Although prokaryotes ordinarily undergo binary fission to produce two identical daughter cells, some are able to undergo alternative developmental pathways that produce daughter cells of distinct cell morphology and fate. One such example is a developmental programme called sporulation in the bacterium Bacillus subtilis, which occurs under conditions of environmental stress. Sporulation has long been used as a model system to help elucidate basic processes of developmental biology including transcription regulation, intercellular signalling, membrane remodelling, protein localization and cell fate determination. This review highlights some of the recent work that has been done to further understand prokaryotic cell differentiation during sporulation and its potential applications.
Understanding the mechanisms that drive cell differentiation and morphogenesis are essential in answering the question of how organisms develop. However, elucidating these mechanisms can be difficult because of the complex and intertwined processes that occur during development (Hartwell and Weinert, 1989; Sasai, 2013). One approach to this problem has been to study the relatively simple developmental programme of endospore formation (‘sporulation’) in the bacterium Bacillus subtilis. During sporulation, a single rod-shaped cell divides asymmetrically, resulting in two genetically identical daughter cells that undergo different cell fates (Stragier and Losick, 1996; Errington, 2003; Piggot and Hilbert, 2004). Sporulation in B. subtilis is a particularly attractive system to look at cell differentiation and morphogenesis not only because of the relative simplicity of the sporulation developmental programme, but also because of the genetic tractability of the system. Bacillus subtilis is naturally competent, and genes necessary for sporulation are often non-essential for normal growth, both of which facilitate the identification of novel factors that participate in this developmental process. As a result, sporulation studies have provided significant insights into basic biological processes such as differential gene expression, membrane remodelling, intercellular communication, subcellular protein localization and morphogenesis.
Bacillus subtilis is ubiquitous in nature and can successfully adapt to various changes in the environment. Under stressful conditions, B. subtilis is able to initiate many survival mechanisms such as motility, uptake of exogenous DNA, biofilm formation and sporulation (Rao et al., 2008; Burton and Dubnau, 2010; Vlamakis et al., 2013). The purpose of sporulation is to produce a largely metabolically inactive dormant cell type called an ‘endospore’ (hereafter, referred to simply as a ‘spore’) that is able to survive harsh environmental conditions until favorable growth conditions are restored (Paredes-Sabja et al., 2011). Bacterial spores are one of nature's most resilient cell types and are able to survive under controlled laboratory conditions for several decades and perhaps even longer in the environment (Jacotot and Virat, 1954; Cano and Borucki, 1995; Vreeland et al., 2000; Setlow, 2007). When the spore senses that environmental conditions are conducive to growth, it is able to germinate and resume its vegetative cell cycle.
Sporulation initiates when the rod-shaped B. subtilis divides asymmetrically, elaborating a ‘polar septum’ that results in two genetically identical but morphologically distinct compartments: a larger ‘mother cell’ and a smaller ‘forespore’, each of which will ultimately experience different cell fates (Fig. 1). Both compartments briefly remain side by side, held together by the external cell wall. The initially flat polar septum then begins to curve as the mother cell swallows the forespore (a process called ‘engulfment’), producing a forespore that resides as a double membrane-bound, roughly spherical organelle inside the mother cell cytosol. The forespore eventually matures into a partially dehydrated, dormant cell that is released into the environment when the mother cell undergoes programmed cell lysis.
Here, we will provide a brief overview of the molecular mechanisms underlying the sporulation process from the decision of a cell to enter the sporulation programme to how the cell undergoes the significant morphological changes in order to become a distinct cell type that is resistant to various environmental insults, with an attempt to highlight primary literature that has been published recently. As a roadmap, we will present the sporulation programme in separate sections in which so-called ‘stages’ of sporulation are numbered ‘0’ to ‘V’, reflecting a classical nomenclature that was based on various morphological landmarks as viewed by electron microscopy (Schaeffer et al., 1963; Piggot and Coote, 1976). It is important to note, though, that as techniques have advanced, it has become clear that sporulation is not composed of distinct stages that occur sequentially. Instead, these stages actually lie along a continuum in which there may be significant overlap in terms of when a ‘stage’ begins and ends.
Stage 0: the decision to sporulate
As with many other developmental programmes, the entry into sporulation is closely regulated and relies on a series of feedback and feed-forward loops. In the bacterial population, sporulation does not occur homogenously, but rather occurs in subpopulations. Presumably, this is a bet-hedging strategy that allows the cell to absolutely confirm the need to sporulate prior to engaging in this highly energy-consuming and, once committed, irreversible developmental programme (Veening et al., 2008).
The first bet-hedging strategy to delay entry into sporulation is the ‘cannibalistic’ behaviour displayed by a subpopulation of cells that are the first to detect the onset of starvation conditions. During cannibalism, this subpopulation of cells kills neighbouring isogenic siblings that have not yet detected the onset of such conditions (Gonzalez-Pastor et al., 2003). Cannibalism is reliant on two secreted killing factors, Skf and Sdp (Liu et al., 2010; Perez Morales et al., 2013), which are produced by cells in a biofilm that produce the biofilm matrix (Lopez et al., 2009). The death of surrounding cells releases nutrients into the environment to support the growth of the subpopulation that produced the toxins. The toxin-producing subpopulation is protected through the concurrent production of a protective factor (Ellermeier et al., 2006). In this way, cannibalism is thought to be a mechanism to delay sporulation and to eliminate cells that are no longer beneficial to the population as it moves towards biofilm formation (Mitri et al., 2011). Consistent with this model, deletion of genes required for cannibalism results in a faster and more homogeneous entry of cells into the sporulation programme (Gonzalez-Pastor et al., 2003).
The transition of B. subtilis from vegetative growth to sporulation is largely governed by the transcriptional master regulator Spo0A, which also regulates biofilm formation (Hamon and Lazazzera, 2001). Spo0A transcriptional activity is activated by a ‘phosphorelay’ system that is governed by five autophosphorylating histidine kinases (KinA-KinE) that respond to different environmental stresses. While ‘limited nutrient availability’ is broadly defined as the signal for entry into sporulation, the identification of specific molecular ligands that activate the histidine kinases has remained elusive. This difficulty is largely due to the wide array of environmental inputs sensed by the bacterium and the somewhat redundant functions of the sensor kinases (LeDeaux et al., 1995). A recent strategy to approach the identification of molecular ligands through co-crystallization successfully identified pyruvate as a potential ligand of KinD (Wu et al., 2013). Pyruvate is involved in numerous metabolic pathways, and it seems reasonable that its levels in the extracellular environment may serve as an indicator of growth conditions. However, it is still unclear whether pyruvate is the physiological ligand of KinD and what effects this interaction may have on sporulation.
Upon activation and autophosphorylation, the phosphoryl group from the histidine kinases is transferred to Spo0A via the phosphotransferases Spo0F and Spo0B, which results in an active phosphorylated Spo0A (referred to as ‘Spo0A∼P’) (Burbulys et al., 1991). Spo0A∼P then goes on to directly regulate the expression of approximately 121 genes (Molle et al., 2003) including activation of genes necessary for sporulation. Counter-balancing the production of Spo0A∼P are several phosphatases including members of the Rap family of phosphatases (Rap A, B, E and H) and the Spo0E phosphatase (Perego et al., 1994). Regulation of the activity of the kinases and phosphatases determines the levels of Spo0A∼P and ultimately whether or not sporulation is initiated. The activity of the Rap phosphatases is regulated by small peptides encoded by phr genes, which are often found in operons with the rap genes (Mueller and Sonenshein, 1992). X-ray analyses have indicated that Phr peptides bind and regulate Rap activity by inducing a conformational change (Baker and Neiditch, 2011; Gallego del Sol and Marina, 2013; Parashar et al., 2013).
The levels of Spo0A∼P are responsible for determining the bacterium's developmental choices. Lower levels of Spo0A∼P result in biofilm formation through promotion of matrix production while higher levels of Spo0A∼P promote sporulation (Fig. 2). The mechanism through which Spo0A∼P is able to regulate these two distinct cell fates is dependent on its regulation of the levels of the matrix gene repressor SinR and its antirepressor SinI (Chai et al., 2011). The sinI regulatory region has numerous Spo0A∼P operator sites that differ in affinity, which allows its expression to be regulated directly by the levels of Spo0A∼P. At lower levels of Spo0A∼P, the high affinity Spo0A∼P operator is bound (Fujita and Losick, 2005) and promotes expression of sinI, leading to matrix production and biofilm formation (Fujita et al., 2005). At higher levels of Spo0A∼P, the lower affinity operators are then able to bind Spo0A∼P, which hinders the expression of sinI and promotes expression of sporulation genes that also have low affinity Spo0A∼P operators (Kearns et al., 2005; Chai et al., 2011). While high levels of Spo0A∼P are important for regulating entry into sporulation, the dynamics through which it achieves high levels of Spo0A∼P is also important. The gradual accumulation of Spo0A∼P appears to exert a temporal control over the Spo0A regulon, which is necessary for robust sporulation (Vishnoi et al., 2013).
Previous studies proposed a model in which there is a threshold level of Spo0A∼P, in addition to the phosphorelay components, that must be crossed in order for sporulation initiation to occur (Fujita and Losick, 2005; Eswaramoorthy et al., 2010a,b). However, recent studies have found significant overlap in Spo0A∼P levels in sporulating and non-sporulating cells (Levine et al., 2012), indicating that there may also be other downstream events that are responsible for the decision to enter sporulation. Consistent with this idea is the observation that entry into sporulation may still be reversible after activation of several Spo0A-regulated spo genes and only becomes irreversible upon activation of σF in the forespore and σE in the mother cell (Dworkin and Losick, 2005). The decision to commit to sporulation instead seems to rely on the ultrasensitive activation of σE (Narula et al., 2012), which occurs after asymmetric septation and σF activation in the forespore.
Stage I: axial filamentation and ensuring correct chromosome copy number
At the onset of sporulation, the cell harbours two chromosomes: one for the mother cell and one for the forespore. The duplicated chromosomes form a condensed serpentine-like structure called the axial filament (Ryter et al., 1966) that stretches from one pole of the cell to the other. The RacA protein is necessary for anchoring the two chromosomes to the cell poles to promote proper chromosome segregation (Ben-Yehuda et al., 2003). RacA binds to GC-rich inverted repeats located around the origin of replication (Ben-Yehuda et al., 2005) and localizes to the cell poles through its interaction with DivIVA, which in turn localizes to the two poles of the cell through the recognition of highly negatively curved membranes (Lenarcic et al., 2009; Ramamurthi and Losick, 2009). In this way, RacA ensures that each daughter cell receives one origin of replication (and, by extension, one chromosome). As such, RacA chromosomal binding sites are functionally analogous to eukaryotic centromeres, and RacA itself, while not physically driving chromosome movement, provides a function analogous to that of the eukaryotic mitotic spindle in maintaining chromosome integrity during eukaryotic cell division.
In addition to proper chromosome segregation, proper chromosome number is also necessary for robust sporulation and has been found to be tightly regulated via at least three proteins: SirA (sporulation inhibitor of replication A), Sda (suppressor of dnaA1) and Spo0A∼P. Transcription of sirA is under the control of Spo0A∼P and occurs upon entry into sporulation. SirA interacts directly with DnaA to inhibit its binding to the origin of replication, which prevents the initiation of additional rounds of DNA replication during sporulation (Rahn-Lee et al., 2011). Sda, on the other hand, inhibits entry into sporulation by binding to the major sporulation histidine kinase KinA during active DNA replication and in response to DNA damage and replication defects (Cunningham and Burkholder, 2009). As a result, entry into sporulation is restricted to the period between rounds of DNA replication (Burkholder et al., 2001; Veening et al., 2009). While Spo0A∼P plays an indirect role in regulating chromosome number through its transcriptional regulation of sirA, it has also been found to play a more direct role through its ability to bind to sites around the origin of replication. Removal of Spo0A∼P binding sites near the origin of replication resulted in an increase in chromosome copy number, indicating that Spo0A∼P binding to these sites acts as an additional mechanism to inhibit active DNA replication during sporulation (Boonstra et al., 2013).
Stage II: asymmetric septation
The sporulation programme is driven by a cascade of compartment-specific sigma factors that is initiated by asymmetric division of the cell. Understanding the activation of compartment-specific sigma factors during sporulation has revealed conserved mechanisms underlying intercellular signalling and the coupling of transcription with morphological changes in the cell. The transition from a medial septum to an asymmetric septum, which divides the cell into a mother cell and forespore, is a morphological hallmark of sporulation. This switch to a polar septum is dependent on two factors: an increase in levels of the cell division protein FtsZ and the production of the SpoIIE protein, which performs a poorly understood function in deploying FtsZ to polar sites (Carniol et al., 2005). After asymmetric division, the first sporulation-specific sigma factor, forespore-specific σF, is activated. Prior to asymmetric division, σF is produced under the regulation of Spo0A∼P, but held in an inactive state by the anti-sigma factor SpoIIAB. After completion of asymmetric division, SpoIIE (which localizes to the polar septum) performs a second function, wherein it dephosphorylates SpoIIAA, which binds and sequesters SpoIIAB, thereby relieving the inhibition of σF (Duncan et al., 1995). Curiously, although SpoIIE is initially produced in both the mother cell and the forespore, activation of σF only occurs in the forespore. Although the biochemical mechanism of σF activation is well understood, the cell biological mechanism that explains the forespore-specific activation of σF is not well known. Part of the answer may be dependent on a preferential localization of SpoIIE on the forespore side of the polar septum (Guberman et al., 2008) and also the temporary genetic asymmetry between the forespore and mother cell that leads to a decrease in the levels of the SpoIIAB anti-sigma factor in the forespore (Dworkin and Losick, 2001).
The temporary genetic asymmetry arises because, at the time of asymmetric septation, the polar septum bisects the axial filament and results in only about a third of the chromosome being harboured in the forespore (Wu and Errington, 1998). The DNA translocase SpoIIIE then pumps the remaining 70% of the chromosome residing in the mother cell into the forespore (Wu and Errington, 1994; Sharp and Pogliano, 2002; Becker and Pogliano, 2007; Burton et al., 2007; Ptacin et al., 2008; Fiche et al., 2013). Prior to translocation of the remaining 70%, the forespore can only express the genes residing on the 30% of the chromosome it harbours initially. This genetic asymmetry has been proposed to play a part in orchestrating the compartment-specific activities that occur throughout sporulation (Frandsen et al., 1999). Another example of this is the mother cell compartment specific reactivation of de novo fatty acid synthesis. Many genes necessary for de novo lipid synthesis are located in the portion of the chromosome initially excluded from the forespore, resulting in the forespore's inability to reactivate lipid synthesis (Pedrido et al., 2013). Reactivation of de novo lipid synthesis is dependent on Spo0A∼P and is required for the mother cell-specific activation of σE, the second sporulation-specific sigma factor (Pedrido et al., 2013).
Similar to σF,σE is produced prior to asymmetric division under the control of Spo0A∼P and is found in both compartments after septation (Fujita and Losick, 2002). However, σE is produced initially as an inactive pro-σE precursor and is specifically activated only in the mother cell. While it was previously established that spoIIGA is required for processing of pro-σE to its mature form (Jonas et al., 1988; Stragier et al., 1988), it was only more recently that its product SpoIIGA was identified as a novel type of aspartic protease (Imamura et al., 2008). Modelling and mutational evidence suggest that SpoIIGA forms dimers similar to the HIV-1 protease and support a model in which SpoIIGA exists in an inactive state that is then activated through a conformational change induced by association with SpoIIR (Hofmeister et al., 1995; Imamura et al., 2008). SpoIIR is produced in the forespore under σF control and is then secreted into the intermembrane space of the septum that separates the mother cell and forespore, where it activates SpoIIGA. SpoIIR's ability to activate SpoIIGA is dependent on the acylation of its threonine residue (T27) and requires de novo fatty acid synthesis (Diez et al., 2012). It is hypothesized that because de novo fatty acid synthesis only occurs in the mother cell, only SpoIIR molecules localized to the mother cell side of the septum would be able to be acylated, and thus, only SpoIIGA molecules at the mother cell membrane are activated, leading to the mother cell-specific activation of σE (Pedrido et al., 2013). The activation of σE then allows transcription of the σE regulon, which includes genes necessary for engulfment.
Stage III: engulfment
After asymmetric division, the polar septum curves around the forespore as the mother cell ‘swallows’ the forespore in a process called engulfment. The result is a double-membrane bound forespore in the mother cell cytosol. Although the dramatic membrane remodelling that occurs during engulfment superficially resembles that of phagocytosis in eukaryotes, the two processes utilize distinct proteins and cytoskeletal elements to achieve this goal. During engulfment, a peptidoglycan degradation machinery composed of SpoIID, SpoIIM and SpoIIP is initially needed for septal wall thinning and subsequently for movement of the engulfing membranes (Abanes-De Mello et al., 2002). Interestingly, cryo-electron micrographs have revealed that a thin layer of peptidoglycan remains during the engulfment process, indicating that the peptidoglycan is not completely removed by the degradation machinery (Tocheva et al., 2013). The residual peptidoglycan may be necessary to serve as a template for subsequent peptidoglycan remodelling events during engulfment and cortex assembly. Recent evidence has indicated that in addition to peptidoglycan degradation, membrane movement during engulfment also relies on active peptidoglycan synthesis (Meyer et al., 2010). This raises the possibility that stiff, newly synthesized, polymers of peptidoglycan may be providing a cytoskeletal role, analogous to that of eukaryotic actin during phagocytosis, to provide the force required for directed membrane movement (Meyer et al., 2010). However, when peptidoglycan is altogether removed by lysozyme treatment, cells are still able to undergo engulfment, albeit at reduced levels. This redundant engulfment mechanism is dependent on a forespore protein, called SpoIIQ, and a mother cell protein, called SpoIIIAH, which reach across the intermembrane space that divides the mother cell and forespore and directly interact, resulting in a ‘zippering’ of both compartments (Blaylock et al., 2004; Doan et al., 2005). In what has been described as a ‘ratchet-like’ mechanism, the engulfing membrane is able to move forward through random thermal motion of the membrane, but reverse movement is restricted, as the engulfing membrane is stapled to the inner forespore by the tight interaction of SpoIIIAH and SpoIIQ (Broder and Pogliano, 2006).
At the end of engulfment, the engulfing membranes must undergo membrane fission to pinch off and release the forespore. The identification of specific factors responsible for membrane fission and fusion in prokaryotes has been historically difficult because of the complexity of the often essential processes that occur concurrently. Unlike most other membrane remodelling events, the membrane fission event that occurs at the end of engulfment is not essential for viability. A recent screen for mutants that were unable to undergo membrane fission at the end of engulfment led to the identification of a mother cell protein called FisB that was enriched at the site of membrane fission during engulfment and was necessary for robust membrane fission. FisB does not resemble or behave like well-studied eukaryotic membrane remodelling proteins like dynamin or the SNARE proteins. Rather, FisB appears to utilize a novel mechanism to promote membrane remodelling through a preferential association with the phospholipid cardiolipin, which is thought to be enriched along negatively curved leaflets of membranes. The current model is that FisB interacts in trans with cardiolipin-enriched membranes at the leading edge of the engulfing membrane to cause a destabilization of both membranes, which in turn could lead to membrane scission (Doan et al., 2013).
At the end of engulfment, the forespore is a free floating cell in the mother cell cytosol. At this time, the SpoIIIAA-SpoIIIAH proteins produced under the control of σE and the SpoIIQ protein under the control of σF are required for the activation of the forespore-specific σG (as noted above, SpoIIIAH and SpoIIQ interact directly with one another to ‘zipper’ the mother cell and forespore together during engulfment). The gene encoding the forespore-specific σG is under the transcriptional control of σF, which ensures that it is only expressed in the forespore. Interestingly, despite being under σF transcriptional control, which occurs after asymmetric septation, activation of σG occurs only after engulfment has finished. How does σG activation occur to coincide with the completion of engulfment? After engulfment, when the forespore is sealed off from the mother cell, the metabolic capacity of the forespore is diminished (Camp and Losick, 2009; Doan et al., 2009). The SpoIIAA-SpoIIIAH and SpoIIQ proteins form a channel between the mother cell and forespore (Meisner et al., 2008) through which the mother cell is able to nurture the forespore through the transfer of what are likely small molecules that enable the forespore to continue expressing genes necessary for sporulation (Camp and Losick, 2009). The structure of the basal components of this ‘feeding tube’ channel has been found to be similar to that of type III protein secretion systems (Levdikov et al., 2012; Meisner et al., 2012). Thus, the activation of σG is thought to occur simply because it is the only sigma factor produced at that specific time and location, and is dependent on the arrival of metabolites delivered from the mother cell. While the feeding tube is necessary for activation of σG, it is still not completely understood how activation of σG is precisely linked to the end of engulfment. Indeed, recent observations have indicated that completion of engulfment is actually linked to the completion of chromosome translocation into the forespore, suggesting that chromosome translocation also contributes to the timing of σG activation (Regan et al., 2012).
Similar to σE, the subsequent mother cell-specific transcription factor σK is produced as an inactive pro-σK protein. Pro-σK is cleaved by SpoIVFB (Lu et al., 1995), which is an intermembrane protease that is initially held inactive in a complex along with SpoIVFA and BofA (Resnekov and Losick, 1998). Activation of SpoIVFB occurs through the action of the σG-regulated SpoIVB processing enzyme. SpoIVB can relieve the inhibition imposed by SpoIVFA and BofA both by cleaving SpoIVFA at multiple sites and by activating the alternate protease CtpB, which can also cleave SpoIVFA (Campo and Rudner, 2006).
Stage IV–V: cortex and coat assembly
The mature spore is encased in two distinct concentric shells: an outer shell, called the ‘coat’, which is composed of roughly seventy different proteins, and an inner shell called the ‘cortex’ made of specialized peptidoglycan (Henriques and Moran, 2007; McKenney et al., 2013). Together, these two shells serve to protect the spore from environmental insults (Setlow, 2006). Among the first, if not the first, coat proteins to localize to the outer forespore surface is a small 26-amino-acid protein [a so-called ‘sprotein’ (Hobbs et al., 2011)], that is exclusively produced in the mother cell, named SpoVM (Levin et al., 1993; van Ooij and Losick, 2003). SpoVM distinguishes the forespore membrane from the mother cell membrane through the recognition of the forespore's positive membrane curvature (Ramamurthi et al., 2009). SpoVM tethers a soluble morphogenetic protein called SpoIVA (Ramamurthi et al., 2006), which is the structural component of the basement layer of the coat, onto the forespore surface (Roels et al., 1992; Price and Losick, 1999). While dynamic cytoskeletal nucleotide-binding proteins like actin and tubulin hydrolyse nucleotides in order to disassemble, SpoIVA, a static cytoskeletal element, instead hydrolyses ATP to drive its self-assembly to form the basement layer of the coat, which acts as a scaffold atop which all other coat proteins are deposited (Ramamurthi and Losick, 2008; Castaing et al., 2013). Curiously, the adenosine triphosphatase (ATPase) domain of SpoIVA resembles that of the TRAFAC class of P-loop guanosine triphosphatases (GTPases). However, SpoIVA, like the myosin/kinesin family of exceptional ATPases in the TRAFAC GTPase class (Leipe et al., 2002), has evolutionarily lost the ability to bind GTP and binds ATP instead.
The coat has been described as having four distinct layers: the basement layer, inner coat, outer coat and crust (McKenney et al., 2010). Each layer's proper assembly is largely dependent on one (or two, in the case of the crust) major morphogenetic protein that defines each layer. For example, deletion of spoIVA results in the mis-assembly of the basement layer (and, by extension, all subsequent layers) (Roels et al., 1992); deletion of safA, cotE, or cotZ and cotY results in the improper assembly of the inner coat, outer coat and crust respectively (Zheng et al., 1988; Chada et al., 2003; Costa et al., 2006; McKenney et al., 2010; Imamura et al., 2011). Another coat protein, SpoVID, has been shown to drive the ‘encasement’ step of coat morphogenesis where coat proteins completely encapsulate the developing forespore (Driks et al., 1994; Wang et al., 2009; McKenney et al., 2010).
A recent study monitoring the dynamics of spore coat assembly found that proteins in the coat can be divided into six classes based on their localization dynamics. Spore coat proteins initially assemble as a scaffold in a focus on the mother cell side of the forespore. The encasement of the forespore by specific classes of coat proteins then occurs in coordinated waves that are largely driven by transcription (McKenney and Eichenberger, 2012). Coat assembly occurs predominantly in the mother cell, where the coat is assembled on the surface of the outer forespore membrane. Interestingly, the discovery that the SpoIIIAH-SpoIIQ complex, which requires the forespore-specific synthesis of SpoIIQ, is necessary for successful encasement suggests that the forespore may also participate in coordinating the assembly of the coat (McKenney and Eichenberger, 2012).
Although the coat is spatially separated from the cortex by the outer forespore membrane, deletion of either spoVM or spoIVA not only abrogates the initiation of coat assembly, but also abolishes cortex assembly (Coote, 1972; Piggot and Coote, 1976; Roels et al., 1992; Levin et al., 1993), indicating that coat and cortex assembly are somehow linked. Many factors participating in assembly of each individual structure have been identified; however, the mechanisms that coordinate temporal assembly of both structures have remained largely mysterious. Recently, a sprotein (37-amino-acids long) named ‘CmpA’, which was encoded by a previously unannotated mother cell-specific sporulation gene, was found to participate with SpoVM in coordinating cortex assembly (Ebmeier et al., 2012). Specifically, a model emerged wherein cortex peptidoglycan assembly (but not vegetative peptidoglycan assembly) is repressed by a hitherto-unspecified inhibitory activity of CmpA. Cells that successfully initiate coat assembly by SpoVM and SpoIVA overcome the inhibition imposed by CmpA by removing the protein, likely by regulated proteolysis, and continue through the sporulation programme to initiate cortex assembly (Ebmeier et al., 2012). Thus, coordination of the assembly of these two large structures appears to be mediated by at least two small proteins, highlighting the general importance of sproteins in biological processes (Hobbs et al., 2011).
The cortex is made of a specialized peptidoglycan that protects the spore from heat and desiccation. The peptidoglycan in the spore resides between the two membrane layers surrounding the forespore and consists of two layers: an inner germ cell wall and an outer cortex. The germ cell wall is a thin layer adjacent to the inner forespore membrane that has a structure similar to that of the vegetative cell wall (Tipper and Linnett, 1976). The cortex, on the other hand, differs in structure from the vegetative cell wall mainly because of a decreased frequency of transpeptidation between glycan chains (Popham and Setlow, 1993b) and the presence of muramic lactam (Warth and Strominger, 1972). These structural changes in the cortex are brought about by the activities of the low-molecular-weight penicillin-binding proteins (PBPs), which often have D,d-carboxypeptidase activity (Popham et al., 1999), and the CwlD and PdaA proteins, which catalyse the production of muramic lactam from muramic acid (Gilmore et al., 2004). During spore germination, the functionally redundant cortex-lytic enzymes SleB and CwlJ (Ishikawa et al., 1998) specifically hydrolyse the cortex peptidoglycan through the recognition of muramic lactam. Mutants with cortexes deficient in muramic lactam are unable to germinate, but can be induced to germinate through exogenous lysozyme treatment (Popham et al., 1996).
It is currently unclear what exactly the functional role of the cortex's unique peptidoglycan structure is. One theory is that the low degree of cross-linking allows the spore to expand and contract in response to environmental changes (e.g. pH, ionic strength or humidity) without germinating (Ou and Marquis, 1970). Other theories suggest that the low degree of cross-linking may allow the spore to contract (Lewis et al., 1960) or expand (Gould and Dring, 1975; Popham et al., 1999) during spore maturation to attain spore dehydration. Interestingly, the degree of cross-linking throughout the cortex is not homogenous, but rather increases progressively towards the outer cortex layers (Meador-Parton and Popham, 2000). Disruption of this cross-linking gradient does not appear to have significant effects on spore core dehydration, suggesting that a broad range of cortex cross-linking is permissible to attain spore core dehydration (Meador-Parton and Popham, 2000).
Synthesis of cortex peptidoglycan occurs through similar mechanisms as vegetative cell wall synthesis. Peptidoglycan precursors are produced and modified in the cytosol of the mother cell by the Mur proteins, which are also responsible for modification of peptidoglcan precursurs during vegetative cell wall synthesis. During sporulation, the production of the Mur proteins is upregulated by σK (Vasudevan et al., 2007). Once properly modified, the peptidoglycan precursors are then tethered to the outer forespore membrane through the formation of the lipid intermediates Lipid I and Lipid II, which are then flipped across the membrane via a Lipid II flippase into the intermembrane space between the outer and inner forespore membranes. Although there are homologs of putative Lipid II flippases that are expressed specifically during sporulation, the identity of the Lipid II flippase during sporulation is currently unknown. The Escherichia coli MviN/MurJ protein has been proposed to be a Lipid II flippase (Ruiz, 2008), and SpoVB was identified as its sporulation-specific homolog (Fay and Dworkin, 2009). However, the discovery that the E. coli FtsW protein, which is a part of the SEDS (shape, elongation, division and sporulation) family, has in vitro flippase activity (Mohammadi et al., 2011) suggests that its sporulation-specific homolog SpoVE (Ikeda et al., 1989) may also be a Lipid II flippase. Thus, both SpoVB and SpoVE are plausible candidates for being sporulation-specific Lipid II flippases. Consistent with this idea, mutations in either spoVB or spoVE abrogate cortex assembly and result in a build up of peptidoglycan precursors in the mother cell (Vasudevan et al., 2007).
After translocation into the intermembrane space, the lipid-linked precursors are assembled into glycan chains via transglycosylation, and peptide cross-links between the glycan strands are formed via transpeptidation. Tranglycosylation and transpeptidation are performed by the high-molecular-weight PBPs to produce the meshwork of peptidoglycan that constitutes the cortex. Some vegetative PBPs (PBP2B and PBP3) are upregulated during sporulation while PBP1, PBP2A, PBP4 and PBP5 are downregulated (Sowell and Buchanan, 1983). PBP2d and PBP2c are expressed in the forespore during sporulation (Popham and Setlow, 1993a; Pedersen et al., 2000) and have been proposed to play partially redundant roles in synthesizing the spore germ cell wall (McPherson et al., 2001).
There are many different factors of the spore that make it able to survive in the environment during harsh conditions. In general, the coat protects the spore from enzymatic assaults such as lysozyme, and the cortex is required for protection from high temperature. The coat's ability to protect the spore from enzymatic assaults has proven useful in resisting predation by bacteriophagous organisms like Tetrahymena (Klobutcher et al., 2006; Laaberki and Dworkin, 2008). The cortex is believed to maintain the spore's partially dehydrated state (Imae and Strominger, 1976; Warth, 1978; Mallidis and Scholefield, 1987; Ulanowski and Ludlow, 1993), and this low water content is associated with resistance to heat (Slepecky and Foster, 1959; Koshikawa et al., 1984; Beaman and Gerhardt, 1986). Other factors that contribute to heat resistance and reduction in spore water content include mineralization (Slepecky and Foster, 1959; Bender and Marquis, 1985; Marquis and Bender, 1985; Atrih and Foster, 2001) and the presence of the small molecule dipicolinic acid (DPA) (Paidhungat et al., 2000; Setlow et al., 2006). Additionally, the spore's DNA is bound by small acid-soluble proteins (SASPs) that protect the DNA from damage. Spores that lack SASPs are more susceptible to DNA-damaging treatments such as exposure to ultraviolet irradiation (Setlow and Setlow, 1987) and hydrogen peroxide (Setlow and Setlow, 1993). A more in-depth discussion of spore resistance mechanisms can be found in Leggett and colleagues (2012).
Interest in the ability of spores to survive extraterrestrial environments dates back several decades to when Hagen and colleagues tested the survival of spores in a simulated Martian environment in an effort to determine the feasibility of extant life on other planets (Hagen et al., 1964). Although the search for extant life is still ongoing, there is significant concern about the contamination of extraterrestrial locations by Earth's organisms carried on spacecrafts (Nicholson et al., 2009). In order to minimize the contamination risk, much research has been done to explore how and under what conditions terrestrial microorganisms may survive and replicate. In particular, understanding how spores, one of Earth's hardiest cell types, are able to withstand the harsh conditions of space may help the space biological research field to minimize terrestrial contamination. A recent study by Moeller and colleagues determined that spore survival in a simulated Mars environment is dependent largely on SASPs, the coat and DPA (Moeller et al., 2012), indicating that survival in Martian environments may depend on the spore's numerous protective factors.
Applications of sporulation studies
Aside from utilizing sporulation as a model system to understand basic biological processes, other applications of studying sporulation derive from the robust nature of the spore. Bacterial cells have been successfully utilized as whole-cell biosensing systems that rely on genetically modified bacteria, which are able to express reporter genes in the presence of an analyte of interest in a dose-dependent matter. Advantages of these systems include low cost, sensitivity, rapid results and the ability to measure the bioavailability of target analytes (Rawson et al., 1989). However, one disadvantage has thus far been a lack of stability of the cells used in whole-cell biosensing systems in the field. A solution to this problem has been to use organisms that are able to undergo sporulation. Once they have formed spores, the biosensors can then be stored easily for extended periods of time until they are ready to be deployed (Date et al., 2010). The harmless nature of B. subtilis and its genetic tractability, which facilitates the introduction of analyte based reporters, makes it particularly attractive for this purpose. Further studies into sporulation may help in the optimization of spore-based biosensors.
The spore's unique features also make it amenable for a multitude of other applications. The spore's outermost layer is composed of proteins, making it easy to decorate the spore with proteins of interest through the incorporation of fusion proteins into the organism's genome. Moreover, such engineered proteins are initially synthesized in the mother cell cytosol and are displayed in the extracellular milieu on the surface of the spore only after mother cell lysis, thereby potentially avoiding protein misfolding issues that may arise when using purified proteins that are subsequently conjugated to a surface. Such display systems may have multiple uses, including the utilization of spores as surface display systems for enzymes or for use as vaccination platforms (Isticato et al., 2001; Mauriello et al., 2004; Hinc et al., 2010).
Since the initial descriptions by Ferdinand Cohn about 140 years ago of heat resistant spores formed by B. subtilis (Cohn, 1877), sporulation has been used as a model system to study a ‘simple’ example of cell differentiation and continues to be used to study fundamental cell biological processes. The non-essential nature of many factors involved in sporulation has greatly facilitated progress made in the field. However, there are still many unanswered questions because of the complex interdependencies and redundancies that are inherent to robust developmental programmes.
Despite decades of study, the basic question of how a precursor cell may differentiate into two morphologically distinct, but genetically identical, daughter cells that exhibit different cell fates remains. For example, although in eukaryotes, membrane remodelling events, such as those involved in organelle morphogenesis, endocytosis and protein trafficking, have been extensively studied, the molecular details underpinning how the architecture of the flat polar septum is altered as the mother cell engulfs the forespore remain an active area of research. Regarding morphogenesis, the spore coat has been a model system for understanding how complex, asymmetric structures may be assembled in an orderly fashion. For years, this structure resisted detailed in vitro investigations because extensive covalent cross-links prevented the extraction of many coat proteins from mature spores. However, advances in bacterial cell biological techniques have revealed detailed interaction networks between the approximately 70 proteins that make up the coat, and an outstanding challenge will be to recreate these networks in vitro in order to ultimately assemble this structure biochemically. At the heart of the study of sporulation remains the differential, but sequential, activation of transcription factors specifically in the mother cell and forespore that can reveal mechanisms by which adjacent cells can communicate with one another. In B. subtilis, the molecular details of how σF, which is activated in the forespore and sets off the cascade of sigma factor activation, have been exquisitely worked out, yet the cell biological mechanisms that can explain how this activation occurs exclusively in the forespore has been largely unclear. A related question is how asymmetric cell division mechanistically arises in the first place. Additionally, the mysterious chemicals that are transferred from the mother cell to the forespore via the ‘feeding tube’ in order to activate σG exclusively in the forespore await discovery and may reveal how activation of a transcription factor may be linked to completion of a morphological event. In total, then, a remarkable developmental process exhibited by a bacterium originally isolated from the soil at the dawn of the era of modern microbiology will likely continue to provide answers to fundamental biological questions. Developing approaches and strategies to unravel these unanswered questions may provide new tools for further understanding other more complex developmental processes as well.
We thank P. Eswaramoorthy for comments on the manuscript. This work was funded by the Intramural Research Program of the National Institutes of Health, National Cancer Institute and Center for Cancer Research.