric
division, the prespore and mother cell contained different
sets of genes in that only the origin-proximal one-third of a
chromosome was present in the prespore, whereas the mother
cell contained one complete chromosome and the origin-distal two-thirds
of another (Fig. 3)
(305). It takes about 15
min before the prespore receives a complete chromosome
(148,
232). It was proposed
that this transient genetic asymmetry could be the key to establishing
compartmentalized gene expression
(84). This proposal was
tested by placing the gene encoding F at
the amyE locus very near the origin of replication,
so that it was present in the prespore at the time
of asymmetric division, and leaving spoIIAB near
the terminus, so that it was initially absent from this compartment
(Fig. 3). This arrangement
of genes supported a modest level of sporulation even in the absence of
the normally essential factors SpoIIAA and SpoIIE
(84). These results
clearly suggested that transient genetic asymmetry could play a role in
compartmentalization of F activity. Fransden and
colleagues favored the existence of a gene near the terminus that
encoded a cytoplasmic inhibitor of SpoIIE
(84), so that transient
genetic asymmetry would deplete this factor from the prespore.
Although this putative factor remains unidentified, the concept of
transient genetic asymmetry has had a dramatic impact on
studies of compartmentalization.
A separate study
addressed the role of transient genetic asymmetry by moving the entire
spoIIA operon from its normal chromosomal location near the
terminus to the amyE locus near the origin of replication
(Fig. 3). Although this
relocation, in itself, had only a mild effect on sporulation (40 to
80% of the parental strain value), when it was combined with the
mutant, cytoplasmic form of SpoIIE, which also had only a mild
effect in itself (9),
there was a synergistic effect, and sporulation was severely reduced
(<1% of that of the parental strain)
(54). Therefore,
targeting of SpoIIE to asymmetric division sites and the
natural chromosomal location of the spoIIA operon are
partially redundant factors that contribute to activation of
F in the prespore. The question remained
how the chromosomal position of spoIIA could compensate for
mislocalization of
SpoIIE.
SpoIIAB degradation.
In a parallel line of
investigation, it was discovered that a C-terminal truncation of
SpoIIAB severely impaired F activation and
sporulation. Immunoblotting revealed that this mutant protein was much
more stable than the wild type, indicating a key role for the
instability of SpoIIAB in regulation of F.
In the absence of its binding partners SpoIIAA and
F, a unique C-terminal motif of SpoIIAB
(215) targets the
protein for degradation by the ClpCP protease complex
(214). It was proposed
that the natural chromosomal position of spoIIA near the
terminus and the instability of SpoIIAB result in the
anti-sigma factor's being temporarily depleted from the
prespore compartment following asymmetric division. By
combining this result with the transient genetic asymmetry experiments,
a holistic picture began to emerge. When SpoIIE is localized
to the asymmetric septum and the spoIIA operon is located near
the origin, SpoIIAA is efficiently
dephosphorylated that it
can overcome the increased concentration of SpoIIAB that is
present in the prespore. Conversely, when the phosphatase
activity of SpoIIE is diminished by its mislocalization, the
natural chromosomal position of spoIIA near the terminus
allows SpoIIAB to be depleted from the prespore by
proteolysis. In both scenarios, F activation and
sporulation occur with high efficiency. Only when the phosphatase
activity of SpoIIE is reduced via impaired localization and
SpoIIAB in the prespore is replenished by the
presence of the spoIIA operon in this compartment is the
activation of F, and thus sporulation, severely
impaired (54,
214).
Although
genetic asymmetry is emerging as an exciting new concept, it is
important to note that the genetic asymmetry is only transient. The DNA
translocase SpoIIIE acts to export the remaining two-thirds of
the chromosome from the mother cell to the prespore, a process
that is estimated to take as little as 15 min
(148,
232). Furthermore, the
half-life of SpoIIAB is about 30 min
(214). Thus, the decline
in SpoIIAB relative to F is relatively
small. Nevertheless, small changes such as this are presumably
sufficient to initiate prespore-specific F
activation. Not only that, F must be activated very
rapidly after septum formation and, following F,
E must be activated in the mother cell if formation
of a second septum at the other end of the organism (the abortively
disporic phenotype) is to be prevented
(124); the second septum
may be formed as soon as 10 min after the first
(77,
232). It is as if the
decision to activate F is balanced on a knife edge;
just a minor reduction in the concentration of SpoIIAB in the
prespore is presumably sufficient to trigger a very rapid
cascade of events that ensure activation of F in
the prespore, and only in the
prespore.
Cell division.
An important question is how the
phosphatase activity of SpoIIE is regulated (if at all) so
that the level of
dephosphorylated
SpoIIAA remains low enough to prevent F
activation in the predivisional cell yet can be turned on so as to
rapidly activate F after asymmetric septation. An
initial study utilizing a conditional mutation (div-355) in
the late-acting essential cell division protein DivIC revealed that
when asymmetric division was blocked, it prevented activation of
F
(166). A second study
found that when cell division was blocked at a very early stage in
sporulating cells by depletion of FtsZ, SpoIIAA was found
largely in the phosphorylated form.
In contrast, when cell division was blocked at a late stage by the
div-355 mutation, there was substantial
dephosphorylation of
SpoIIAA. However, in neither circumstance did
F become active. These results suggested a two-step
checkpoint: interaction with FtsZ triggered the phosphatase activity of
SpoIIE, but the resulting
dephosphorylated
SpoIIAA did not activate F until
asymmetric division was complete. This checkpoint could be either
enforced or bypassed by modification of SpoIIE. A mutant
allele of spoIIE, spoIIE48, caused a phenotype
similar to that of div-355 in that there was substantial
dephosphorylation of
SpoIIAA and yet F activation was blocked.
Conversely, replacement of the N-terminal transmembrane domains of
SpoIIE with those of the E. coli MalF protein
resulted in delocalized protein that caused hyper-F
activity even when asymmetric division was prevented
(150).
SpoIIE
exhibits many properties expected of a "surveillance"
protein that senses asymmetric division: it localizes to asymmetric
division sites (10,
14) in an FtsZ-dependent
manner (168), assists in
the formation of polar Z rings
(19,
149), and interacts with
FtsZ (186). Consistent
with the checkpoint model, a number of studies have found missense
mutations in SpoIIE that uncouple asymmetric division from
F activation
(32,
73,
111). Indeed, one study
tested the in vitro phosphatase activity of the mutant proteins and
found that it was similar to that of the wild-type protein
(73), further supporting
the concept that SpoIIE is subject to a
phosphatase-independent regulatory step that couples asymmetric
division to F
activation.
SpoIIAB sink.
Separate studies have clearly
established that when asymmetric division was prevented,
dephosphorylated
SpoIIAA accumulated but F remained
inactive (73,
150).
However, how the cell prevented the
dephosphorylated
SpoIIAA from activating F in the
predivisional cell remained mysterious. A recent study has provided
evidence that SpoIIAB, it its ADP-bound state, can serve as a
"sink" absorbing SpoIIAA in the cell until a
threshold level is reached
(32). Precisely why
asymmetric division allows the threshold level of
dephosphorylated
SpoIIAA to be crossed in the prespore is unknown;
some speculate that the phosphatase activity of SpoIIE is
stimulated by asymmetric division
(73), whereas others
think that equivalent inheritance of SpoIIE in the two
compartments creates a favorable
SpoIIE/SpoIIAA-PO4 ratio in the
prespore (32).
Depletion of SpoIIAB from the prespore compartment as
a consequence of transient genetic asymmetry
(54,
214) would logically
contribute to decreasing the threshold level in this compartment.
Interestingly, it was also found that in vivo, the spoIIE48
mutation substantially impairs
dephosphorylation of
SpoIIAA (32),
rather than not affecting phosphatase activity, as previously thought
(150). Indeed, a screen
for suppression of a similar SpoIIE mutation identified
mutations in SpoIIAA, SpoIIAB, and SpoIIE
itself, all of which restored sporulation by increasing the level of
dephosphorylated
SpoIIAA in the cell
(32). Therefore, the
second step of the checkpoint model, in which SpoIIE prevents
dephosphorylated SpoIIAA from liberating
F
(150), appears unlikely.
Rather, it appears that the SpoIIAB-ADP sink fulfills this
role in the cell.
Biochemical studies.
Another area of ongoing research
is the nature of the complexes that SpoIIAB forms with its
binding partners. The basic model is that SpoIIAB inhibits
F activity directly by binding to it and also
indirectly by acting as a serine kinase that inactivates
SpoIIAA by phosphorylating it
(199,
207). The phosphatase
activity of SpoIIE functions to reverse this reaction
(49), enabling
SpoIIAA to attack the SpoIIAB-F
complex and allowing F to become active. However,
additional biochemical and genetic experiments have revealed more
subtle and complex interactions between these factors.
One of the
products of F activation is the catalytically
inactive SpoIIAB-ADP complex, and it has been reported that
the replacement of ADP by ATP in this complex is an especially slow
reaction (206), so that
SpoIIAB-ADP functions to sequester SpoIIAB in an
inactive form. In addition, similar experiments have shown that an even
longer-lived intermediary is SpoIIAA-SpoIIAB-ADP
(162). Although the
experiments were performed in vitro, subsequent genetic analysis has
revealed that different mutant SpoIIAA proteins incapable of
forming the SpoIIAA-SpoIIAB-ADP complex in vitro
cannot activate F in vivo; the mutant strains are
Spo–
(161), indicating that
this mechanism of sequestering SpoIIAB is physiologically
relevant. However, as described above, the same complex has been found
to impair F activation in the predivisional cell
(and possibly mother cell) by absorbing
dephosphorylated
SpoIIAA (32).
Understanding how the
SpoIIAA-SpoIIAB-ADP complex
functions both to inhibit F activation by absorbing
dephosporylated SpoIIAA and also to promote
F activation by absorbing SpoIIAB will
require further study.
Structural studies of the complex formed
by SpoIIAB and F have allowed further
elucidation of the mechanism by which SpoIIAA functions.
First, it was determined that the inhibitory complex had the
stoichiometry
SpoIIAB2:1F
(30). Next, determination
of the crystal structure of a complex of the Bacillus
stearothermophilus SpoIIAB and F
revealed that only one of the two SpoIIAB molecules in the
complex bound F
(31). Because of the high
degree of conservation of these molecules among sporeformers
(277), it is reasonable
to assume that the B. subtilis homologues form a similar
complex.
It had previously been thought that since the same
residues of SpoIIAB contact both SpoIIAA and
F
(92), SpoIIAA
competed with F to bind SpoIIAB. However,
these new results required a reevaluation of how SpoIIAA
interacted with the SpoIIAB-F
complex. Experiments were performed with SpoIIAB
heterodimers consisting of wild-type SpoIIAB and
SpoIIABR20E, a mutant deficient in binding SpoIIAA
(92). The experiments
revealed that SpoIIAA interacted with the molecule of
SpoIIAB not bound to F and induced release
of F from the other SpoIIAB molecule, most
likely by steric displacement
(115). Another
interesting result from this study was that a mutant of
SpoIIAB deficient in its kinase activity
(SpoIIABR105A) resulted in excessive levels of
F activity. This result is notable in light of a
previous study that found, counterintuitively, that the kinase activity
of SpoIIAB was essential for F activation
(91). It is currently
thought that the phosphorylation
reaction takes place following, and therefore can be uncoupled from,
F
liberation.
Summary.
A great deal of genetic and biochemical evidence has
been obtained about the pathway regulating F
activity. What originated as a relatively simple biochemical model
(Fig. 4A) has become far
more complicated (Fig.
4B). Presently, there
appear to be a number of overlapping mechanisms: localization of
SpoIIE to the asymmetric septum and its regulation by
interaction with division proteins
(9,
10,
14,
74,
75,
111,
150,
186),transient genetic asymmetry
(54,
84), proteolysis of
SpoIIAB (214),
and a sink to absorb
dephosphorylatedSpoIIAA (32)
that also sequesters SpoIIAB
(161). The major
challenge for the future is to understand how all of these mechanisms
(and potentially others) are integrated so that activation
of F is tightly coupled to asymmetric division
and completely compartmentalized to the
prespore. The large number of contributing mechanism
highlights how critical it is for the developing organism to
efficiently regulate the earliest-acting cell-specific transcription
factor.
F RegulonClassical genetic analysis has revealed at least four spo loci directly regulated by
F:
spoIIR, required for activation of E in
the mother cell (143,
179); spoIIIG,
encoding the late prespore-specific transcription factor
G
(282,
283); spoIIQ,
required for expression of spoIIIG and for engulfment under
certain conditions (178,
284); and
spoIVB, a regulator of the late mother cell-specific
factor K
(39,
96). The gene encoding a
spore catalase required for hydrogen peroxide resistance,
katX, is part of the F regulon
(12), as is gpr,
encoding a protease specific for SASPs (see below)
(285). In addition,
three other genes with roles during sporulation have been identified as
being regulated by F: bofC, an additional
regulator of K activity
(97), and two regulators
of F activity, lonB
(4,
263) and rsfA
(306). Therefore,
primary functions of F are to couple
prespore- and mother cell-specific gene expressionand to direct synthesis of the late prespore transcription
factor G.
The F regulon
has been partially defined by chip array analysis. The analysis
revealed 66 genes that were active during the middle part of
sporulation and whose expression depended upon both Spo0A and
F
(71). However, the
approach identified genes responsive to both
F as well as the mother
cell-specific transcription factor E, so
more detailed analysis may be necessary to more fully define all of the
genes controlled by
F.
E
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TopPreviousNextReferences |
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We now turn to the next compartmentalized transcription factor,
E, which is
activated specifically in the mother cell following asymmetric
septation. Activation of E depends upon receipt of
a signal from the prespore, providing the first example of
communication between the two compartments during development.
Activation of both E and F is
coupled to asymmetric division. However, their activation mechanisms
are very different. F is synthesized in an active
state and held inactive by an anti-sigma factor, in contrast,
E is synthesized in an inactive state and activated
by proteolytic cleavage.
The first sporulation-specific
factor be purified was E, initially known as
29
(103). Its isolation
provided the first evidence that alternative factors were
involved in sporulation and led to the hypothesis that a cascade of
factors drove the developmental process
(181). Immunoblotting
experiments revealed that E was synthesized
specifically during sporulation. The anti-E
antiserum also detected a slightly larger protein, called P31, that was
synthesized earlier than and thought to be a precursor of
E
(292).
A
combination of genetic and protein analysis revealed that
E and P31 (now called pro-E)
were the products of the spoIIG locus
(279,
292). Pulse-chase and
microsequencing experiments revealed that pro-E was
processed into E by proteolytic removal of 27
residues from its N terminus
(159,
278). Subsequently, the
spoIIG locus was found to be a two-gene operon: the first gene
(spoIIGA) is essential for the processing reaction
(136,
147,
193), and the second
gene, spoIIGB, encodes pro-E, which is
processed in a SpoIIGA-dependent manner into active
E. It was proposed that SpoIIGA was the
processing enzyme and that its ability to act on its substrate
(pro-E) required the appearance of the sporulation
septum, thereby constituting a developmental checkpoint
(278). Mutational
analysis revealed that mutants of pro-E that were
poorly processed could be suppressed by mutations in
SpoIIGA, providing support for a direct role for the
SpoIIGA protein in processing
(225). However, this
role has yet to be verified by in vitro experiments. Activation of
E was also found to depend on F
activity (143,
179). The pathway
regulating E activation is illustrated in Fig.
5.
 View larger version (14K): [in a new window] |
FIG. 5. Regulation
of E activation. Parallel vertical lines separating
the prespore (right) from the mother cell (left) represent the
asymmetric septum. Broken arrows represent transcriptional activation,
and solid arrows represent posttranslational regulation.
Spo0A-PO4 is present in the predivisional cell as well as
both compartments and therefore is represented above the sporulation
septum. Pro-E is synthesized in a
Spo0A-PO4-dependent manner and therefore is present
in both compartments; however, recent study has indicated that
Spo0A-PO4-dependent transcription is largely confined to the
mother cell after asymmetric division. This distinction is represented
here by a thick line, indicating a high level of expression, in the
mother cell and a thin line, indicating a low level of expression, in
the prespore. Pro-E, which is membrane
bound, is processed into the active form, E, by
the inferred membrane-bound protease SpoIIGA.
SpoIIGA becomes active in response to
SpoIIR, whose expression is activated by
F. SpoIIGA is presumably present in both
compartments, but E becomes active only in the
mother cell, at least in part because of the higher concentration of
pro-E in this compartment, as well as because of
degradation of pro-E in the prespore. The
prespore specificity of SpoIIR expression may
contribute to but is not critical for mother cell-specific activation
of
E.
|
The spoIID locus was found to be exclusively transcribed by E-
E
(246). Immunoelectron
microscopy of cells containing a spoIID-lacZ fusion showed
signal exclusively in the mother cell
(48). This
compartmentalization of E-directed gene expression
has been confirmed many times in subsequent studies (reviewed in
references 231 and
281). As with
F in the prespore, a major question to be
answered was how the activity of E was confined to
a specific compartment. The spoIIG operon is expressed before
septation (93,
218), suggesting that
posttranslational regulation played a role in its compartmentalization.
Since E activation was dependent on
F activity
(135), it was suggested
that a F-controlled gene encoded a signal from the
prespore that triggered processing of
pro-E to E in the mother cell
(182,
268).
Intercompartmental signaling.
The link between
F activity and E activation was
identified as the spoIIR (or csfX) locus
(143,
179). Mutation of
spoIIR blocked the processing of pro-E to
E. It was found to be the only
F-directed locus that was required for
E activation, and artificial induction of
spoIIR rendered E activation independent
of F
(179,
314). The
SpoIIR protein contains a putative signal sequence, suggesting
that it may be secreted from the prespore into the
intermembrane space separating it from the mother cell. As evidence for
secretion, it was found that conditioned medium from bacteria
engineered to express SpoIIR could trigger processing of
pro-E to E when added to
protoplasts expressing the spoIIG operon
(119). It is thought
that SpoIIR acts from the prespore to trigger
SpoIIGA-directed proteolysis of pro-E to
E, although no direct biochemical evidence yet
exists to support this hypothesis.
Although the signal
transduction pathway linking the compartments has now been identified,
the basis for mother cell-specific activation of E
remains unclear. One possibility is that the prespore-specific
location of spoIIR expression, in itself, directs that
E be active only in the mother cell. To test this
possibility, spoIIR was removed from F
control and expressed prior to asymmetric division from the
spoIIE promoter in strains with no F.
Despite this switch in time and location of SpoIIR formation,
E was activated only in the mother cell of the
organisms that underwent asymmetric division
(314). Interpretation
was complicated because about half of the population did not form an
asymmetrically located septum, and those organisms displayed
uncompartmentalized E activity. Apparently, if an
asymmetric septum was formed before E activation,
E activity was confined to the mother cell;
however, if E activation preceded septation, it
prevented septation
(314), a result
consistent with the E-directed block in division
that normally prevents the abortively disporic phenotype
(57,
124,
232). Overall, the
results indicate that compartmentalization of E
activity in the mother cell can occur independently of
F and of compartmentalized spoIIR
expression.
Protein localization.
Compartmentalization studies
now focused on the subcellular location of the processing reaction. It
was found that fusion proteins of SpoIIGA and
pro-E to GFP localize to the asymmetric septum
(72,
138). Fractionation
experiments revealed that pro-E is found primarily
in the membrane, whereas processed E is found
primarily in the cytoplasm
(118). These results
suggested that the processing reaction took place at the membrane;
consistent with this conclusion, it was found that a
pro-E mutant that failed to localize to the cell
membrane was not processed
(139).
Immunofluorescence with anti-E antibodies (which
also interact with pro-E) revealed a signal at the
cell membrane prior to asymmetric division, a concentrated
signal at the asymmetric septum upon division, and finally a
signal dispersed in the cytoplasm thereafter
(118). Similar
immunofluorescence experiments revealed very little signal in the
prespore; most of the signal was confined to the mother cell
after asymmetric division
(234). In addition,
pro-E artificially produced in the
prespore was not processed
(140) unless
SpoIIGA was also expressed in this compartment
(141). These studies
suggested that exclusion of either SpoIIGA or
pro-E from the prespore was a mechanism of
compartmentalization. In support of this model, a fusion to GFP of the
N-terminal 55 residues of pro-E, encompassing the
prosequence, localized to the sporulation septum and, in protoplasts
formed after asymmetric division, was found predominantly in the mother
cell (138). These
results suggested that pro-E is sequestered to the
mother cell face of the asymmetric septum.
However, a study
assaying the expression pattern and stability of a full-length,
functional pro-E-GFP fusion (as opposed to
the truncated version in the previous studies) found that the fusion
protein localized nonspecifically to the cell membrane. As in the
previous studies, fluorographs showed a strong signal associated with
the septum (86), but a
similar signal was observed with the membrane stain FM4-64, indicating
that there is no specific concentration of this protein at the
asymmetric septum, in contrast to previous interpretations
(118,
138). Rather, the strong
signal probably resulted from the two septal membranes' being
viewed end on, as disks
(86). In addition, it was
found that the fusion protein was preferentially degraded in the
prespore and the spoIIG promoter was largely active
in the mother cell after asymmetric division, providing two different
mechanisms to concentrate pro-E in the mother cell
(86). These results
provide an alternative explanation of the earlier report of
pro-E localizing to the mother cell face of the
septum (138), namely, a
large increase in mother cell-specific synthesis of
pro-E and prespore-specific degradation of
pro-E following asymmetric division. Since previous
studies had shown that that spoIIG transcription commenced
prior to asymmetric division
(93,
218), a major question
to be addressed was how spoIIG transcription became largely
confined to the mother cell after asymmetric
division.
Role of Spo0A.
In a continuation of the study discussed above,
Fujita and Losick (87)
found that a substantial increase in Spo0A activity followed asymmetric
division and was confined to the mother cell. This result explains the
increase in transcription that they had observed
(86) for the
Spo0A-dependent (257,
258) spoIIG
promoter. Disrupting the pattern of Spo0A activity by expression of a
Spo0A inhibitor in the mother cell or a constitutively active form of
Spo0A in the prespore severely reduced spore formation,
indicating that preferential mother cell-specific Spo0A activity is
important for development. A challenge now is to understand how the
increase in Spo0A activity is coupled to asymmetric division
and confined to the mother cell. Fujita and Losick propose
that transient genetic asymmetry excludes important
phosphorelay genes from the prespore at the time of
asymmetric division. However, a recent study found that
dephosphorylated
SpoIIAA, thought to be found largely in the prespore
(171), inhibits
Spo0A-dependent transcription
(6). This result suggests
a supplemental or alternative mechanism to transient genetic asymmetry,
that dephosphorylated
SpoIIAA generates a feedback loop that prevents expression of
Spo0A-dependent genes, such as spoIIG, from occurring in the
prespore. Further studies will be necessary to determine the
role that this feedback inhibition plays in compartmentalization of
spoIIG transcription and of E
activity.
Timing of activation.
Another focus of study has been the importance of
timing of E activation during sporulation. Although
it was known that pro-E processing required
asymmetric septation
(278) and
F-dependent spoIIR expression
(143,
179), how these events
coordinated proper timing of E activation was
unknown. It was found that expression of spoIIR in the
predivisional cell, rather than in the prespore, had only a
mild effect on spore formation
(314) and on the timing
of pro-E processing and E
activity (86). Premature
spoIIG expression from a constitutive rather than its normal
Spo0A-induced promoter had little if any effect on these properties.
However, constitutive expression of both spoIIG and
spoIIR resulted in early processing and poor sporulation
(86). Therefore, correct
timing of expression of both spoIIG and of spoIIR
contributes to the proper timing of E activation;
they provide partly redundant mechanisms to coordinate morphogenesis
and gene regulation.
Another aspect of timing to consider is that
cells that fail to activate E frequently undergo
asymmetric division at both poles of the cell and subsequently fail to
sporulate. This second division results in an abortively disporic
phenotype in which there is a three-chambered structure consisting of
two prespores separated by a large central compartment
(124,
228). These two
asymmetric divisions occur in rapid succession, with as little as 10
min separating them (77,
232). Genetic and cell
biological experiments have revealed that three
E-directed genes that are required for engulfment
of the prespore by the mother cell are also responsible for
preventing the second division in the mother cell
(57,
232). The rapid
succession of the two divisions indicates that the
E-dependent mechanism to impair asymmetric division
must be activated very rapidly after the first division to prevent the
second. Moreover, the first division is needed in order to activate
F, so that spoIIR is transcribed and hence
E is activated
(143,
179). Thus, some
E must become active in the mother cell very
shortly after septation, probably before the large postseptation
increase in Spo0A-directed spoIIG transcription can have an
effect.
Regulation by gene position.
Similar to the role that the
chromosomal position of the spoIIA operon plays in
F activation
(54), one of the ways in
which the cell ensures proper timing of E
activation is the chromosomal location of the spoIIR gene. The
spoIIR gene is located very near the origin of replication, so
that it is initially present in the prespore at the time of
asymmetric division and will be immediately transcribed by RNA
polymerase with F
(143,
179) (Fig.
3). Placing
spoIIR at a location near the terminus, so that it must be
imported into the prespore by the DNA translocase
SpoIIIE (17,
303,
305) to become
accessible to F, curtailed its expression
(148,
320). This relocation
also resulted in a severe reduction in E activity
and in sporulation, and many cells in the population exhibited the
abortively disporic phenotype. Therefore, the chromosomal position of
spoIIR is important for the proper timing of
E activation, prevention of the second asymmetric
division in the mother cell, and efficient sporulation
(148,
320).
In summary,
much is now known of the signal transduction pathway governing mother
cell-specific gene expression (Fig.
5). The discovery that
Spo0A activity is largely confined to the mother cell after septation
(87) is an important
recent contribution to our understanding of compartmentalization of
E activity. The mechanism for the partial
compartmentalization of Spo0A remains unknown. Genetic asymmetry,
already known to contribute to compartmentalization of
F activity and timing of E
activation (54,
148,
320), has been suggested
as a mechanism to partially compartmentalize Spo0A activity
(87). Alternatively, or
additionally, differential localization of phosphorelay components
could play a role; a precedent for this scenario can be found in
Caulobacter crescentus development
(196). In addition, the
role of a novel inhibitory feedback loop involving SpoIIAA
(6) has yet to be
explored. The protein(s) responsible for degradation of
pro-E in the prespore remains unknown;
functional analysis of the F regulon should
facilitate its
identification.
E RegulonMembers of the
E regulon that are
important for sporulation include spoIID, spoIIM, and
spoIIP, which are required for engulfment and to prevent a
second asymmetric division from occurring in the mother cell
(1,
37,
57,
85,
228,
232,
246,
271,
272); spoIVA,
cotE, and spoVID, which encode scaffold proteins for
spore coat assembly (18,
245,
273,
317,
318); the
spoIIIA operon, which is required for activation of the
late-prespore specific sigma factor G
(125,
146); sigK, the
composite gene for the late mother cell-specific transcription factor
K
(155,
280); and
spoIVCA, the gene for the recombinase that generates
sigK via a chromosomal rearrangement
(155,
235,
256). In addition,
E activates transcription of spoIIID,
which encodes a regulator of some E-dependent genes
(102). As a result,
early mother cell-specific gene expression is divided into an initial
phase, when certain genes responsive to E alone are
expressed, including spoIIID, and a later phase, when
SpoIIID represses some E-controlled genes
and activates transcription of additional
E-controlled genes.
The main functions of
E are to prevent asymmetric division in the mother
cell, to trigger engulfment of the prespore, to initiate spore
coat assembly, and to direct synthesis of the late mother cell-specific
transcription factor K. It should be noted that
functional conservation of E has been observed in a
range of species of Bacillus and Clostridium
(7). In Bacillus
thuringiensis, the cry1A(a) gene, encoding a protoxin
crystal protein, is expressed in the mother cell under the control of
E as well as K
(2), and in
Clostridium perfringens, the enterotoxin gene cpe is
most likely also under the control of E and
K
(315).
The
E regulon has been defined by microchip array in
two independent studies
(58,
75). One study found that
253 genes (in 121 operons) are regulated by E, and
46 of these were present in all endospore-forming bacteria whose
genomes have been sequenced and absent from the genome of the related
nonsporeformer Listeria monocytogenes. Null mutations in 12 of
98 previously undefined genes or operons caused substantial defects in
sporulation; fusions of several of the encoded proteins to GFP showed
localization to the outer prespore membrane
(58). The other study
found 171 genes under the control of E, and
functional analysis of this group found five novel genes required for
efficient sporulation
(75). The two studies
agreed well and, in addition to many novel genes, identified most of
the previously described E-directed
genes.
TopPrevious NextReferences |
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The major morphological event that follows activation of
F and E
is engulfment of the prespore by the mother cell. After
asymmetric division, the prespore and mother are adjacent and
are in direct contact with the medium. Completion of engulfment results
in the prespore's being entirely surrounded by the mother
cell and so not in direct contact with the medium (Fig.
1). It should be noted
that the word engulfment has been used in two senses in studies of
spore formation, which at times can cause confusion. The first sense
means the process of engulfment, and the second means the completion of
engulfment (228). In
terms of mutant designation, substages IIii and IIiii are in the
process of engulfment
(124), whereas stage III
denotes completion of engulfment
(124,
228). The activation of
the late cell-specific sigma factors (G and
K) is coupled to completion of engulfment, as
activation of the early factors (F and
E) is coupled to asymmetric division
(231). Therefore, a
discussion of engulfment is important for an understanding of
compartmentalization, because there is compartmentalization between
pre- and postengulfment (in the sense of completion of engulfment) gene
expression. The process of engulfment starts with changes in the sporulation septum. The first observed change is loss of wall material from the center of the septal disk, followed by loss of much of the wall material of the septum. After this autolysis has occurred, the points of attachment of the septal membrane to the peripheral cell membrane migrate to the cell pole. Engulfment is completed by membrane fusion, resulting in the detached prespore's being entirely within the mother cell (Fig. 1) (124, 178, 227). Expression of both prespore-specific and mother cell-specific loci is required for engulfment. spoIID (37, 228), spoIIM (271), and spoIIP (85) mutants display autolysis and membrane flexibility at the center of the spore septum but not its periphery. spoIIQ mutants display a block just prior to completion of engulfment (178). Loci expressed before septation are also involved in engulfment (a spoIIB spoVG double mutant displays aberrant septal wall autolysis) (190, 224), and the spoIIIE locus has been shown to be required for the membrane fusion event (264, 266).
Transcription
of the spoIID
(246), spoIIM
(272), and
spoIIP (85) loci
is directed by E, suggesting that initiation of
engulfment is driven by proteins synthesized in the mother cell.
Consistent with a direct role in engulfment, GFP fusions with all three
of the encoded proteins localized to the asymmetric septum and
engulfing prespore membrane
(1,
57). In addition, these
proteins degraded partial septa and prevented a second division in the
mother cell (57,
232). SpoIID
bears homology with a modifier of cell wall hydrolases
(157,
180). All of this
indirect evidence suggests that the proteins function to degrade
peptidoglycan, both to enable membrane migration around the
prespore and to maintain asymmetry by preventing division in
the mother cell. A recent study has shown that purified SpoIID
degrades bacterial cell wall in vitro
(1), providing a
biochemical link with the genetic and cytological data regarding this
class of proteins. In addition, this study revealed that leaky
spoIID and spoIIP mutants were impaired in both
autolysis and membrane migration, suggesting that these two processes
are mechanistically linked
(1).
The spoIIB gene, which is expressed in the predivisional cell (190), was initially thought to be required for engulfment and sporulation (37, 228). It later became clear that it is only required in strains in which a second gene expressed in the predivisional cell, spoVG (319), has been disrupted (190). Single mutations in either spoIIB and spoVG cause a very mild defect, whereas the double mutant displays little autolysis and sporulates poorly (190, 224). SpoIIB localizes to the asymmetric septum and bears weak homology with an amidase, suggesting a direct role in engulfment (190, 224). The function of SpoVG in engulfment is unclear; it appears to play some role in inhibiting asymmetric division (195, 224). The defects in double spoIIB spoVG mutants can be partially suppressed by mutation of a third gene, spoVS (241). However, the complex relationships between these genes have yet to be fully explored.
After autolysis and membrane migration around the prespore, the final stage of engulfment is membrane fusion. The early engulfment processes are largely driven by proteins in the mother cell. However, the SpoIIQ protein, which is expressed in the prespore from an E-
F-directed gene, is
required for membrane fusion. SpoIIQ is predicted to be
largely extracellular and has homology with endopeptidases, suggesting
a direct role in engulfment. Consistent with this suggestion, an
epitope-tagged SpoIIQ protein localized to the center of the
asymmetric septum (178).
Subsequently, SpoIIQ was found to be necessary for engulfment
when sporulation was induced by nutrient exhaustion in rich medium but
not when sporulation was induced by nutrient replacement in
minimal medium (284).
SpoIIQ is nevertheless required for sporulation in both
conditions, one reason being that spoIIIG, encoding
G, is not transcribed in its absence. The link
between SpoIIQ and spoIIIG transcription is unclear
but most likely indirect
(284). A novel cytology-based screen revealed that the SpoIIIE protein, which is required for DNA transfer into the prespore (17, 303), is also required for membrane fusion (264). This result was surprising in light of earlier studies that found that spoIIIE mutants formed apparently fully engulfed, though unstable, prespores (121, 228, 275). While SpoIIIE initially localizes to the center of the asymmetric septum (304), it then travels as a focus along the engulfing membrane, finally coming to rest at the extreme pole of the cell, concomitant with membrane fusion at that location (264). In addition, a SpoIIIE mutant in which the putative ATP binding site is disrupted was found to be defective for DNA translocation but competent for membrane fusion, thereby uncoupling the two functions of the protein (264). Potentially, SpoIIIE establishes a checkpoint to ensure that chromosome segregation is completed prior to membrane fusion.
A final, puzzling finding is that mutations in the genes encoding the E1ß and E2 subunits of the pyruvate dehydrogenase complex block sporulation just prior to and after the completion of engulfment, respectively. Although changes in medium composition can rescue other aspects of the defects in these mutants, they cannot restore sporulation, suggesting a sporulation-specific function independent of their enzymatic activity (90). More work will be necessary to determine what role these proteins play in engulfment.
Although several proteins involved in engulfment have been identified, biochemical analysis of the process has only recently been initiated. Several questions need to be addressed. What is the mechanism by which engulfment proteins, and the associated autolysis, are confined to septa so as not to damage the cytoplasmic cell wall? What ensures that autolysis starts in the middle of the septal disk and not its periphery, as during vegetative division? Does the chromosome's traversing the septum provide an initiating target for the autolysis? Lastly, it is likely that a major focus of study will be how the cell couples the completion of engulfment to the late stages of cell-specific gene expression, and this is discussed below.
G
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Completion of engulfment of the prespore by the mother cell signals the next phase of compartment-specific gene expression. However, synthesis of the associated
factors commences earlier.
F directs synthesis of the late prespore
transcription factor G, and E
directs synthesis of the late mother cell transcription factor
K. Since G is made only in the
prespore and K is made only in the mother
cell, compartmentalization between prespore and mother cell is
not a major focus of study. Rather, the focus of study is
compartmentalization in the sense of activation after completion of
engulfment but not before. G and
K are both subject to complex regulation at
different levels, and their activities are tightly coordinated with
each other and with cellular morphogenesis. Activation of
K depends on G, so it is
G activation that is most immediately tied to
completion of engulfment, providing another developmental checkpoint to
coordinate morphogenesis and gene regulation.
The first insight
into the role of G was provided by the study of
small acid-soluble proteins (SASPs). These are present in high
concentrations in the mature spore, and members of a major class, the
/ß SASPs, bind to and protect DNA in the spore
(213). Immunoelectron
microscopy revealed that, during spore formation, several SASPs were
found almost exclusively in the engulfed prespore
(83). This finding
suggested the existence of a prespore-specific transcription
factor responsible for the compartmentalized pattern of SASP
expression. Prespore-enriched fractions of sporulating cells contained
a protein that supported in vitro transcription of sspE
(encoding SASP-
) and, when sequenced, was discovered to be the
spoIIIG gene product and named G
(283). The
spoIIIG gene had been previously identified as being located
immediately downstream of spoIIGB, encoding a product with
substantial homology to factors, and being essential for
spore formation (142,
194). Consistent with
the immunoelectron microscopy data, genetic analysis revealed that
spoIIIG is expressed in a prespore-specific
pattern (93,
142). The pathway for
G activation is illustrated in Fig.
6.
 View larger version (12K): [in a new window] |
FIG. 6. Regulation
of G activation. Two concentric semicircles
represent the inner and outer membranes of the engulfed
prespore. Broken arrows represent transcriptional activation,
and solid arrows represent posttranslational regulation. The structural
gene for G, spoIIIG, is transcribed
exclusively in the prespore under the control of
F (SpoIIAB [AB] is presumably
inherited from the predivisional cell). Although not represented here,
transcription also requires expression of SpoIIQ in the
prespore and an unknown gene in the mother cell. Activation of
G does not occur until engulfment is complete.
Activation requires release of inhibition by SpoIIAB. This
release depends on the E-directed expression of the
spoIIIA operon in the mother cell. Activation of
G also requires SpoIIIJ, which is
expressed vegetatively and localizes to the prespore membrane
but need only be expressed in the prespore. It seems likely
that some mechanism distinct from SpoIIAB inhibition keeps
G inactive prior to engulfment. The mechanism
responsible for coupling activation to completion of engulfment remains
unclear.
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Transcriptional regulation of the spoIIIG locus is complex and may play an important role in coupling
G activation to engulfment. The
spoIIIG locus is transcribed from the upstream spoIIG
promoter during the early part of sporulation, but no detectable
G protein is produced from this transcript
(194,
282), most likely
because of a predicted RNA hairpin in the intergenic region
(194). Moving
spoIIIG to an ectopic locus does not impair spore formation,
indicating that readthrough from the spoIIG promoter is not
critical to G expression
(282); the role of this
transcript remains obscure. The spoIIIG locus is also
transcribed from its own promoter, recognized by RNA polymerase with
either F or G; this transcript
is translated, leading to active G during the later
stages of sporulation
(282). Since the
activity of F is confined to the prespore
(189), the presence of a
F-dependent promoter ensures that
G is present, and therefore active, only in the
prespore (83,
142).
Transcription
from the spoIIIG promoter differs from that of other
F-directed promoters in a number of ways. First,
its expression depends upon E activation
in the mother cell
(218), suggestive of a
signaling pathway linking the two compartments. Transcription of
spoIIIG also depends upon expression of SpoIIQ in the
prespore, suggesting a potential link between
G activation and engulfment
(284). Lastly,
consistent with dependence upon these two events, expression occurs
later than that of other known F-dependent
promoters (144). The
importance of this complex transcriptional regulation in sporulation is
unclear but most likely assists in proper timing of
G activation relative to early compartmentalized
gene expression and morphogenesis. Because G can
direct transcription of its own structural gene, tight regulation is
presumably required to prevent inappropriate initiation of an
autocatalytic loop.
GSimilar to other sporulation
factors, G is subject to posttranslational
regulation. The first evidence was that spoIIIJ, a
vegetatively expressed gene
(60), and the
spoIIIA operon, expressed in the mother cell
(43,
125), were both required
for G activation but not spoIIIG
transcription (60,
146). The anti-sigma
factor for F, SpoIIAB
(38,
51,
260), has emerged as
also acting on G and indeed mediating the effects
of spoIIIJ and spoIIIA. Fractionation studies
revealed that SpoIIAB disappeared from the prespore
at the time of G activation. In addition,
overexpression of G resulted in toxicity that could
be suppressed by simultaneous expression of SpoIIAB
(151), and disruption of
spoIIAB led to excessive levels of G
activity (38). These
results suggested that SpoIIAB inhibited G
as well as F during sporulation. As direct evidence
for this hypothesis, it was shown that SpoIIAB bound
G in vitro, and a mutant form of
G that SpoIIAB bound poorly in vitro,
SpoIIIGE155K, bypassed the requirement for spoIIIA
for G activation in vivo but not for sporulation
(146). A recent study
has shown that this mutant form of G bypasses the
requirement for spoIIIJ for G activation
but, once again, not for sporulation
(262). The ability of
this mutant to restore G activity but not
sporulation in these backgrounds indicates that either the
spoIIIA and spoIIIJ locus plays an additional role in
sporulation or that the restored G activity is not
properly regulated.
These results suggested a model in which
SpoIIAB holds G inactive until a signal is
received from SpoIIIJ and at least one product of the
spoIIIA operon. The question then is how SpoIIIJ and
the products of the spoIIIA operon function to activate
G. Analysis has been hampered by the fact that the
eight products of the spoIIIA operon, all predicted to be
membrane bound, are not homologous to any known proteins
(281). However, it was
found that although SpoIIIJ is expressed vegetatively
(60), expression only in
the prespore, not in the mother cell, was sufficient for spore
formation. In contrast, expression of the spoIIIA operon in
the prespore did not support sporulation
(262). SpoIIIJ
is a homologue of the E. coli YidC protein
(205), which is required
for insertion of proteins into the lipid bilayer
(254,
261). Consistent with
its expression during vegetative growth
(60), spoIIIJ is
essential if its paralogue, yqjG, is disrupted
(205), and depletion of
both encoded proteins decreases the stability of membrane-bound and
secreted proteins (288).
In addition, SpoIIIJ localizes to the asymmetric septum and
engulfing prespore membrane
(205,
262). One possible role
in G activation is that SpoIIIJ is
required for proper insertion and/or maintenance of a receptor in the
inner prespore membrane that recognizes a signal from the
mother cell.
The SpoIIAB pathway in part explains
postengulfment G activation, but several
observations suggest that there must be some additional
G regulator that, minimally, prevents activation
prior to engulfment (66,
262). Thus, the
spoIIIGE155K allele that renders G
activation independent of SpoIIIJ and the products of the
spoIIIA operon do not affect the timing of
G activation
(146,
262). Furthermore,
F and G are very similar and
are both subject to regulation by SpoIIAB, with
F having much higher affinity for SpoIIAB
in vitro; for both, binding is disrupted by SpoIIAA
(66). It is difficult to
see why G is held inactive when
F is active if SpoIIAB is the only direct
regulator (66). A major
feature of G regulation is that it only becomes
active upon completion of engulfment
(275,
276). The molecular
basis for this linkage is unknown.
The complex transcriptional
and posttranslational regulation presumably ensures that
G becomes active only in the prespore and
only following engulfment. One possible reason for the complexity is
that because the spoIIIG promoter is transcribed by RNA
polymerase with G
(282), even a small
amount of G activity can lead to an autocatalytic
loop that causes excessive levels of G-dependent
transcription. In support of this hypothesis, a mutation in
lonA, encoding a putative Lon protease, resulted in high
levels of G activity under nonsporulating
conditions, in which F-directed transcription of
spoIIIG does not occur
(259). The complexity of
the control is indicated by the observation that expression was
postexponential and not constitutive in the lonA mutant;
furthermore, neither expression in sporulating conditions nor spore
formation was affected by the lonA mutation
(259). Another reason
for a multitude of controls may be the presence of the transcript of
spoIIIG that originates from the upstream spoIIG
promoter (194,
282). The
spoIIG promoter is strongly activated in the mother cell by
Spo0A (86,
87), and the cell must
inactivate any G that is produced from this
transcript in order to prevent a feedback loop
(282) and
G activation in this compartment.
In summary,
the mechanism of coupling G activation to the
completion of engulfment is largely unknown. The mechanism underlying
the dependence of spoIIIG transcription on
E activation
(218) is also unknown.
Likewise, the specific role of the eight products of the
spoIIIA operon, all of which are thought to be membrane bound
and are not obviously homologous to any known proteins, is unclear. The
role of SpoIIQ in both engulfment
(178) and
spoIIIG transcription
(284) is intriguing. One
reason for our ignorance may be that genetic redundancy, as seen in
F and E regulation, is
preventing the isolation of strains with a single mutation that are
blocked in G activation or that have uncoupled
activity from engulfment. Functional analysis of the
F
(71) and
E
(58,
75) regulons may help to
overcome this problem and identify additional factors involved in
G
regulation.
G RegulonAt least three classes of genes are present in the
G regulon, those involved in sporulation, in
germination, and in protecting the spore from DNA damage. The genes
important for sporulation include spoIIIG, leading to an
autocatalytic loop (142,
282); spoIVB, a
serine peptidase that signals from the prespore to the mother
cell to activate K
(39); the spoVA
operon, which is required for dipicolinic acid uptake into the
prespore from the mother cell
(63,
202,
289); spoVT, a
regulator of G-dependent gene expression
(13); and bofC,
a regulator of pro-K processing
(97,
296). The germination
genes include the gerA operon, involved with germination in
response to alanine; the homologous gerB operon, involved with
germination in response to a panel of germinants
(L-asparagine, glucose, fructose, and potassium ions)
(213); and
pdaA, which is required for the formation of muramic
-lactam, a unique component of spore cortex peptidoglycan
(89). The genes for
protection from DNA damage include splB, encoding
spore-photoproduct lyase, which helps protect spore DNA from
UV damage (68,
219); yqfS,
encoding a type IV apurinic/apyrimidinic endonuclease
(253,
293); and ssp
genes, which encode SASPs, the predominant proteins of the spore core
(107). Some of the SASPs
bind the DNA in the spore and help protect it from exposure to heat, UV
radiation, desiccation, and several other adverse conditions and also
provide a source of amino acids upon germination. Transcription of all
the ssp genes (sspA through sspP), except
sspF and sspG, is directed by G
(107). Therefore, three
main functions of G are to couple late
prespore and mother cell gene expression, to protect the spore
from hazardous conditions, and to prepare the spore for germination.
For detailed discussions of spore cortex formation and germination, we
refer the reader to references
201,
213, and
286. K
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Following the completion of engulfment and activation of
G in the prespore,
K becomes active in the mother cell.
K is only synthesized in the mother cell, so
morphological coupling and compartmentalization into the postengulfment
mother cell are the main focus of discussion. As is the case for
E and G, activation of
K depends upon intercompartmental signaling
(182,
231). Regulation of
K is similar to that of E in
that K is synthesized as an inactive precursor,
pro-K, that is processed into its active form upon
receipt of a signal from the prespore. However, the mechanisms
of regulation are very different. K was first
identified as a factor that could direct transcription in
vitro of the late sporulation genes spoIVCB and cotD
(152). However, the gene
encoding K was unknown. Genetic studies focused on
the spoIIIC locus, which appeared to encode a small protein
with high similarity to the C-terminal region of bacterial
factors but lacking any corresponding N-terminal region
(64). Interestingly, a
different gene, spoIVCB, was found to encode a
protein similar to the N-terminal region of a factor. These
unusual findings were explained when it was shown that the region
separating the two genes, approximately 48 kb in size, was excised
during sporulation, resulting in the formation of a single, composite
gene, sigK, encoding a full-length factor,
K
(280). SpoIVCA appears
to be the protein responsible for removal of the skin (sigma K
intervening) element. It has substantial similarity to the Hin family
of site-specific recombinases, binds to the processing site in vitro
(235), and is the only
sporulation-specific protein required for excision to occur in vivo
(155). Transcription of
both spoIVCA and the sigK gene is confined
to the mother cell, initiated from a E-dependent
promoter requiring SpoIIID
(102,
156,
255,
256). Since the rearrangement generating sigK occurs only in the mother cell chromosome, which is not inherited, it could potentially be a novel mechanism of compartmentalizing gene expression. However, the presence of an intact sigK gene during growth and sporulation had no effect on development (155). Consistent with the dispensability of skin excision, other strains of B. subtilis and other species of sporeformers lack any inserted element in their sigK gene (2, 255, 277) with the notable exception of Clostridium difficile (see below) (104). However, similar to some other sporulation-regulatory elements, the role of skin excision was obscured by a genetic redundancy.
K is synthesized as an inactive
precursor, pro-K
(40,
152,
185). Removal of the
prosequence (resulting in constitutively active K)
combined with artificial removal of the skin element resulted
in some K activity during vegetative growth and
impaired sporulation
(211). The vegetative
expression is most likely because, like that of spoIIIG in the
prespore (282),
expression of the sigK gene is autocatalytic, being driven by
a K-dependent promoter
(211). Interestingly,
the sigK gene in Clostridium difficile also contains
an insertion element, but it differs from the skin element in
size, orientation, sequence, and site of insertion. The sigK
gene of C. difficile differs from its B. subtilis
counterpart in that it does not encode a prosequence and
K appears to be synthesized in an active state.
Consistent with the skin element and prosequence playing
redundant roles in regulation, it was found that expression of an
intact sigK gene in trans in C. difficile
resulted in poor sporulation
(104). Presumably,
regulation of K occurs primarily via chromosomal
rearrangement in this organism, whereas in B. subtilis there
is redundancy in regulation; either prosequence processing or
skin excision suffices to ensure the proper time and location
of K activation. Consistent with this reasoning,
the chromosome of C. difficile lacks obvious homologues to
known pro-K processing components
(277).
K ProcessingBased on the nucleotide sequence of sigK and the N-terminal sequence of
K, it was inferred
that that the N-terminal 20 amino acid residues are removed from the
sigK gene product of B. subtilis to generate active
K
(152,
280). Immunoblotting
revealed that pro-K appeared approximately an hour
earlier than processed K, and processing depended
upon gene expression in the prespore
(40,
185). Therefore, similar
to regulation of E
(159,
278,
279), it appeared that
intercompartmental signaling resulted in an inactive proprotein's
being processed into an active factor. This
signaling pathway has been identified and characterized in detail (Fig.
7).
 View larger version (17K): [in a new window] |
FIG. 7. Regulation
of K activation. Two concentric semicircles
indicate the inner and outer prespore membranes surrounding
the engulfed prespore. Broken arrows represent transcriptional
activation, and solid arrows represent posttranslational regulation.
SpoIVB is expressed in the prespore under the control of
G and is thought to be inserted into the inner
prespore membrane, where it undergoes autoproteolysis.
G also directs expression of BofC, which is an
inhibitor of SpoIVB. In the mother cell, BofA, SpoIVFA, SpoIVFB, and
pro-K are all produced under the control of
E. SpoIVFB is thought to be the processing enzyme
that acts upon pro-K to generate active
K. BofA inhibits SpoIVFB. This inhibition is
mediated by SpoIVFA, which acts to bring these proteins in contact with
one another. Signaling by SpoIVB relieves inhibition of SpoIVFB,
possibly by proteolysis, and so triggers pro-K
processing. Pro-K is tethered to the outer
prespore membrane by its N terminus, and it is thought that
the processing reaction with SpoIVFB occurs within the
membrane.
|
K processing
(41,
127). The first,
spoIVF, is a bicistronic operon. spoIVFA encodes an
inhibitor of pro-K processing, whereas
spoIVFB encodes a factor critical for processing
(40,
41,
185). The second locus,
bofA, also encodes an inhibitor of pro-K
processing (243). The
three encoded proteins, SpoIVFA, SpoIVFB, and BofA, identified in these
early studies remain the three main mother cell-expressed proteins
known to be involved in pro-K processing. SpoIVFB
is considered the enzyme that cleaves the prosequence from
pro-K, and BofA and SpoIVFA act to negatively
regulate this process
(248).
There
are multiple lines of evidence suggesting that SpoIVFB is
the processing enzyme. Most compelling among these is the finding that
expression of SpoIVFB is sufficient to trigger a low level of
pro-K processing during vegetative growth in B.
subtilis as well as in E. coli
(184,
242). SpoIVFB has been
shown to share two motifs with mammalian site 2 protease, a zinc
metalloprotease whose catalytic center is most likely membrane
embedded. Mutations in conserved residues in these motifs in SpoIVFB
abolished pro-K processing, suggesting that
pro-K processing occurs within the membrane
(247,
311). Consistent with
this conclusion, biochemical and cytological experiments have indicated
that pro-K, SpoIVFB, SpoIVFA, and BofA all localize
to the cell membrane
(100,
240,
249,
294,
312). Models of how BofA
and SpoIVFA effect the negative regulation of SpoIVFB action have
undergone a series of changes, perhaps reflecting the complexity of the
process (41,
100,
153,
242,
249). The most recent
study suggests that SpoIVFA acts to bring BofA and SpoIVFB together in
a heteromultimeric complex localized to the outer prespore
membrane and that BofA is the direct inhibitor of SpoIVFB processing
activity (249) (Fig.
7).
Prespore signaling.
Processing of
pro-K into mature K was blocked
in a strain lacking the late prespore transcription factor
G
(40,
185), suggestive of
signaling between the compartments. Several lines of evidence indicate
that the signal is encoded by the spoIVB locus. Disruption of
spoIVB blocks pro-K processing, and
transcription of spoIVB is largely dependent upon
G
(39,
96). Transcription of
spoIVB is the only function of G that is
required for pro-K processing
(95). The SpoIVB protein
has a serine peptidase domain and a PDZ domain for
protein-protein interactions; both domains are important for
pro-K processing and sporulation
(116,
297).
It is
thought that SpoIVB is inserted into the inner prespore
membrane, where it undergoes autoproteolysis
(297) to release
signaling fragments that can diffuse across the intermembrane space and
interact with the SpoIVFA-SpoIVFB-BofA complex inserted in the outer
prespore membrane
(249). A recent study
found that SpoIVB degrades SpoIVFA in vitro
(45), providing a
possible mechanism of action. If this reaction occurs in vivo, it would
release SpoIVFB from inhibition by BofA and trigger
pro-K processing (Fig.
7)
(249). Interestingly,
spoIVB mutants that show pro-K processing
but are Spo– have been obtained, suggesting that
SpoIVB has an additional, unknown function in sporulation
(212). Two other factors
that play accessory roles in regulating pro-K
processing have been identified. The BofC protein, expressed in the
prespore, inhibits SpoIVB autoproteolysis and thus
pro-K processing
(296). The CtpB protein
is similar to SpoIVB in that it contains PDZ and serine peptidase
domains, although it is expressed in the mother cell under the control
of E. CptB is required for optimal efficiency of
pro-K processing
(216).
In summary,
the signaling pathway regulating K activation is
now well understood, and yet a number of issues remain. Processing of
pro-K has not been achieved in vitro. The proposal
that SpoIVFA is proteolyzed by SpoIVB
(45) needs to be tested
in vivo. In addition, it is not known how the mother cell processing
components localize specifically to the outer prespore
membrane. It has been proposed that SpoIVFB is initially inserted into
both the cell and prespore membranes and subsequently diffuses
to and is retained by SpoIVFA only in the latter
(250). However, the
mechanism underlying SpoIVFA targeting to the outer
prespore membrane remains
unknown.
K RegulonThe known genes in the B. subtilis
K regulon are involved in formation of the spore
coat, spore maturation, and regulation of
K-dependent transcription. The regulon includes 14
cot genes, which encode proteins that make up the protective
spore coat (47,
109); spoVD and
spoVK, required for spore maturation
(42,
69); and the structural
gene for K itself, leading to an autocatalytic loop
(155). Expression of the
transcriptional regulator gerE is directed by
K, dividing K-dependent
promoters into three classes: those solely regulated by
K are expressed throughout the late mother
cell-specific stage, those that are repressed by GerE and are expressed
early after K activation, and those that require
GerE and K for activation and are expressed later
(316). Detailed reviews
of coat formation have appeared elsewhere
(46,
47,
109). As mentioned
previously, in other species expression of toxin genes is directed by
K: in B. thuringiensis, the
cry1A(a) gene, encoding a protoxin crystal protein, is
expressed in the mother cell under the control of K
as well as E
(2), and in C.
perfringens, the enterotoxin-encoding gene cpe is most
likely under the control of K as well as
E
(315). |
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The primary emphasis of our discussion has been on the spatial compartmentalization of gene expression associated with two key morphological events, completion of septation and completion of engulfment. However, an important aspect of development is temporal regulation of gene expression. Temporal control is exercised in part by coupling activation of
factors to
morphological events: F, and hence
E, to septation and G,
and hence K, to engulfment, as discussed above. The
two prespore factors, F and
G, have overlapping promoter
specificities (4,
107), suggesting three
temporal promoter classes in the prespore: those
recognized by F only, those recognized by
F and G, and those recognized
by G only. A similar argument can be made for the
mother cell, where E and K also
have overlapping promoter specificities
(107).
Additional
layers of temporal control are exercised by activator and repressor
proteins under the control of and associated with these
factors. Such regulators have been more fully characterized for the
mother cell. The SpoIIID protein acts as an activator of some
and a repressor of other genes in the E regulon and
is itself a member of that regulon. Thus, SpoIIID divides the
regulon into an early phase, when some genes are expressed, and a late
phase, when other genes are expressed, and some genes of the first
phase are turned down or off
(102,
125,
152,
154). GerE effects a
similar division of the K regulon
(122,
316). Further temporal
division of the K regulon was shown to result from
the combined action of GerE and SpoIIID: SpoIIID
repressed and so delayed the expression of some GerE-activated genes
but not others (123).
GerE also appears to be regulated, in part, through the action of the
spore coat protein SpoVIF
(158).
Different
temporal classes are also found in the F and
G regulons, but the mechanisms do not seem so
clearcut (107). For
example, the spoIIIG locus is expressed about 40 min later
than other members of the F regulon
(144) and requires
expression of spoIIQ
(284) and some
component(s) of the E regulon
(218). The gene for a
repressor-activator, RsfA, has been identified in the
F regulon
(306), but its role is
less well defined than that of SpoIIID or GerE. Finally, in
the G regulon, the sspF locus is
transcribed about 1 h after other members of the regulon
(217). Two transcription
regulators of G-directed transcription have been
identified, SplA and SpoVT
(13,
68); they do not appear
to have the substantial roles exercised by SpoIIID or GerE in
their regulons and are not known to regulate sspF. In summary,
there is a complex temporal progression of gene expression in both the
prespore and the mother cell.
Analysis of expression of
the F-directed spoIIR locus indicated a
very short period of transcription
(148,
320), suggesting that
transcription of some F-directed genes stops before
G becomes active. This supposition was confirmed by
using a two-part probe to test for compartmentalization of gene
expression within individual cells
(173). The result also
provides clear evidence for spo gene expression being switched
off during spore formation. It is not known if the temporal
compartmentalization applies to other F-directed
promoters, or if it is tied to a morphological event such as the
completion of engulfment, and the mechanism remains unknown. A similar
switch was detected in the mother cell between the
E-directed cotEP1 promoter and
K-directed gerE expression. The mechanism
is also unknown but is distinct from SpoIIID-directed
repression (173). In
general, the mechanisms of turning gene expression off during spore
formation are much less well understood than those for turning
expression on; their importance remains to be
established.
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Sporulation has been studied almost exclusively in rod-shaped bacteria. However, one way to study the relationship between morphogenesis and gene expression is to extend studies to organisms with different shapes, notably cocci. Although coccal mutants of B. subtilis have been too sick to form spores (108), the coccal species Sporosarcina ureae is a naturally occurring sporeformer. S. ureae fits, by most criteria, into the genus Bacillus (70). The spores formed by S. ureae are structurally very similar to those of bacilli and clostridia (120). The sporulation septa of S. ureae resemble the sporulation septa of bacilli and clostridia in their ultrastructure and are very different from vegetative septa (244). However, the sporulation division in S. ureae is medially located with respect to cell poles, in contrast to the gross asymmetry of its location for bacilli and clostridia (244, 313). Nevertheless, compartmentalization of gene expression is observed in S. ureae, and it is inferred that spatial asymmetry is not required for compartmentalization of gene expression in this species (313). Thus, one plausible factor in the establishment of compartmentalized gene expression, gross volume asymmetry, is eliminated.
Despite the symmetry of location of the sporulation division, S. ureae has a homologue of the spoIIIE gene (35, 36) that encodes a DNA translocase critical for sporulation in B. subtilis (17, 303). This homologue is also essential for sporulation in S. ureae and can complement B. subtilis spoIIIE mutants (35, 36). Presumably the DNA translocase function of SpoIIIE is still required in S. ureae. Although the cells formed at division are similar in volume, successive division planes are at right angles to each other (22, 313). This change in direction requires that the chromosome reorient at each division. This reorientation may set the stage for a distinction in chromosome movement between the prespore and the mother cell. Conjecturally, transient genetic asymmetry (54) is still a factor in the establishment of compartmentalized gene expression in S. ureae. This conjecture raises related questions. Does trapping of the chromosome terminus region in a particular cell at septation determine that the cell will be the mother cell? Is that location predetermined by division history, or is the choice random? One intriguing possibility is that in the absence of volume asymmetry, genetic asymmetry becomes the predominant determinant of compartmentalization, so that its disruption would have a much more dramatic effect than is observed in B. subtilis (54).
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The spatial compartmentalization between prespore and mother cell of the activities of
F, E, G,
and K is, when first established and for the
duration of spore formation, essentially complete. However, there are
several mutant backgrounds in which this compartmentalization is
disrupted. In all known examples, the mutants are impaired in their
ability to form spores. This result is strong circumstantial evidence
for the conclusion that complete compartmentalization of the activities
of the four factors between prespore and mother cell
is essential for spore formation, although it does not establish the
conclusion unequivocally. The barrier that ordinarily prevents
activation of K before completion of engulfment
appears to be important but not essential; mutants that show activation
before completion of engulfment show reduced rather than no spore
formation (40,
211). Below we discuss
different mutations that disrupt the prespore-mother cell
compartmentalization. The analysis of spoIIAB mutations
(38,
260) was an important
landmark in the elucidation of the regulation of F
activity. Otherwise, mutations disrupting prespore-mother cell
compartmentalization have not been studied extensively; it may be that
additional types of mutation can be identified and substantially more
can be learned through their study.
The components for
F activation are present before septum formation,
but F normally only becomes active following
septation. However, as discussed earlier, control of
F activation appears to be on a knife edge. Thus,
mutations that inactivate SpoIIAB and cause
hyper-F activity
(38,
260) result in
uncompartmentalized F activity. Indeed, the
sporulation septum is not formed
(38). Similarly, certain
mutations in spoIIE result in hyper-uncompartmentalized
F activity and also prevent septation and spore
formation (73,
111). Clearly, in these
mutants F becomes active before septation, and
indeed a F-directed gene appears to prevent
formation of the sporulation septum
(111).
Mutations
that disrupt the spoIIIE locus result in loss of
compartmentalization
(171,
173,
234,
303-305).
In contrast, many missense mutations in spoIIIE do not disrupt
compartmentalization but rather result in
hyper-prespore-specific expression of
F-directed genes located in the origin-proximal
third of the chromosome and no expression of such genes located
elsewhere in the chromosome
(303-305).
Both classes display a similar block in DNA translocation. However the
mutant SpoIIIE protein of the latter class (class I) is
located in the middle of the spore septum, as is wild-type
SpoIIIE; no localization, and often no SpoIIIE
protein, is detected with the former class (class II) of mutant
(304). These
observations led to the suggestion that SpoIIIE might form an
effective seal around the DNA as it traversed the septum. In the
absence of this seal, in class II mutants, small molecules, such as
F or SpoIIAA, could traverse the septum,
resulting in a loss of compartmentalization
(304). However, a
separate study found that ß-galactosidase could not diffuse
across the septa of class II mutants and proposed that persistence of
SpoIIE in the mother cell rather than a hole in the septum was
responsible for the loss of compartmentalization
(234). Our recent
studies support the existence of a small hole permeable to
factors or their regulators and to GFP
(110).
A further
puzzling observation was that whereas F-directed
gene expression was uncompartmentalized in class II spoIIIE
mutants, it was again compartmentalized when a spoIIG mutation
was introduced into such mutants
(171,
234,
309), blocking
E activity. Given that
E-directed genes are required to cause septal wall
autolysis during engulfment
(1,
37,
57,
85,
228,
232,
246,
271,
272), it seemed
plausible that similar activity might enlarge a breach in
the septum resulting from loss of SpoIIIE. Indeed, E-directed transcription
of spoIID and spoIIP is required to disrupt
compartmentalization in spoIIIE class II mutants
(110). Presumably,
autolytic activity associated with SpoIID and SpoIIP
enlarges the putative hole sufficiently to allow passage of
F or its regulators and of GFP and possibly of
E.
Mutations in the spoIIIA and
spoIIIJ loci have been known for some time to prevent
activation of G and hence K
(60,
146,
281). These mutations
have recently been found to also cause a loss of compartmentalization
of F and E activities
(172). The
activities of these factors are initially compartmentalized
in the mutants, and compartmentalization is only lost aftercompletion of engulfment of the prespore by the mother cell.
Thus, it appears to be a secondary consequence of the sporulation
defect rather than the cause of the stage III blockage. It is likely
that the loss of compartmentalization is a consequence of instability
of the engulfed mutant prespores
(172).
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Spore formation in B. subtilis is a simple developmental process (Fig. 1) that lends itself to genetic and cell biological analysis. B. subtilis monitors its intracellular and extracellular environment, integrating this information into a phosphorelay that regulates the initiation of sporulation. When conditions are appropriate, the action of the phosphorelay results in the phosphorylation and so activation of the master response regulator Spo0A. Active Spo0A and the transition regulator
H trigger global changes in gene
regulation, setting the stage for formation of the axial filament and
the asymmetrically located sporulation division. Formation of the axial
filament requires the DNA-binding proteins RacA and Soj as well as the
division protein DivIVA (Fig.
3). The asymmetric
division requires greatly increased expression of ftsZ and
induction of spoIIE, and as a consequence the cytokinetic
protein FtsZ spirals from mid-cell to sites near the poles; division
occurs at one of these sites, yielding the smaller prespore
and the larger mother cell.
Compartmentalized activity
of two sporulation-specific sigma factors commences very
soon after the sporulation division, and it is now well established
that compartmentalization is, within the limits of detection, complete:
F in the prespore and
E in the mother cell (Fig.
2). The first to become
active is F. A complex regulatory pathway has been
elucidated for F, centered on the anti-sigma factor
SpoIIAB, the anti-anti-sigma factor SpoIIAA, and the
protein phosphatase SpoIIE, which activates SpoIIAA
by dephosphorylating it (Fig.
4A). Activation is
associated with completion of the sporulation division septum and is
confined to the prespore. When the sporulation septum is first
formed, only the origin-proximal one-third of a chromosome is present
in the prespore. This genetic asymmetry is resolved by
SpoIIIE, which transfers the remaining portion of the trapped
chromosome into the prespore (Fig.
3). It takes perhaps 15
min for the origin-distal two-thirds, including the
genes for SpoIIAA, SpoIIAB, and
F, to be transferred. The
transient genetic asymmetry during this time, the
instability of SpoIIAB, the long-lived
SpoIIAA-SpoIIAB-ADP complex
sequestering
dephosphorylated
SpoIIAA, and the localization of the SpoIIE
phosphatase to the septum have been shown to be important contributors
to the prespore specificity of F
activation (Fig. 4B),
although there is considerable redundancy in their contributions. What
is not yet clear is why activation occurs so rapidly after septation
and why F activity is absolutely confined to the
prespore. It is as if the system is very delicately balanced
with respect to the prespore, and a slight push ensures
prespore-specific activation, yet the system is robust in that
normally there is no activation before septation and none in the mother
cell.
Activation of the next factor, E in the
mother cell, requires prior activation of F.
Activation is by cleavage of a prosequence from
pro-E, probably through the action of the putative
protease SpoIIGA. The activation of E must
occur rapidly (<10 min?) after formation of the sporulation
septum in order to prevent the formation of a second asymmetrically
located septum. Minimally, in that time F becomes
active and directs transcription of spoIIR; SpoIIR
triggers the processing of pro-E;
E directs transcription of spoIID,
spoIIM, and spoIIP, whose products act to prevent the
formation of the second septum. It is not clear how
E activation is achieved so rapidly and exclusively
in the mother cell, although prespore-specific proteolysis of
pro-E is probably a contributory factor.
Postseptation enhancement in the mother cell of Spo0A-directed
transcription of spoIIG (encoding pro-E)
is an important contributing factor to the production of
E in the mother cell (Fig.
5), but it is not clear
why Spo0A activity (and perhaps phosphorelay activity) is greatly
enhanced in the mother cell and curtailed in the prespore, nor
is it clear if this increased transcription could account for the
rapidly induced, E-directed suppression of
septation.
Activation of pro-E requires
expression of the F-directed spoIIR locus.
This exemplifies a recurring theme, that compartment-specific gene
expression is controlled by intercompartmental regulatory signals, a
theme often referred to as crisscross regulation. Mother cell-specific
activation of E does not require that
spoIIR be expressed in the prespore. Thus, the mother
cell specificity of E activity can be independent
of the prespore specificity of F action.
These sigma factors direct compartmentalized gene expression but not
the process of compartmentalization.
Completion of engulfment of
the prespore by the mother cell starts the next phase of
compartmentalized gene expression. It is associated with the action of
a second pair of sporulation-specific sigma factors,
G and K (Fig.
2) The first of these to
become active, G, does so exclusively in the
prespore. Its prespore specificity can be explained,
at least in part, by F-directed transcription of
spoIIIG, the structural gene for G.
However, it is not clear why activation occurs only after completion of
engulfment. A chain of regulators involves the products of
spoIIIA (made in the mother cell) and spoIIIJ (needed
only in the prespore) antagonizing the
anti-G activity of SpoIIAB (Fig.
6), and yet the action of
SpoIIAB is inadequate to explain why G is
not activated before completion of engulfment and why its activation is
tied to that event. It is thought that a critical regulator remains to
be identified. The morphological coupling of the activation of
F and G to completion of
septation and engulfment, respectively, is clear; the mechanisms are
not.
The final factor to be activated during
sporulation, K, is also subject to complex
regulation. First, in B. subtilis strain 168, though not in
other strains or species (with the exception of C. difficile),
there is mother cell-specific excision of an insertion element in
sigK, the structural gene for pro-K. There
is then E-directed transcription of sigK,
also ensuring the mother cell specificity of K.
Finally, as with its predecessor in the mother cell,
E, K is activated from an
inactive prosequence form. It requires a complex of proteins
synthesized in the mother cell together with a
G-directed signal from the
prespore (Fig.
7). Mechanistically, the
activation of pro-K is very different
from that of pro-E. The final processing
enzyme is thought to be a membrane-embedded metalloprotease,
SpoIVFB.
The compartment-specific factors provide the
bare framework of compartment-specific gene expression. However,
within each regulon are several temporal classes of
genes, some requiring additional activators and some subject to
repressors. Some genes are clearly turned off before others become
active. For key regulators, timing is critical. For example,
accelerating the expression of K or delaying the
expression of spoIIR greatly impairs spore formation.
The concept of transient genetic asymmetry emphasizes the importance of
timing in the establishment of compartment-specific gene
expression.
Compartmentalization of gene expression during bacterial spore formation is but part of the process of forming a heat-resistant, dormant spore. In our discussion of each of the sigma factors, we have only briefly mentioned the roles that their regulons play in building the mature spore. There is more to be done to understand compartmentalized gene expression during spore formation. There is very much more to be done to fully understand how this "simple" cell differentiation is achieved.
Work by the authors was supported in part by Public Health Service grants GM43577 (to P.J.P.) and T32AI07101 (to D.W.H.).
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Temple University School of Medicine, 3400 N. Broad St., Philadelphia, PA 19140. Phone: (215) 707-7927. Fax: (215) 707-7788. E-mail: piggotp{at}temple.edu.
Present address: Department of Anatomy and Cell Biology, Columbia University, New York, NY 10032.
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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
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