Spores of Bacillus species can remain dormant and resistant for years, but can rapidly ‘come back to life’ in germination triggered by agents, such as specific nutrients, and non-nutrients, such as CaDPA, dodecylamine and hydrostatic pressure. Major events in germination include release of spore core monovalent cations and CaDPA, hydrolysis of the spore cortex peptidoglycan (PG) and expansion of the spore core. This leads to a well-hydrated spore protoplast in which metabolism and macromolecular synthesis begin. Proteins essential for germination include the GerP proteins that facilitate germinant access to spores' inner layers, germinant receptors (GRs) that recognize and respond to nutrient germinants, GerD important in rapid GR-dependent germination, SpoVA proteins important in CaDPA release and cortex-lytic enzymes that degrade cortex PG. Rates of germination of individuals in spore populations are heterogeneous, and methods have been developed recently to simultaneously analyse the germination of multiple individual spores. Spore germination heterogeneity is due primarily to large variations in GR levels among individual spores, with spores that germinate extremely slowly and termed superdormant having very low GR levels. These and other aspects of spore germination will be discussed in this review, and major unanswered questions will also be discussed.
Spores of Bacillus species are metabolically dormant (but see below) and have minimal levels, if any, of common high-energy small molecules such as ATP and reduced pyridine nucleotides (Setlow and Johnson 2012). These spores are also extremely resistant to a variety of external stresses including heat, desiccation, UV and γ radiation, and a variety of chemical agents including many common microbiocides (Setlow 2006; Setlow and Johnson 2012). Spores of some Bacillales species, as well as some of those of their close relatives, the Clostridiales, are also agents of significant food spoilage, food borne disease and a number of other human and animal diseases (Setlow and Johnson 2012). While spores are dormant and by themselves cannot cause deleterious effects, spores constantly sense their environment. Then, if conditions that are potentially conducive to cell growth are detected, spores can ‘return to life’ rapidly in the processes of spore germination followed by outgrowth that sequentially convert the dormant spore into a growing cell (Paidhungat and Setlow 2002; Setlow 2003). As most dormant and resistant properties of a spore are rapidly lost in germination, this process has long been considered a possible way to establish simple methods for spore eradication. Consequently, there continues to be much applied interest in means to trigger or prevent spore germination. In addition, the process of germination is a simple yet fascinating biological signal transduction system, and one in which a variety of methods can be applied to study the mechanisms of this biological event from many different perspectives. Spore germination has been studied for many years, and in recent years, significant progress has been made in elucidating the mechanisms involved in germination, in particular in examining this process at the single spore level. These latter studies have revealed an extraordinary amount of heterogeneity in germination rates among individuals in spore populations and have elucidated many of the reasons for this germination heterogeneity (Setlow et al. 2012).
This review will discuss the overall mechanism of spore germination as it takes place in spores of Bacillus species, primarily the model spore-former Bacillus subtilis. Results from studies with spores of other Bacillus species, including Bacillus anthracis, Bacillus cereus and Bacillus megaterium, while more limited, indicate that general conclusions about spore germination drawn from studies with B. subtilis spores are also true for spores of other Bacillus species (Hornstra et al. 2006; Christie and Lowe 2007; Giebel et al. 2009; Ghosh and Setlow 2009, 2010; Setlow et al. 2009; Carr et al. 2010; Heffron et al. 2010; Kong et al. 2011; Luu et al. 2011; Li et al. 2013; Gupta et al. 2013; Ramirez-Peralta et al. 2013; Gupta, S., and Christie, G., personal communication). The mechanism of germination of spores of Clostridium spores, while not as well studied as that of Bacillus spores, also exhibits many similarities to germination of Bacillus spores, albeit with some notable differences (Paredes-Sabja et al. 2011; Xiao et al. 2011), and these will be noted when appropriate. This review will focus on spore germination, with this process defined as the initial events in the ‘return to life’ of a dormant spore that almost certainly do not require simultaneous energy (e.g. ATP) production. In contrast to spore germination, outgrowth will be defined as events taking place after completion of germination that do require ATP production, and thus metabolic activity. However, details of spore outgrowth itself, as well as a period between germination and outgrowth that has been determined ‘spore ripening’ (Segev et al. 2013), will not be discussed. Importantly, a number of major unanswered questions about the mechanism of spore germination will also be presented. Throughout this review, I have generally cited more recent references for various topics. I apologize in advance to authors whose earlier work was not cited.
Sporulation and Spore Properties
Spores of the Bacillales and Clostridiales genera are formed in sporulation, a process that in the Bacillales is generally induced by starvation for one or more nutrients (de Hoon et al. 2010; Higgins and Dworkin 2012; Setlow and Johnson 2012). The sporulation process is characterized most often by an unequal cell division generating a larger mother cell and a smaller forespore each with its own genome, and the mother cell ultimately engulfs the forespore giving a cell within a cell. As sporulation proceeds, the forespore matures due to gene expression in both the mother cell and forespore compartments. This maturation includes synthesis of many gene products unique to the developing and mature spore that assemble into a number of layers that are also unique to spores (Fig. 1). Almost all of these unique layers appear to play at least some role in the spore germination process. Thus, the exosporium (in spores that have this structure) and coats can contain enzymes such as alanine racemase and purine nucleoside hydrolase that may modulate spore germination by modifying specific germinants (Henriques and Moran 2007; Chesnokova et al. 2009; McKenney et al. 2013). Indeed, this alanine racemase appears essential for normal sporulation in B. anthracis by generating d-alanine and thus inhibiting germination of developing forespores within the sporangium that is triggered by endogenous l-alanine (Chesnokova et al. 2009). However, this is not the case in B. subtilis spores (Butzin et al. 2012). The spore coats also contain other proteins involved in spore germination, most notably the GerP proteins that appear to facilitate the access of exogenous molecules that trigger spore germination, termed germinants, to their targets further within the spore (see below; Behravan et al. 2000; Carr et al. 2010; Butzin et al. 2012).
In contrast to roles in germination for the outermost spore layers, there is no known role for the outer spore membrane in germination, but this is not the case for the spore cortex. The cortex is also extremely important in maintaining spore dormancy, as it appears to prevent the expansion of the spore's central core, the site of spore DNA, RNA and most metabolic enzymes, and which has a very low water content (see below; Gerhardt and Marquis 1989; Setlow 2006). The cortex is primarily composed of a thick layer of peptidoglycan (PG) with a repeating disaccharide backbone and a cross-linking oligopeptide structure similar to that of growing cell and spore germ cell wall PG (Popham 2002). However, the cortex PG has several cortex-specific modifications, most notably conversion of many of the muramic acid residues in the polysaccharide backbone to muramic acid-δ-lactam (MAL), a modification not present in growing cell or germ cell wall PG. Notably, a major and essential event in spore germination is the degradation of the cortex by cortex-lytic enzymes (CLEs) that require MAL for PG recognition and cleavage (Setlow 2003). These CLEs are also located at least in part at the cortex/coat boundary, and there may be other proteins associated with the cortex as well. The germ cell wall that lies beneath the cortex is also composed primarily of PG, but the structure of this PG appears to be essentially identical to that of growing cell wall PG. During the final stage of spore germination, the germ cell wall expands without new PG synthesis and becomes the cell wall of the outgrowing spore.
The inner membrane (IM) that separates the germ cell wall from the central spore core also plays a major role in spore germination, as many important germination proteins are located in the IM either as integral or peripheral membrane proteins (Setlow 2003). This membrane has extremely low permeability to small hydrophilic compounds, and even rates of water movement across the IM are much slower than across plasma membranes of growing cells and germinated spores (Setlow 2003, 2006; Westphal et al. 2003; Sunde et al. 2009). The IM has other unique properties, in that lipid molecules in this membrane are largely immobile, with lipid mobility restored upon completion of spore germination (Cowan et al. 2004). The IM also has a much higher viscosity that that of growing cell or fully germinated spore membranes (Loisan et al. 2013). Despite these rather novel IM properties, the phospholipid and fatty acid composition of the IM is essentially identical to that of growing cells, and major alterations in this membrane's phospholipid or fatty acid composition have only minimal effects on spore properties (Cortezzo et al. 2004; Griffiths and Setlow 2009).
The spore core also is the site of major sporulation-specific events that ultimately have significant impact on spore germination. Late in sporulation, the developing spore core accumulates a huge depot (approx. 20% of spore core dry weight) of a spore-specific small molecule, pyridine-2,6-dicarboxylic acid (dipicolinic acid, DPA; Fig. 2). DPA is synthesized in the mother cell and accumulated in the spore core as a 1 : 1 chelate with divalent cations, primarily Ca2+ (CaDPA). The water content of the spore core is also reduced significantly as spore maturation continues, falling to 25–50% of core wet weight in the mature spore (Gerhardt and Marquis 1989). In addition to DPA accumulation and a decrease in water content, the spore core pH falls from approx. 7·8 in the sporulating cell to approx. 6·5 late in sporulation but before CaDPA accumulation. The spore core-specific changes in the high CaDPA content, low core water content and lower core pH all contribute to spore dormancy, with the low core water content undoubtedly the major factor. Indeed, at least one spore core protein is immobile in the dormant spore core, undoubtedly because of the core's low water content (Cowan et al. 2003). All of these core-specific changes are reversed in the early min of spore germination, as all CaDPA is excreted, core water content rises to 80% of wet weight, core pH rises to approx. 8·0, and the immobile protein noted above becomes freely mobile (Cowan et al. 2003; Setlow 2003). These latter events are the major ones early in spore germination, and an understanding of the mechanisms of these events will be essential to obtain a thorough understanding of the mechanism of spore germination itself.
Overview of Spore Germination
While spores are dormant and resistant as described above, they are sensitive to molecules in the environment, and if appropriate molecules and/or conditions are sensed, the dormant and resistant state is rapidly lost as spore germination is initiated. The germination process takes place in several stages (Fig. 3), and some events that take place prior to germination can also have major effects on the overall germination process as described below.
As noted above, the molecules that trigger spore germination are called germinants and encompass a wide variety of generally low molecular weight compounds, as well as at least one environmental condition, high hydrostatic pressure (HP; Setlow 2003; Setlow and Johnson 2012). Small molecule germinants are normally divided into nutrient and non-nutrient germinants, and most often these two types of molecules trigger germination by different methods. However, this is a bit imprecise because some non-nutrients trigger germination in the same way as nutrient germinants, and actual metabolism of nutrient germinants plays no role in their triggering of germination, as they are merely signalling molecules. However, it is likely that for most nutrient germinants, their presence signals that the spore's environment is likely to allow cell growth, while most, but not all, non-nutrient germinants may subvert the nutrient-germination process in some fashion.
Nutrient germinants include a number of specific nutrient molecules including but not exclusively amino acids, sugars and purine nucleosides, and the specific active molecules are very species and strain specific (Gould 1969). Active nutrient germinants are also stereospecific as while l-alanine is often a germinant, d-alanine is often a strong inhibitor of l-alanine germination. Nutrient germinant specificity is due to the proteins that bind these germinants, termed ‘germinant receptors’ (GRs), and these are discussed in detail below. As noted above, metabolism of the nutrient germinants is not involved in the germination process. In addition to the small molecules noted above, it is not uncommon for specific monovalent cations, most often K+, to be obligatory co-germinants for nutrient germinants, and in few cases, specific K+ salts alone can also trigger spore germination.
The best studied non-nutrient germinants are CaDPA and cationic surfactants, in particular dodecylamine. These compounds trigger germination for spores of many Bacillus species, and the mechanism of their stimulation of spore germination is very different than that of the nutrient germinants as discussed below, and almost always, GRs are not involved. Additional non-nutrient germinants include (i) specific PG fragments that appear to trigger germination through activation of a spore protein kinase in a process that is not well understood (Shah et al. 2008), (ii) specific bile salts that trigger germination of spores of Clostridium difficile (Burns et al. 2010a), presumably signalling the environment of the mammalian large intestine, and (iii) lysozyme or other PG hydrolases, but only if the spore coat permeability barrier has been breached. The final non-nutrient germinant, and one that has significant applied importance, is pressures of 1000s atmospheres of HP that trigger germination of Bacillus and many Clostridium spores, although the susceptibility of spores of different species to HP germination can vary significantly (Reineke et al. 2013; Sarker et al. 2013). In general, an HP of 50–350 megaPascals (MPa) triggers spore germination by activation of spores' GRs, while higher pressures trigger spore germination by direct activation of CaDPA release from spores, most likely by activation of a CaDPA channel composed of SpoVA proteins (see below; Setlow 2003). Pressures ≥ 500 MPa and generally elevated temperatures are used in a number of food processing applications to generate pasteurized and in some cases sterile products with long shelf life and with much better sensory and nutritional qualities than are obtained by conventional high-temperature processing.
Events prior to spore germination
Even before the addition of a germinant to a spore population, there are events or treatments that can alter the germination of the resultant spores. These include the sporulation conditions such as medium composition, damage to or removal of spores' outer spore layers, pretreatment with agents such as heat and oxidizing chemicals, and perhaps some metabolic activity even in dormant spores. In general, richer sporulation media give spores of individual strains that germinate better than do spores prepared in poorer media, although with B. subtilis, sporulation temperature has minimal effects (Hornstra et al. 2006; Ramirez-Peralta et al. 2012a,b). Removal of spores' outer layers can also have no effects on spore germination, although spores' outer layers often contain enzymes that can degrade or modify germinants as noted above, and decoating treatments will remove these enzymes. Removal of spores' outer layers can also increase spore germination with some agents, in particular dodecylamine (Setlow et al. 2003). However, treatments that remove spore coats using denaturing agents, often at high pH and temperature, can remove or inactivate important germination proteins such as one or both of the redundant CLEs, CwlJ and SleB, in Bacillus spores, the only CLE, SleC, in Clostridium spores as well as the protease(s) that activates SleC in Clostridium spores (Atrih and Foster 2001; Makino and Moriyama 2002; Setlow 2006; Paredes-Sabja et al. 2009a,b; Burns et al. 2010b). As expected, such treatments, in particular in Clostridium spores, can have significant effects on spore germination by abolishing PG cortex hydrolysis and giving spores with greatly decreased apparent viability. However, spores in which there is no cortex hydrolysis by endogenous CLEs can invariably be readily recovered if lysozyme is added to plating media. Removal of spore coats also eliminates the requirement for GerP proteins for spore germination with nutrient germinants (Behravan et al. 2000; Carr et al. 2010; Butzin et al. 2012).
Another pretreatment that can have significant effects on spore germination is activation, which generally promotes germination by increasing both its rate and extent (Keynan and Evenchick 1969). This generally applies only to nutrient germination, and the activation treatment that is most often used is a short (15–60 min) exposure to high temperature in water. However, spores can also become activated during long-term storage in water at moderate temperatures. Spores of different species generally exhibit different requirements for heat activation with some spores showing minimal requirements, while others exhibit major increases in apparent viable counts upon appropriate activation. Interestingly, superdormant (SD) spores of several Bacillus species have higher temperature requirements for heat activation than do dormant spores (Ghosh et al. 2009).
While heat activation as a procedure to stimulate spore germination has been known for more than 50 years, the mechanism of this effect is not clear. However, a variety of evidence including the reversibility of the heat activation process, the fact that it is almost always GR-dependent germination that is sensitive to heat activation, and limited studies on the effects of heat activation on spore protein structure are consistent with heat activation causing some conformational change(s) in spore protein, perhaps spores' GRs (Zhang et al. 2009).
A third factor that can markedly alter spore germination is pretreatment by agents such as oxidizing agents or heat giving significant spore killing (Coleman et al. 2007; Wang et al. 2011c, 2012; Setlow et al. 2013). Almost always such treatments result in spores that germinate more slowly than untreated spores, although even spores that are dead by the criterion of being able to give a colony on a plate will still germinate, albeit often very slowly. Where this has been studied, the defect in the germination of such treated spores is primarily in greatly increased times between addition of a germinant and initiation of rapid CaDPA release (termed Tlag values; see below). However, in some cases, the actual time for the release of 90% of a spore's CaDPA depot is also increased.
Another factor that appears to be able to modify spores' germination properties is enzymatic and possibly metabolic activity in the dormant spore core. Recent work (Segev et al. 2012) indicates that there can be enzymatic activity in dormant spores' core, most notably the degradation of ribosomal RNAs. This rate of this activity was very dependent on the specific conditions under which spores were incubated, and this activity had significant effects on rates of spore germination and outgrowth.
Events in spore germination
Spore germination begins upon mixing spores with a germinant (Figs 3 and 4). These germinants then have to pass through the spores' outer layers to access their targets in the cortex or IM, and there likely are special proteins (i.e. GerP proteins; see below) to ensure that this permeation is rapid (see below). The first measureable event following addition of a germinant is what is termed commitment whereby the germination process will continue even if the germinant is removed. This process has only been well studied with nutrient germinants and in only a few species, and what is involved in the process of commitment is not understood (Yi and Setlow 2010). Another early germination event that takes place at around the same time as commitment is the release of monovalent cations including Na+, K+ and H+ (Swerdlow et al. 1981; Setlow 2003; van Beilen and Brul 2013). Presumably these ions ultimately come from the spore core, and this is certainly the case for the H+, and the H+ release raises spore core pH to approx. 8. How these monovalent cations are released, how any channels for these ions are gated and what anions are also released are not known. It is also not known whether this monovalent cation release is causally related to the commitment step in spore germination,
A few min after commitment and monovalent cation release, the spore core's huge CaDPA depot is also released (Fig. 3). Under normal conditions, release of CaDPA from an individual wild-type spore takes only approx. 2 min (Kong et al. 2011); all CaDPA is released in this process and is replaced by water, thus raising core water content slightly (see below). The CaDPA release is almost certainly via channels in the spore's IM composed of at least in part SpoVA proteins (see below), but how water is taken up is not known, and Bacillus species generally lack aquaporins. The structure of the SpoVA CaDPA channel and how this channel is gated are also not known. Overall, it appears likely that the channels involved in release of low molecular weight compounds from the spore core early in spore germination are selective, and not simply a mechanosensitive channel that releases all molecules below a certain molecular weight (Wahome and Setlow 2006; Vepachedu and Setlow 2007b; Vepachedu et al. 2007c; Setlow et al. 2008). With completion of CaDPA release and the attendant water uptake into the spore core, Stage I of germination is complete (Setlow et al. 2001).
A major feature of Stage I is the enormous heterogeneity in the process, with some spores taking ≤10 min to complete Stage I, while others take many hour or even days (Setlow et al. 2012). The reason for this heterogeneity appears to be huge variation between individual spores in the time, termed ‘Tlag’, between germinant addition and initiation of rapid CaDPA release. However, it seems most likely that this huge variation is actually in the time between germinant addition and spore commitment, as the time between commitment and Tlag appears to be relatively constant, even for spores with widely different Tlag times (Yi and Setlow 2010). Many causes of the wide variation in Tlag values between individual spores are known, with a major one being variations in spore's levels of GRs as described below. In contrast to the highly variable Tlag values in individuals in spore populations, the actual time period in which ≥90% of CaDPA is released, defined as ΔTrelease, is relatively constant at approx. 2 min (Kong et al. 2011).
Spores that have completed Stage I can be isolated by use of mutations that block further events in germination, and these Stage I-germinated spores are stable for at least 24 h (Setlow et al. 2001). Stage I-germinated B. subtilis spores have an increased core water content (from 35% wet weight in dormant spores to 45% in Stage I-germinated spores) and a decreased wet heat resistance, although nowhere near the low heat resistance of a fully germinated spore. Importantly, there is no detectable cortex PG hydrolysis in Stage I, although there is likely a small amount, because loss of the CLE CwlJ alone in spores of B. subtilis and B. megaterium increases ΔTrelease times in individual spores 5- to 10-fold (Peng et al. 2009; Setlow et al. 2009; Zhang et al. 2012b). The Stage I-germinated spores still do not accumulate ATP, presumably because the water content in these spores is not sufficient for significant enzyme action in the spore core.
As is not surprising, the Stage I events, in particular CaDPA release, trigger the second and final part of spore germination termed ‘Stage II’ (Fig. 3). The major event in Stage II is the hydrolysis of the PG cortex by CLEs that specifically recognize cortical PG via the MAL modification, leaving the germ cell wall PG intact. Bacillus spores have two redundant CLEs: CwlJ and SleB (Setlow et al. 2003, 2009; Giebel et al. 2009; Heffron et al. 2010). SleB is a lytic transglycosylase, and CwlJ may also have this mode of action (see below). There is some evidence that B. megaterium spores have a third redundant CLE that is also sufficient for spore germination (Christie et al. 2010b). However, this CLE has not yet been identified.
In contrast to the multiple CLEs in Bacillus spores, Clostridium spores that have been studied have only a single essential CLE, termed ‘SleC’, with an unknown mechanism of action. In addition to these CLEs, additional enzymes active only on cortex PG have been identified in spores of Bacillus and Clostridium species (Makino and Moriyama 2002; Lambert and Popham 2008; Paredes-Sabja et al. 2009b; Lambert et al. 2012). However, none of these additional enzymes alone appear capable of catalysing sufficient cortex PG breakdown to allow completion of spore germination, although they can readily digest large cortex fragments generated by CwlJ, SleB or SleC. Interestingly, while CwlJ and SleB are present in Bacillus spores in a form that can be active, at least in vitro, SleC in Clostridium spores is present as a zymogen that is activated by one or more CspB proteases early in germination (Makino and Moriyama 2002).
Hydrolysis of the cortical PG and excretion of much of the fragments appear to relax the constraint upon the expansion of the spore core that then takes up more water and expands, likely with a remodelling of the germ cell wall which is not understood (Setlow 2003; Fig. 3). The IM surface area also increases 1·5- to 2-fold in this process and does so without new membrane synthesis (Cowan et al. 2004), again via a process that is not understood. The end result of Stage II of germination is that the spore core now contains approx. 80% wet weight as water, and enzymes become active in the core (Paidhungat and Setlow 2002). This leads to degradation of a novel group of small, acid-soluble proteins in the spore core and initiation of metabolism and macromolecular synthesis in the core. Completion of Stage II of germination also leads to at least partial breakdown of the spore coat and exosporium (if the latter is present), presumably by proteolysis, and eventual escape of the outgrowing spore from the spore coat and exosporium (Plomp et al. 2007; Steichen et al. 2007).
Components of the Spore Germination Apparatus
As is not surprising, a number of proteins are required for spore germination that are not present in growing cells (Paidhungat and Setlow 2002; Setlow 2003; Paredes-Sabja et al. 2011; Xiao et al. 2011). These proteins are synthesized only during sporulation, some in the developing spore and some in the mother cell, and this synthesis is regulated at the transcriptional level. This developmental and compartment-specific transcription during sporulation is driven by modification of RNA polymerase specificity by changes in sigma factors and other regulatory proteins associated with RNA polymerase at various times in development and in the forespore and mother cell compartments (de Hoon et al. 2010; Higgins and Dworkin 2012). There are five unique types of germination proteins, and these include GerP proteins, GerD, GR proteins, SpoVA proteins and CLEs as described below.
The GerP proteins are a group of small proteins encoded in an operon that is expressed only in sporulation in the mother cell compartment, and these proteins are most likely in the spore coat (Behravan et al. 2000; Carr et al. 2010). GerP proteins do not have any major sequence similarity to known proteins. Analyses in B. anthracis, B. cereus and B. subtilis indicate that loss of one or all of these proteins significantly reduces GR-dependent germination as well as CaDPA germination, but with no effect on dodecylamine germination. However gerP spores do not appear to have a general coat defect (Butzin et al. 2012). The germination phenotype of gerP mutants is eliminated if the spores are decoated either chemically or genetically. This finding led to the suggestion that the gerP proteins are needed to facilitate the movement of some germinants across spores' outer layers to gain access to targets below the coat or cortex layers. Consistent with this suggestion is that (i) the gerP phenotype can be suppressed by elevated nutrient germinant concentrations well above what is normally saturating and (ii) gerP spores germinate normally with pressures of approx. 150 MPa that trigger germination by activating GRs. However, there is no knowledge of how GerP proteins facilitate germinant access across spores' outer layers, and obvious homologs of the B. subtilis GerP proteins are not encoded in genomes of all Bacillales and Clostridium species (Setlow, P., 2013, unpublished results).
GerD is an approx. 180-residue protein present in spores of Bacillus species and is synthesized only in the forespore in parallel with GR and SpoVA proteins 1–2 h prior to forespores' CaDPA accumulation (Paidhungat and Setlow 2002; Setlow 2003). GerD is almost certainly a peripheral IM protein in the spore that is largely held in the IM by a diacylglycerol anchor (Igarashi et al. 2004; Pelczar and Setlow 2008; Mongkolthanaruk et al. 2009). GerD exhibits no clear sequence similarity to known proteins. However, this protein is predicted to be largely hydrophilic and is on the outer surface of the IM where it is largely if not completely in a single small focus termed the germinosome where the GR proteins are also found (Griffiths et al. 2011; Korza and Setlow 2013). GerD is essential for GR assembly in the germinosome in spores, although GRs are present in the IM of ΔgerD spores. In the absence of GerD, rates of GR-dependent germination are greatly decreased (Pelczar et al. 2007; Gupta, S., and Christie, G., personal communication), although why this is the case is not clear. However, the essential role of GerD in GR-dependent germination appears confined to the Bacillales, as GerD is not present in spores of the Clostridiales (Paredes-Sabja et al. 2011; Xiao et al. 2011). In B. subtilis, there are approx. 4000 GerD molecules/spore and its level is constant in different sporulation media, although may vary between individual spores in populations (Griffiths et al. 2011; Ramirez-Peralta et al. 2012a; Stewart and Setlow 2013).
The GRs are what recognize and respond to nutrient germinants, with a variety of different germinants recognized by GRs as described above. Spores of Bacillus species invariably have multiple GRs with different and sometimes overlapping germinant specificity (Ross and Abel-Santos 2010; Paredes-Sabja et al. 2011; Xiao et al. 2011). As GerD, GR proteins are also synthesized in the forespore and in parallel with GerD and SpoVA proteins. GRs contain three proteins termed A, B and C, and these are most likely subunits that interact to form a functional GR; a likely fourth GR D subunit has also been recently identified in Bacillales and Clostridiales species (see below). It is common that all three GR subunits are encoded in a single operon. In these cases, individual A, B and C proteins are associated primarily with a single GR, but there are clearly many exceptions to this, especially because some GR subunits are expressed monocistronically. Indeed, in at least one case, a GR protein from a monocistronic gene has been shown to interact at least functionally with GR proteins expressed from a rather far removed genetic locus, and there is other evidence for at least some functional interaction between individual proteins of different GRs (Igarashi and Setlow 2005; Christie et al. 2008; Stewart et al. 2012). There is clear sequence homology between A subunits of different GRs both within and across species and between B subunits and C subunits. However, there is minimal sequence homology with proteins in available databases. Most Clostridiales spores also have GRs that are clearly closely related to those of Bacillales, including putative D subunits, although a few Clostridium species have no GRs yet still germinate well with specific small molecule signals (Paredes-Sabja et al. 2011; Xiao et al. 2011). The most notable of the latter species is C. difficile whose spores germinate with specific bile salts, undoubtedly reflecting this species' preferred niche in the mammalian large intestine. Where it has been studied, all three GR subunits are required for GR function in Bacillus species, although as loss of any one subunit often renders the others unstable, this conclusion is not as definitive as one would like. Indeed, there is some evidence with spores of Clostridium perfringens that individual GR subunits may retain some germination-triggering activity (Banawas et al. 2013, in press).
Bioinformatic analysis suggests that the GR A proteins have 4–6 transmembrane (TM) segments, plus a large N-terminal hydrophilic segment and a small hydrophilic C-terminal segment (Wilson et al. 2012; Korza and Setlow 2013). The A protein has been localized in spores' IM by a variety of methods, and as noted above, GRs are present largely or completely with GerD in the germinosome. A large fraction of the A subunit of B. subtilis spores' GerA GR, presumably the N-terminal hydrophilic segment, is on the IM's outer surface (Wilson et al. 2012; Korza and Setlow 2013). The GRs' B subunits are predicted to be largely hydrophobic, with 10–12 TM segments, plus some small hydrophilic loops. The hydrophobic character of the B subunits has greatly slowed studies on these proteins, although they are almost certainly also in the IM along with the GRs' A and C subunits. As GerD, GRs' C subunits are IM lipoproteins, with these proteins on the IM's outer leaflet (Ross and Abel-Santos 2010; Paredes-Sabja et al. (2011); Xiao et al. 2011; Wilson et al. 2012; Korza and Setlow 2013). The structure of most of the B. subtilis GerB GR's C subunit has been determined by X-ray crystallography (Li et al. 2010), and the protein has a novel structure, so the structure did not give specific insight GR C protein function. As expected, mutations of highly conserved residues in the A, B and C subunits most often reduce or eliminate the function of the affected GR, even if the other GR proteins are still stable (Christie et al. 2008; Cooper and Moir 2011; Li et al. 2011a; Mongkolthanaruk et al. 2011). However, loss of one GR's subunit by a suitable deletion and even some point mutations can result in loss of the GRs' other subunits, presumably by their degradation. However, in some cases, analysis of specific mutants in GR proteins has given some hints as to locations of germinant binding regions in the GR proteins (Christie et al. 2010a; Mongkolthanaruk et al. 2011).
Very recently evidence was presented for a fourth GR protein that modifies GR function, termed ‘D subunit’, in spores of at least B. megaterium and B. subtilis (Paredes-Sabja et al. 2011; Ramirez-Peralta et al. 2013). Genes for these putative D subunits are present within or just upstream of operons encoding other GR subunits, are transcribed in parallel with the adjacent or encompassing GR operon and encode small approx. 75 residue proteins with two predicted TM domains, but no obvious sequence homology. In B. megaterium and B. subtilis spores, loss of these putative D-genes has either positive or negative effects on GR function, although not on levels of GR A, B and C subunits. Similar putative GR D subunits are encoded in many other Bacillales species' GR coding regions and in Clostridiales species as well.
Levels of GRs in spores vary significantly depending on sporulation conditions, with rich media leading to higher GR levels than with poor media (Hornstra et al. 2006; Ramirez-Peralta et al. 2012b). In rich media, levels of individual GRs in B. subtilis range from 600 to 1100 molecules per spore (Stewart and Setlow 2013), and these levels can be 3- to 8-fold lower in spores made in a poor medium. In B. subtilis, regulation of GR subunit genes is complex, with regulation by medium composition in some unknown fashion and by at least two auxiliary transcription factors, SpoVT and YlyA, all of which precisely control levels of GR subunits in individual sporulating cells (Ramirez-Peralta et al. 2012a; Traag et al. 2013). Probably as a result of this complex regulation, GR levels appear to vary significantly not only between spore populations made differently, but also between individual spores in a population, with stochastic effects on regulatory protein levels and GR expression levels likely also contributing to the wide variations in GR levels in individuals in spore populations (Griffiths et al. 2011; Setlow et al. 2012). The precise level of specific GRs in spores is almost certainly the major factor determining the rate of germination of spores of different populations, and also of different individuals in a single population (Zhang et al. 2010, 2011, 2012a, 2013) as discussed further below under SD spores.
Bacillus subtilis spores' GRs have been by far the best studied and these comprise five potential GRs, all encoded by tricistronic operons, although in one case, a putative GR D subunit is also a part of the operon (Setlow 2003; Ramirez-Peralta et al. 2013). In B. subtilis, two of the five GR subunits have no known germinants although the genes are expressed, and loss of these two GR operons does not affect spore germination with any known germinant. Of the other three GRs, GerA responds to l-alanine or l-valine alone and is strongly inhibited by d-alanine. As d-alanine can be generated by a sporulation-specific alanine racemase present in spores' outer layers, this enzyme has been suggested to modulate spore germination during sporulation by d-alanine synthesis as noted above. While GerA alone can trigger B. subtilis spore germination, neither of the other two important B. subtilis GRs, GerB and GerK, alone trigger germination efficiently. Rather these two GRs cooperate to respond to a germinant mixture termed ‘AGFK’, composed of l-asparagine (or l-alanine), D-glucose, D-fructose and K+. It appears likely that GerB binds the amino acid and probably the D-fructose, and GerK binds the D-glucose, while both GerB and GerK GRs probably bind K+. In the presence of D-glucose and K+, the GerK GR can also stimulate GerA function significantly, as can GerB plus l-asparagine, D-fructose and K+. This cooperation between GRs is a general feature and can also be seen in the synergy between low concentrations of germinants that target different GRs that respond to individual germinants, as these germinant mixtures generally give much more rapid germination than would be predicted by the activities with the individual germinants alone, although negative effects of one GR on the activity of another have also been seen (Atluri et al. 2006; Luu et al. 2011; Yi et al. 2011; Stewart et al. 2012; Gupta et al. 2013). However, the mechanism of this positive or negative cooperativity between GRs is not known, although it could be due to GR–GR association such as seen in the interaction between sensory proteins in bacterial chemotaxis.
As would be expected, loss of all GRs from Bacillus subtilis spores greatly reduces their germination with nutrient germinants, although not with CaDPA or dodecylamine. However, GR-less B. subtilis spores still exhibit a very slow rate of germination of approx. 0·1% per day, although the mechanism of this spontaneous germination is not clear (Paidhungat and Setlow 2002; Setlow 2003). Perhaps some component downstream of GRs in the signalling process fires occasionally. At the other end of the spectrum, forespore-specific overexpression of GRs 5–12-fold in B. subtilis results in more rapid spore germination, although the increased germination rates are by no means linear with GR levels (Cabrera-Martinez et al. 2003). Interestingly, if GerA or GerK GR expression is under too strong a forespore-specific promoter, the developing spore appears to germinate within the sporangium soon after GR synthesis begin, although why this happens is not clear.
The SpoVA proteins are a group of up to seven proteins that are expressed in the developing forespore in parallel with GRs and GerD (Paidhungat and Setlow 2002; Setlow 2003; Paredes-Sabja et al. 2011; Xiao et al. 2011). In B. subtilis, there are seven proteins, SpoVAA, B, C, D, Eb, Ea and F, which are encoded in a single operon in this order. Other Bacillales and Clostridiales species can have fewer proteins (although SpoVAC, SpoVAD and SpoVAEb are always present) and in some cases with more than one transcriptional unit. Bioinformatic analyses predict that SpoVA proteins other than SpoVAD and SpoVAEa are membrane proteins, generally integral membrane proteins, and at least SpoVAD and SpoVAEa, the two hydrophilic SpoVA proteins, have been localized to spores' IM and on this membrane's outer surface and are present at 6–8000 molecules per spore (SpoVAD) or 750 molecules per spore (SpoVAEa; Korza and Setlow 2013; Perez-Valdespino, A., 2013, unpublished results; Stewart and Setlow 2013).
In B. subtilis, loss of any SpoVA protein, except SpoVAEa or SpoVAF, gives an asporogenous phenotype, as while DPA is synthesized normally in sporulation, it is not taken up and the DPA-less spores germinate in the sporangium and lyse (Errington 1993; Perez-Valdespino, A. 2013, unpublished results). A C. perfringens ΔspoVA strain also does not accumulate DPA in spores, although these DPA-less spores are stable (Paredes-Sabja et al. 2008). Several temperature-sensitive spoVA B. subtilis mutants have also been isolated, and these accumulate DPA relatively normally at the permissive temperature, but DPA release from these spores during nutrient germination at the nonpermissive temperature is slowed considerably (Vepachedu and Setlow 2004; Wang et al. 2011a). These results led to the suggestion that SpoVA proteins make up the channel for DPA accumulation during sporulation, perhaps as CaDPA, as well as CaDPA release in spore germination. This suggestion was strengthened recently by a number of additional results in B. subtilis including the demonstration that (i) SpoVAD specifically binds the natural isomer of CaDPA in vitro, and this binding is eliminated as is CaDPA uptake in sporulation by mutation of conserved residues in the likely CaDPA binding pocket (Li et al. 2011b), (ii) SpoVAC expressed in E. coli acts as a mechanosensitive channel (Velasquez-Guzman et al. 2012), (iii) dodecylamine may well directly activate a SpoVA protein channel (Vepachedu and Setlow 2007b), and (iv) loss of SpoVAEa greatly reduced GR-dependent germination, but with no effects on CaDPA or dodecylamine germination (Perez-Valdespino, A., 2013, unpublished results).
While the results given above are certainly consistent with SpoVA proteins forming a CaDPA channel in the spores' IM, there is no information on the precise structure or organization of this channel, how it is gated, how the energy requiring process of CaDPA uptake into developing spores takes place, and how the same channel can act in both CaDPA uptake in sporulation that will require energy to accumulate CaDPA against a concentration gradient, as well as the likely energy independent CaDPA efflux in spore germination.
Spores of Bacillus species have two redundant CLEs, CwlJ and SleB, and both require the cortex-specific modification MAL for recognition and cleavage of PG (Setlow 2003; Giebel et al. 2009; Setlow et al. 2009; Heffron et al. 2010; Paredes-Sabja et al. 2011). SleB appears to be a lytic transglycosylase, and CwlJ has been predicted to have a similar catalytic activity, although this has not been demonstrated either in vivo or in vitro (Li et al. 2012, 2013). CwlJ is synthesized in the mother cell compartment of the sporulating cell and is located near the coat–cortex boundary. The enzyme is synthesized in its mature form and is thus present in the spore in a form that could be active. CwlJ localization in the spore requires its partner protein GerQ (originally YwdL) that in some Bacillus species is encoded as a cistron of a bicistronic operon with cwlJ, but in other Bacillus species, including B. subtilis, gerQ and cwJ are monocistronic. Most if not all SleB is synthesized in the forespore and is found in the IM (Boland et al. 2000; Chirakkal et al. 2002), however, some SleB is also found at the coat–cortex boundary. As CwlJ, SleB is present in spores in its mature form and also requires a partner protein, YpeB, for its localization in spores. YpeB has a likely membrane insertion sequence, and most of both YpeB and SleB are associated with the spore's IM, with both proteins on the outer surface of the IM (Korza and Setlow 2013). However, association of SleB and YpeB has not been demonstrated in vitro.
The crystal structure of the catalytic domain of SleB from B. cereus and B. anthracis has been determined at high resolution, and the structures are almost identical and very similar to those of lytic transglycosylases, including a large cleft for PG binding plus a catalytic glutamate residue (Jing et al. 2012; Li et al. 2012). SleB also has a PG-binding domain that is important for catalytic activity in vivo but not in vitro (Heffron et al. 2011; Li et al. 2013). Interestingly, YpeB can significantly inhibit SleB activity in vitro, and this has been suggested to be important for regulation of SleB activity in vivo (Li et al. 2013). CwlJ lacks a PG-binding domain, but its catalytic domain has extensive sequence similarity to SleB, and this similarity extends to CwlJ's predicted structure plus a putative catalytic glutamate residue that is essential for CwlJ function in vivo (Li et al. 2013).
A variety of evidence has shown that CwlJ is activated during germination by the CaDPA released in Stage I of germination (Setlow 2003). CwlJ is also activated by exogenous CaDPA, but only with the natural DPA isomer. The germination by exogenous CaDPA requires high CaDPA concentrations, while low concentrations are rather ineffective. This likely precludes triggering of the germination of nearby dormant spores by CaDPA released from one germinating spore (Zhang et al. 2011). In contrast to CwlJ, CaDPA alone does not activate SleB, and its mechanism of activation in germination is not known. It has been suggested that SleB's activity is determined by changes in strain on cortex PG due to changes in core water content (Setlow 2003), but the evidence for this suggestion is relatively weak. This is certainly a major unknown about CLEs, as is why CwlJ does not become active in sporulation when CaDPA is taken up or why SleB does not act on cortex PG shortly after the enzyme is made, as this is well before CaDPA uptake.
In contrast to spores of Bacillales species, most Clostridiales spores do not have CwlJ and SleB as CLEs, but rather have a single CLE termed SleC (Makino and Moriyama 2002; Paredes-Sabja et al. 2011; Xiao et al. 2011). SleC is synthesized in the mother cell and is in spores' outer layers where it is readily extracted from spores during decoating treatments (as is CwlJ in Bacillus spores). While there is no evidence for a partner protein for SleC, more important is the fact that SleC is synthesized as a preproprotein. The presequence is cleaved shortly after synthesis and is presumably crucial for translocation of SleC made in the mother cell across the outer forespore membrane to its location below the outer spore membrane where it is present in the dormant spore as the proprotein that is inactive. This proprotein is activated by proteolytic cleavage by a CspB protease, of which spores can have one or several, in the first min of spore germination. The CspB(s) are subtilisin-like proteases that are also synthesized in the mother cell and are localized in the same region of the spore as is pro-SleC. Presumably CspB action is triggered somehow by Stage I germination events, but in most cases how CspB is activated is not clear. The exception is with spores of C. difficile in which it appears likely that CspB is directly activated by bile salts (Francis et al. 2013), thus obviating the need to have any GRs recognizing bile salts for the germination of spores of this and similar species that lack GRs.
Overall information flow in germination of spores of Bacillus species
When all information on the various germination proteins in Bacillus spores is put together, this allows construction of a flow diagram of the signal transduction pathways in germination of spores of Bacillus species (Fig. 4). This includes pathways for germination with all germinants except PG fragments, although there are still a number of steps in which there is a lack of knowledge.
Analysis of the Germination of Individual Spores
It has been known for more than 40 years that the germination of individual spores in populations is extremely heterogeneous, with some spores germinating rapidly and others slower, and with some small fractions of the population germinating extremely slowly – not for many hour or even days (Setlow et al. 2012). These latter spores have been termed SD and are of major concern to the food industry, as has been the overall heterogeneity in the germination process itself. The study of spore germination heterogeneity has been simplified in the past 5 years by the continued development of sophisticated methods for simultaneous analysis of the germination of hundreds of individual spores in a population, and this technology is being continuously refined (Eijlander et al. 2011; Kong et al. 2011, 2013; Stringer et al. 2011; Wang et al. 2011b). Techniques that have been used for these analyses involve simultaneous analysis of multiple individual spores by fluorescence microscopy, Raman microspectroscopy, phase contrast microscopy or differential interference contrast (DIC) microscopy and have coupled these analytical methods with sophisticated image analysis. These analyses have been carried out with spores of both Bacillus and Clostridium species, and the conclusions using DIC microscopy, in particular, have been very similar. Thus, the loss in spores' phase contrast or DIC image intensity takes place in two steps: loss of approx. 75% of the total image intensity takes place in approx. 2 min in parallel with CaDPA release, as shown by examination of individual spores' Raman spectra that are dominated by CaDPA-specific peaks. The remainder of the loss in spores' DIC image intensity takes place after CaDPA release, and this loss is slow (10–20 min) and is due to spore cortex hydrolysis and the expansion of the spore core with attendant core water uptake. Use of both Raman microspectroscopy and DIC microscopy to monitor the germination of hundreds to thousands of individual spores of a number of Bacillus species as well as C. perfringens has shown that after addition of germinants to spores, individuals in the population exhibit an extremely variable lag period, Tlag, in which there is minimal if any change in DIC image intensity (Kong et al. 2011; Wang et al. 2011b; Fig. 5; shown for two characteristic spores). However, at the end of Tlag, rapid CaDPA release begins and almost all CaDPA is excreted by Trelease, which is the time for release of ≥90% of CaDPA; ΔTrelease = Trelease-Tlag, and is approx. 2 min and relatively constant for individual spores in a spore population. Following Trelease, there is the much slower fall in DIC image intensity that ends at Tlys, with ΔTlys = Tlys-Trelease being 10–20 min depending on conditions and strain/species (Fig. 5). As with ΔTrelease, ΔTlys is also relatively constant for individual spores in a population germinating under the same conditions.
The work noted above indicates that the major variation between the germination of individual spores in a population is in the value of Tlag for individual spores which can vary tremendously, although more specifically it is the time needed for individual spores to become committed to germinate that varies. However, times to commitment have not been directly measured for large numbers of individual spores, but are only inferred from measurements of times for commitment in germinating spore populations, and in general, commitment takes place a few min prior to Tlag (Yi and Setlow 2010).
The major question that arises from the knowledge that it is variations in Tlag that are responsible for variations in the rates of germination of individual spores in populations is what determines Tlag values. A variety of work has determined that factors that do or do not affect average spore Tlag values are generally as follows (Zhang et al. 2010, 2012a,b; Wang et al. 2011a; Setlow et al. 2012; Zhou et al. 2013): (i) germination temperatures above and below an optimum temperature give longer Tlag values, (ii) heat activation lowers Tlag values, (iii) increasing germinant concentrations decrease Tlag values, but only up to saturating germinant concentrations, and then there are no further effects, (iv) changes in CLE or SpoVA protein levels have no significant effects on Tlag values, (v) the lack of GerD greatly increases Tlag values, (vi) the presence of a combination of germinants that trigger different GRs that can act alone (i.e. – synergy) decreases Tlag times, and (vii) and most importantly, increasing GR levels decrease Tlag times for germination with that GR's cognate germinant, while lower levels of that GR increase Tlag values for germination with that GR's cognate germinant. In contrast to effects on Tlag values, the only factor that has major effects on ΔTrelease values (other than temperature (Zhou et al. 2013) is the presence of the CLE CwlJ, as its absence increases ΔTrelease values 5- to 10-fold (Peng et al. 2009; Setlow et al. 2009; Zhang et al. 2012b).
The results noted above suggest that a likely reason for spores being SD is that these are spores with low GR levels, much lower than those in the great majority of the population. This suggestion was tested by isolation of SD spores from populations of spores of several Bacillus species as those spores that had not germinated even after many hour of germination with saturating nutrient germinant concentrations (Ghosh and Setlow 2009, 2010). This resulted in isolation of 1–2% of the starting dormant spores as the SD ones, although this percentage was higher with spores made in a poor medium, was lower when multiple germinants targeting multiple GRs were used, was higher when subsaturating germinant concentrations were used or no heat activation was applied prior to germination and was very low when spores with elevated GR levels were used. While the SD spores isolated in this fashion germinated normally with the non-GR-dependent germinants CaDPA and dodecylamine, SD spores isolated by germination with a germinant targeting one GR germinated extremely poorly with the original germinant (Table 1; Ghosh and Setlow 2009, 2010). The SD spores also did not germinate rapidly with a germinant targeting a different GR, although this latter germination was significantly faster than that with the original germinant used to isolate the SD spores (Table 1). Invariably, the germination defect in SD spores was greatly increased Tlag values compared with those for the initial dormant spores (Zhang et al. 2012a). Despite the severe germination defect in SD spores, when SD spores were germinated by CaDPA and then resporulated, the germination of the resultant spores with nutrient germinants was normal. Interestingly, SD spores of several Bacillus species required significantly higher temperatures for optimal heat activation than did dormant spores (Ghosh et al. 2009), although the meaning of this observation is not clear.
Table 1. Germination of superdormant (SD) Bacillus subtilis spores with various germinantsa
Spores of B. subtilis PS533 (wild-type) or its isogenic gerB* derivative which germinate with l-asparagine alone were germinated twice for 5 h each with various germinants to isolate SD spores. The SD spores were then germinated with various nutrient germinants and the % spore germination in 2 h was determined by phase contrast microscopy. Data are taken from Ghosh et al. (2012).
Wild-type spores (PS533).
GerB* spores. These spores have a specific amino acid change in their GerB germinant receptor (GR) that renders L-asparagine germination with this GR independent of the GerK GR and glucose, fructose and K+ (Setlow 2003).
Most importantly, analysis of the levels of various GRs in dormant and in SD B. subtilis spores prepared with different germinants showed that SD spores prepared with a particular germinant had levels of the GR targeted by that germinant that were up to approx. eightfold lower than in the initial dormant spores, while GR levels targeted by germinants not used originally were only approx. twofold lower than in the original dormant spores (Table 2). However, GerD and SpoVAD levels were identical in dormant and SD spores (Ghosh et al. 2012). Overall, these results indicate that the major reason for superdormancy in spores is a low level of GRs, although it is clear that (i) the actual rate of spore germination is not linear with spore GR levels for reasons that are unclear, and (ii) levels of different GRs tend to exhibit some covariance such that spores with very low levels of one GR also exhibit somewhat lower levels of other GRs. This latter observation is consistent with the generally similar overall regulation of the expression of operons encoding different GRs.
Table 2. Relative germinant receptor (GR) levels in superdormant (SD) Bacillus subtilis spores isolated with various nutrient germinantsa
SD spores isolated by germination with:
l-Valine via the GerA GR
AGFK via the GerB and GerK GRs
l-asparagine via the GerB* GR
Level of GR protein in SD spores/levels in dormant spores
SD spores of wild-type or gerB* B. subtilis strains were isolated by germination with various GR-dependent germinants: l-valine via the GerA GR, AGFK via both the GerB and GerK GRs, and l-asparagine via the GerB* GR. Levels of GR subunits were determined in the SD spores and the initial dormant spores by Western blot analysis of several amounts of spore inner membrane protein. The data were taken from the study by Ghosh et al. (2012).
Conclusions and Questions
Clearly much has been learned in recent years on the process and mechanism of spore germination in Bacillus species, and with spores of Clostridium species as well. Most important, however, is that in addition to all the questions noted above, some really major fundamental questions remain, answers to which are essential to our thorough understanding of the mechanisms of spore germination in both Bacillus and Clostridium species. These major questions include the following: (i) What do GRs actually do? Given their IM location, the novel properties of this membrane and the rapid movement of compounds across the IM early in germination, it is tempting to speculate that GRs transport something, although it is not CaDPA. It is also attractive to think that GRs somehow can trigger the opening of the SpoVA CaDPA channel when GRs bind a germinant, and while there is some suggestive evidence for this, the direct evidence is quite weak at present (Vepachedu and Setlow 2007a). (ii) What is happening in commitment and the period prior to Tlag that leads to opening of CaDPA channels? The answer to this question is also crucial to understanding the mechanism of initiation of Stage I of germination, but again we have no real idea of what is going on in this period of germination, other than that GR–germinant interaction is going on. Interestingly, HP treatment of B. subtilis spores at 140–150 MPa that acts through GRs also shows the same phenomenon of commitment and a dependence on GR levels as does nutrient germination (Kong, L., Doona, C.J., Setlow P., and Li, Y.-Q., 2013, unpublished results), so perhaps this is an intrinsic property of the GR system for triggering spore germination. (iii) How is the SpoVA channel organized and regulated? Once again there are many more unknowns than knowns, but the answer to this question could have significant applied importance, as an answer might suggest ways to artificially open the CaDPA channel and thus germinate whole spore populations very rapidly. While obviously many questions remain about spore germination, progress in the past few years has been extremely exciting, and if this continues, perhaps the next iteration of such a review article on spore germination will have answers to these fundamental questions.
Work in the author's laboratory has received generous support over the past years from the National Institutes of Health, the U.S. Department of Agriculture and the U.S. Army Research Office. Work over the past 4 years has been supported by a U.S. Department of Defense Multi-disciplinary University Research Initiative through the U.S. Army Research Laboratory and the U.S. Army Research Office, under contract number W911NF-09-1-0286. The author is also extremely grateful to all past and present lab members and collaborators who have shared the journey through spore germination.