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Microbiology and Molecular Biology Reviews, June 2004, p. 234-262, Vol. 68, No. 2
1092-2172/04/$08.00+0     DOI: 10.1128/MMBR.68.2.234-262.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Compartmentalization of Gene Expression during Bacillus subtilis Spore Formation

David W. Hilbert{dagger} and Patrick J. Piggot*

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

   SUMMARY
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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, {sigma}F in the prespore and then {sigma}E in the mother cell, and then later, following engulfment, {sigma}G in the prespore and then {sigma}K in the mother cell. The coupling of the activation of {sigma}F to septation and {sigma}G to engulfment is clear; the mechanisms are not. The {sigma} factors provide the bare framework of compartment-specific gene expression. Within each {sigma} regulon are several temporal classes of genes, and for key regulators, timing is critical. There are also complex intercompartmental regulatory signals. The determinants for {sigma}F regulation are assembled before septation, but activation follows septation. Reversal of the anti-{sigma}F activity of SpoIIAB is critical. Only the origin-proximal 30% of a chromosome is present in the prespore when first formed; it takes {approx}15 min for the rest to be transferred. This transient genetic asymmetry is important for prespore-specific {sigma}F activation. Activation of {sigma}E requires {sigma}F activity and occurs by cleavage of a prosequence. It must occur rapidly to prevent the formation of a second septum. {sigma}G is formed only in the prespore. SpoIIAB can block {sigma}G activity, but SpoIIAB control does not explain why {sigma}G is activated only after engulfment. There is mother cell-specific excision of an insertion element in sigK and {sigma}E-directed transcription of sigK, which encodes pro-{sigma}K. Activation requires removal of the prosequence following a {sigma}G-directed signal from the prespore.


   INTRODUCTION
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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.

 
The cell type-specific, compartmentalized programs of gene expression result from the cell type-specific activity of RNA polymerase sigma factors: {sigma}F and then {sigma}G in the prespore and {sigma}E and then {sigma}K in the mother cell (Fig. 2) (231). Compartmentalization of gene expression in each cell type is coupled to morphogenesis, with {sigma}F and {sigma}E becoming active after asymmetric division and {sigma}G and {sigma}K becoming active after engulfment of the prespore by the mother cell. Thus, the activation of {sigma}G and {sigma}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 {sigma}F, {sigma}E, {sigma}G, and {sigma}K, respectively. The prespore membranes are not stained in the {sigma}G and {sigma}K images because engulfment is complete and the prespore membranes are now inaccessible to the lipophilic FM4-64 stain. {sigma}F, active in the prespore, is the first compartmentalized {sigma} 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-{sigma}E into active {sigma}E in the mother cell. RNA polymerase with {sigma}E transcribes the spoIIIA operon, whose products then signal across the prespore membrane to activate {sigma}G, expressed in the prespore under the control of {sigma}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 {sigma}G) requires an unknown signal from the mother cell as well as the SpoIIQ protein, expressed in the prespore under the control of {sigma}F. Once {sigma}G becomes active, it causes expression of SpoIVB, which is inserted into the inner prespore membrane. SpoIVB triggers processing of pro-{sigma}K, which is synthesized in the mother cell from the {sigma}E-directed sigK gene. The processing enzyme is thought to be SpoIVFB, which also expressed in the mother cell under the control of {sigma}E but does not act upon pro-{sigma}K until it receives the SpoIVB signal from the prespore.

 
In this article we review the process of spore formation. We focus primarily on B. subtilis but include discussion of other species where we think it appropriate. We discuss in detail the genetic and biochemical experiments that have led to the discovery and characterization of cell-specific programs of gene expression. Since sporulation follows a distinct series of morphological and genetic stages, we detail steps in the order that they naturally occur, as though following a single cell through the entire developmental process. The cell-specific changes in gene expression that occur during sporulation are coupled to morphogenesis. The two major phases of compartmentalization are associated with two major morphological events, completion of septation and completion of engulfment. Consequently, we start with a brief description of the morphological changes during sporulation. We discuss in depth the events leading to the asymmetrically located sporulation division, which primes the organism for the compartmentalization of gene expression that follows the division. Compartmentalized gene expression is associated with the activation of the four sporulation-specific {sigma} factors. We discuss the activation of each. We pay particular attention to regulation of {sigma}F because it is the first {sigma} 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.


   MORPHPOLOGICAL STAGES OF SPORULATION
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In order to discuss compartmentalization of gene expression, we first review the morphological changes in the developmental process, with which changes in gene expression are associated. Formation of heat-resistant spores from vegetative cells of B. subtilis takes about 7 h at 37°C. The morphological changes during sporulation were initially characterized by electron microscopy (145, 251). The basic sequence of changes is similar for all species of Bacillus and Clostridium that have been studied (79) and is illustrated in Fig. 1. Identification of successive stages by Roman numerals follows the convention introduced by Ryter (251) and now generally used. The vegetative cell is designated stage 0. Formation of an axial filament of chromatin, where two copies of the chromosome condense and elongate to form a filament that stretches across the long axis of the cell, is defined as stage I (21, 29, 308). Subsequently, the cell divides at a subpolar site, resulting in the formation of two unequally sized daughter cells.

Completion of septation is designated stage II. At the time of asymmetric division, only approximately one-third of a chromosome is present in the smaller prespore (also called the forespore), but the remaining two-thirds is rapidly pumped in by the DNA translocase SpoIIIE (17, 303), resulting in two cells with unequal volumes but identical genomes. Next, the polar septum undergoes septal thinning, followed by bulging of the prespore into the mother cell and migration of the septal membrane around both sides of the prespore. When the migration is complete, the membranes fuse at the cell pole, pinching off the prespore and releasing it within the mother cell as a free-floating protoplast that is surrounded by two membranes, one derived from each of the two cells; completion of engulfment is designated stage III (227). Engulfment is followed by the deposition of two peptidoglycan layers, the primordial germ cell wall and the cortex, in the space between the membranes surrounding the prespore (stage IV) (81). Following this deposition, a complex structure of proteins on the outside surface of the prespore, known as the coat, is constructed (stage V) (47, 109). This stage is followed by maturation of the spore (stage VI), when it gains resistance to UV radiation and high temperature (208). Lastly, the mother cell lyses (stage VII), releasing the mature spore into the environment. Germination and outgrowth, followed by a resumption of the vegetative growth cycle, occur when the spore finds itself in a nutrient-rich environment (213). Sporulation mutants are denoted by the stage in the process at which they are blocked (e.g., spoII mutants complete asymmetric septation but fail to complete engulfment). The names for sporulation loci include the stage of blockage caused by mutation and a distinguishing letter designation (e.g., spoIIA) (228, 231).

The profound morphological changes that occur during sporulation are coupled to global changes in gene expression, which are effected by activation of alternative RNA polymerase {sigma} factors (Fig. 2) (228, 231). Activation of {sigma}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, {sigma}F becomes active in the prespore, rapidly followed by activation of {sigma}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 {sigma} factors. Upon completion of engulfment, {sigma}G becomes active in the prespore and {sigma}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).


   INITIATION OF SPORULATION
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Gene expression becomes compartmentalized immediately after the spore septum has formed. To understand how this compartmentalization happens, it is important to explore the events leading to it. In this section we briefly discuss the activation of the master sporulation response regulator Spo0A and the alternative {sigma} factor {sigma}H. For more focused reviews on the initiation of sporulation, we refer the reader to references 27 and 223.

The Phosphorelay

Spo0A is the master regulator for entry into spore formation. It is activated by the phosphorelay, a more complex version of the classic two-component system (26). In turn, Spo0A-PO4 activates transcription of genes required for axial filament formation and for asymmetric division. It also activates transcription of the genes encoding the early compartmentalized {sigma} factors, {sigma}F and {sigma}E, as well as their regulators. Sporulation is initiated in response to a number of external and internal signals that are integrated into the phosphorelay, including signals for nutrient starvation, cell density, and cell cycle progression (27, 223, 291).

At least five kinases are involved in the phosphorelay: KinA, KinB (290), KinC (160), KinD (134), and KinE (67), of which KinA and KinB are the primary kinases for initiation of sporulation. In response to unidentified stimuli, they autophosphorylate and then donate their phosphate groups to the response regulator Spo0F. Spo0F lacks an output domain and is incapable of activating transcription; it serves only as an intermediary in the phosphorelay. The phosphotransferase Spo0B transfers the phosphate from Spo0F-PO4 to Spo0A (26). The phosphorelay is also subject to negative regulation: for example, the phosphatases Spo0E, YisI, and YnzD dephosphorylate Spo0A, thereby preventing its activation (210, 221). In order to ensure that sporulation occurs only under the appropriate conditions, the phosphorelay must integrate different intracellular and extracellular signals. The mechanisms responsible for this integration are described below.

Cell density. Efficient sporulation requires high cell density (101). When cell density is low, the Rap (response regulator aspartyl phosphatase) proteins RapA, RapB (222), and RapE (133) dephosphorylate Spo0F-PO4, preventing Spo0A activation. The rapA and rapE genes are cotranscribed with a downstream open reading frame encoding the signaling peptide precursors PhrA and PhrE, respectively, which are processed and exported out of the cell. As cell density increases, the processed peptides are imported by the oligopeptide permease (Opp) and inhibit the activity of RapA and RapE; similarly, the processed product of PhrC (CSF [competence- and sporulation-stimulating factor])inhibits RapB. Inhibition of the Rap proteins prevents dephosphorylation of Spo0F-PO4 and allows phosphorylation of Spo0A and the initiation of sporulation when cell density is high (133, 220).

Nutrient starvation. In addition to high cell density, nutrient starvation is also required for the initiation of sporulation. A dramatic drop in the concentration of GTP and GDP correlates with the onset of sporulation, and inhibition of GMP synthesis by decoyinine treatment induces sporulation in the absence of nutrient starvation (200). CodY has recently been identified as the key sensor of guanine nucleotide levels. Disruption of the codY gene allows sporulation to occur in the presence of excess nutrients, and the ability of the CodY repressor to bind DNA correlates with the GTP concentration. As a consequence, when GTP levels drop upon entry into stationary phase, CodY-regulated genes are derepressed (239). Microchip array analysis has identified phrA, phrE, and kinB, all positive regulators of the phosphorelay, as targets of CodY repression (204). Therefore, one way that nutrient starvation is integrated into the decision to sporulate is transcriptional regulation of phosphorelay components via CodY. In addition, sporulation is also subject to catabolite repression (117) and requires a functioning Krebs cycle (131), although the molecular basis of these dependencies is unknown.

Cell cycle. In addition to factoring extracellular conditions such as cell density and nutrient availability into the decision to sporulate, the intracellular environment is monitored as well. Damage to DNA and blocking of either the initiation or progression of DNA replication prevent the initiation of sporulation (126, 128, 129, 132, 165). These conditions lead to the expression of Sda (suppressor of dnaA) because of the presence of binding sites in the sda promoter region for the repressors DnaA and LexA, which no longer repress when DNA replication is blocked or DNA is damaged, respectively. Sda impairs KinA autophosphorylation, blocking the phosphorelay (28). As a result, a developmental checkpoint is established that only allows cells with undamaged, replicating chromosomes to proceed into development.

An additional mechanism is thought to link chromosome partitioning status to sporulation. Spo0J and Soj (suppressor of spo0J) (also known as Spo0JB and Spo0JA, respectively) are members of the plasmid-partitioning families of proteins ParB and ParA, respectively (130). Spo0J colocalizes to cell poles with the chromosomal origin of replication (175), binds to sites near the chromosomal origin (174), and is required for optimal efficiency of chromosome segregation (130). In the absence of Spo0J, Soj binds to the promoter regions of at least four Spo0A-responsive genes (spo0A, spoIIA, spoIIE, and spoIIG) and represses their transcription (33, 191, 237, 238). This effect appears to be mediated by dynamic protein localization; when Spo0J is present, Soj oscillates between sites near the poles of the cell, presumably preventing stable DNA-protein interaction and transcriptional repression. However, in the absence of Spo0J, Soj remains static and represses developmental transcription (191, 238). Although it is tempting to speculate that Spo0J and Soj sense chromosome partitioning status and regulate the initiation of sporulation accordingly, direct evidence for this model is lacking. However, consistent with a role in monitoring the cell cycle, recent studies have linked these proteins to cell division, to initiation of DNA replication, and to axial filament formation (11, 163, 209, 308).

Spo0A Regulon

The sum of all of these interactions determines if enough Spo0A-PO4 has been generated to initiate sporulation. Spo0A-PO4 can either activate or repress transcription by binding to a 7-bp sequence, TGNCGAA, where N is any nucleotide, in or near promoters recognized by the vegetative {sigma} factor {sigma}A and the alternative {sigma} factor {sigma}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 {sigma}F and {sigma}E, respectively; and spoIIE, encoding a bifunctional protein phosphatase that is required for asymmetric division and {sigma}F activation (231). In addition, the gene encoding the effector of axial filament formation, racA, is under the control of Spo0A (21, 308). Thus, Spo0A activates the synthesis of factors required for chromosome remodeling, asymmetric division, and the compartmentalized gene expression that immediately follows. The racA gene was identified by functional analysis of the Spo0A regulon (21), and such analyses will most likely reveal additional genes involved in sporulation.

Role of {sigma}H

In addition to the phosphorelay and the main vegetative {sigma} factor {sigma}A, the transition-state regulator {sigma}H is also required for the initiation of sporulation. Regulation of {sigma}H synthesis and activity is not well understood but involves both posttranscriptional and posttranslational mechanisms (106, 177). {sigma}H regulates a number of phosphorelay genes: {sigma}H-dependent transcription of spo0A is essential for sporulation (270), and kinA, kinE, and spo0F also have {sigma}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 {sigma}H (197). Other {sigma}H-regulated genes are important for later events in sporulation: {sigma}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 {sigma}F and its regulators, is under the control of {sigma}H (302). A substantial portion of the {sigma}H and Spo0A regulons overlap: for example, spoIIA and racA are regulated by both transcription factors. The {sigma}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).


   AXIAL FILAMENT FORMATION
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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).

Genetic Control

Standard genetic analysis failed to identify effectors of axial filament formation, and only recent genomic and cell biological studies have allowed identification of the components involved. Despite their name, none of the classic spo0 mutations clearly prevented axial filament formation (228): spo0H mutants formed axial filaments (29, 94), whereas spo0A, spo0B, and spo0F mutants underwent an additional symmetric division during sporulation, and it was not clear if that was preceded by axial filament formation (29, 53). Some insight into the process was derived from analysis of the SMC (structural maintenance of chromosomes) protein, required for chromosome compaction and partitioning (25, 176). Mutants lacking this protein are able to activate Spo0A but are unable to form axial filaments (99), suggesting that an effector of axial filament formation is either missing or nonfunctional in this background. It was also found that asymmetric division did not occur in the absence of axial filament formation, suggesting the existence of a checkpoint that functioned to couple the two events (99). Such a checkpoint would ensure that asymmetric division would trap the origin-proximal region of a chromosome in the prespore, providing a template for transcription by E-{sigma}F (RNA polymerase core enzyme, E, associated with {sigma}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 {sigma}F is depicted as being active in the prespore and {sigma}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.

 
RacA. Genomic approaches made it possible to elucidate the role that DivIVA played in axial filament formation. One approach identified Spo0A-regulated genes by microchip array (71), systematically disrupted them, and screened the mutants for changes in chromosome segregation (21). In a separate study, {sigma}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 {approx}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.


   ASYMMETRIC DIVISION
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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.

FtsZ Ring Switching

Dynamic repositioning. During vegetative growth, the essential prokaryotic tubulin homologue FtsZ forms a ring (the Z ring) at mid-cell, where division subsequently occurs (298). However, during sporulation, activation of Spo0A triggers the formation of Z rings near both poles of the cell (167). The switch has recently been characterized by deconvolution microscopy. The use of this technique revealed that, during sporulation, a Z ring initially forms at mid-cell but the FtsZ then redeploys to sites near both poles through the formation of a dynamic helical intermediate (19). This intermediate resembles the helical structures formed by two bacterial actin homologues, Mbl and MreB (137); one possibility is that FtsZ relocates by tracking along these structures.

Genetic control. Early studies showed that the ftsAZ operon contained three promoters, one of which was activated during sporulation by {sigma}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 {sigma}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 {sigma}H-dependent promoter of ftsAZ resulted in severe impairment of polar Z ring formation and asymmetric division. Conversely, if ftsAZ overexpression was combined with expression of spoIIE, asymmetric division could be triggered during vegetative growth (19). Therefore, spoIIE induction and increased ftsAZ expression play overlapping roles during sporulation in ensuring polar Z ring formation and asymmetric division.

SpoIIE. The discovery of a role for SpoIIE in polar Z ring formation (19, 149) reinforced previous ultrastructural studies that had implicated this protein in asymmetric division. These studies found that null mutations in spoIIE resulted in rare asymmetric septa that were aberrantly thick, whereas several point mutations blocked sporulation but allowed the formation of typical sporulation septa at normal frequency (16, 228). Consistent with this role, it was found that SpoIIE localizes to asymmetric division sites (10, 14) in an FtsZ-dependent manner (168). FtsZ and SpoIIE also interact in vitro and in yeast two-hybrid assays (186). However, how this interaction assists polar Z ring formation and asymmetric division is unknown. Some possibilities include anchoring an end of the FtsZ helix to the cell membrane, antagonizing MinCD near the pole of the cell, and recognizing a polar marker (19). In addition to SpoIIE and increased ftsAZ expression, there is evidence that MinCD and SpoVG may play minor roles in selecting the asymmetric division site (15, 195).

Abortively Disporic Phenotype

In spo+ strains, surface annular structures (cloisons) and Z rings appear at both polar sites (19, 167, 251), indicating that the cell has two potential polar division sites. However, a septum is normally formed at only one of these sites (251). In certain mutants, both potential polar division sites are utilized, resulting in a three-chambered organism consisting of two smaller prespores separated by a larger central compartment. This is the abortively disporic phenotype, which is associated with mutations in some spoII loci (228, 252). Mutants that demonstrate the abortively disporic phenotype can initiate sporulation but have defects in activating the mother cell-specific transcription factor {sigma}E (124). Three proteins expressed in the mother cell under {sigma}E control, SpoIID, SpoIIM, and SpoIIP, are required to prevent the second asymmetric division (57, 232). Although it was puzzling why B. subtilis would generate two potential division sites during sporulation, recent studies have provided some insight. Cells with anucleate prespores are frequently observed in racA mutant cells, which are deficient in axial filament formation. However, a substantial proportion of these cells undergo a second asymmetric division at the other end of the cell, and if they successfully capture DNA in the second prespore, they form spores (21, 308). This result suggests that the ability to divide at both asymmetric division sites is a failsafe mechanism to ensure successful sporulation even in the absence of axial filament formation.

Which End of the Cell?

An unanswered question is how the cell determines at which end of the cell it will divide. Although FtsZ rings form at both potential polar division sites, the next known protein to assemble, FtsA, localizes to only one of them (76), indicating that FtsA may play some role in selecting which of the two potential division sites is utilized. Chromosome segregation into the prespore or mother cell appears to be essentially random with respect to time of replication, and so chromosome age is presumably not a factor in determining which end becomes the prespore (43, 65). In most circumstances and in a range of species, spores are formed almost exclusively at the older pole of the cell, clearly suggesting that the pole is a determinant of asymmetry and compartmentalization (52, 112, 113). However, when the sporulation procedure involves centrifugation (with a force of perhaps 5,000 x g) and 25 mM Mg2+, spore position is essentially random with respect to pole age (52). Thus, polar determinism can be lost without losing asymmetric division or compartmentalization.

Differences between Sporulation Septum and Vegetative Division Septum

The asymmetrically located sporulation division is often considered the defining early morphological event in sporulation. The machinery for asymmetric division is similar to that used for vegetative division (61). However, there are several distinctive features of the sporulation division (114, 227) in addition to those described above. Since the division is critical to the compartmentalization of gene expression that follows, it is useful to summarize those distinctive features. (i) The sporulation division septum is much thinner than the vegetative division septum. (ii) The two cells that result from the sporulation division do not separate from each other, as occurs following vegetative division. Rather, the mother cell engulfs the prespore. (iii) Autolysis of the wall material (peptidoglycan) within the sporulation septum begins in the center of the septum, and ultimately there is apparently complete loss of wall material. In contrast, autolysis of the wall material of the vegetative septum begins at the periphery of the septum and proceeds inwards. Moreover, there is little loss of wall material—the split septum provides the wall for the poles of the nascent cells (227). (iv) Prior to the sporulation division, the two chromosome origins and associated proteins move to the extreme poles of the cell rather than to a subpolar location, as in vegetative division (21, 175, 300, 308). (v) The septum is asymmetrically located, with respect to the cell poles, during sporulation but not during vegetative growth (251) (vi) Several proteins become associated with the sporulation septum that are not associated with the vegetative division septum (10, 14, 72). (vii) Complete partitioning of a chromosome into the prespore occurs after septation (303, 310), so that there is genetic asymmetry between the prespore and the mother cell when they are first formed (54, 84, 305). (viii) After the sporulation division, different programs of gene expression are initiated in the two daughter cells (231). These programs are driven by activation of cell-specific {sigma} factors that direct RNA polymerase to transcribe different genes, which encode factors responsible for establishing the very different fates of the prespore and the mother cell.


   TRANSFER OF DNA INTO THE PRESPORE
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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.


   COMPARTMENTALIZATION OF GENE EXPRESSION
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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 {sigma} factors whose activation is coupled to landmark morphological events. Asymmetric division triggers the activation of {sigma}F in the prespore, followed by activation of {sigma}E in the mother cell. Later in sporulation, the completion of engulfment of the prespore by the mother cell leads to the activation of {sigma}G in the prespore and {sigma}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 {sigma} 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 {sigma} factors is compartmentalized to be compelling, our understanding of the mechanisms of compartmentalization is still incomplete. A variety of regulators of {sigma} 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 {sigma} factors.

Developing Evidence that Gene Expression Is Compartmentalized

The different fates of the prespore and the mother cell suggested that different genes are expressed in the two compartments. This suggestion was supported by biochemical characterization of extracts enriched for the contents of the prespore or the mother cell (5, 55, 88, 269). The first clear evidence that expression of spo loci was compartmentalized came from the study of genetically mosaic bacteria (43, 226). The mosaics were obtained by transforming spo mutants at the start of sporulation so that only one of the two copies of the chromosome became spo+. After division, mutant and wild-type alleles were distributed randomly into the prespore and the mother cell. Since the mother cell is destroyed and only the chromosome in the prespore is inherited upon spore germination, it was possible to infer the location of spo locus expression. For several loci, the spores obtained gave rise to spo mutant progeny, indicating that only the mother cell chromosome required a spo+ allele for sporulation to occur. Other loci that were tested yielded only spo+ spores, indicating that, for sporulation to occur, the allele on the prespore chromosome had to be transformed to spo+ (43, 226, 230). Subsequently, determination of ß-galactosidase activity in prespore- and mother cell-enriched extracts from strains expressing spo-lacZ transcriptional fusions provided strong support for differential gene expression between mother cell and prespore (reviewed in reference 59).

Direct evidence that the expression of particular genes was completely compartmentalized was obtained by the use of immunoelectron microscopy with antibodies to small acid-soluble proteins (SASPs), which revealed that these proteins are found exclusively within the prespore (83). The utility of this technique was expanded by using antibodies to ß-galactosidase on samples from strains expressing spo-lacZ transcriptional fusions (48, 189). The experiments demonstrated that the activities of {sigma}F and {sigma}G were confined to the prespore and those of {sigma}E and {sigma}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 {sigma}F and {sigma}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 {sigma}G and {sigma}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 {sigma} factor {sigma}A continues to be active in both prespore and mother cell throughout sporulation (173).


   ACTIVATION OF {sigma}F
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spoIIA Operon

Studies of the spoIIA locus have been critical to our understanding of compartmentalization of gene expression. The locus was found to be a tricistronic operon (80, 229) that was transcribed prior to asymmetric septation (93, 218) in a Spo0A- and {sigma}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 {sigma} factor (62, 274), named {sigma}F (183). The first targets identified for E-{sigma}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-{sigma}F-directed genes analyzed subsequently (231).

Posttranslational Regulation of {sigma}F

Although the spoIIA operon is expressed prior to asymmetric division (93, 218), {sigma}F does not become active until after asymmetric division (93, 142). It seemed likely that {sigma}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 {sigma}F activity, and mutation of SpoIIAB increased {sigma}F activity. Mutation of SpoIIAA blocked {sigma}F activation, but activity could be restored to these strains by mutation of SpoIIAB. Taken together, these results indicated that SpoIIAB antagonized {sigma}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 {sigma}F, blocked sporulation prior to asymmetric septation, and caused extensive lysis (38). Given that the activity of {sigma}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 {sigma}F regulation. It was shown that the negative role of SpoIIAB was direct in that it bound {sigma}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 {sigma}F activity, and mutation to an aspartic acid (mimicking phosphorylation) blocked {sigma}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 {sigma}F both directly, as an anti-{sigma} factor, and indirectly, by inactivating the anti-anti-{sigma} 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 {sigma}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 {sigma}F activation is illustrated in Fig. 4A.



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FIG. 4. Models of {sigma}F regulation. AA, AB, and E refer to SpoIIAA, SpoIIAB, and SpoIIE, respectively. The anti-{sigma} factor SpoIIAB binds {sigma}F as a dimer but is represented here as a monomer for simplicity. (A)Basic model of {sigma}F regulation. The anti-{sigma} factor SpoIIAB binds {sigma}F and holds it inactive. This inhibition can be reversed by the anti-anti-{sigma} 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-{sigma} factor) and active when dephosphorylated by SpoIIE. Once dephosphorylated, SpoIIAA can bind SpoIIAB and liberate {sigma}F, activating prespore-specific transcription. In this model, the phosphorylation state of SpoIIAA is directly correlated with {sigma}F activity, and the fate of SpoIIAB after {sigma}F liberation and the nucleotide binding status of SpoIIAB are not considered. (B) Integrated model of {sigma}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 {sigma}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 {sigma}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 {sigma}F. The mother cell (not shown) is presumed to resemble the predivisional cell.

 
Mechanisms of Compartmentalization

Although the known factors involved in {sigma}F regulation have been identified, it is still not clear why {sigma}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 {sigma}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 {sigma}F activity to the prespore. However, it was not clear how the proposed differential ATP/ADP ratio was established in vivo, and direct evidence for the regulatory roles of these nucleotides was lacking. Subsequent in vitro studies of the interaction betwe