Title: Bacterial spore structures and their protective role in biocide resistance
Abstract: Journal of Applied MicrobiologyVolume 113, Issue 3 p. 485-498 REVIEW ARTICLEFree Access Bacterial spore structures and their protective role in biocide resistance M.J. Leggett, M.J. Leggett Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, UKSearch for more papers by this authorG. McDonnell, G. McDonnell STERIS Ltd, Basingstoke, UKSearch for more papers by this authorS.P. Denyer, S.P. Denyer Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, UKSearch for more papers by this authorP. Setlow, P. Setlow Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this authorJ.-Y. Maillard, J.-Y. Maillard Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, UKSearch for more papers by this author M.J. Leggett, M.J. Leggett Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, UKSearch for more papers by this authorG. McDonnell, G. McDonnell STERIS Ltd, Basingstoke, UKSearch for more papers by this authorS.P. Denyer, S.P. Denyer Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, UKSearch for more papers by this authorP. Setlow, P. Setlow Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT, USASearch for more papers by this authorJ.-Y. Maillard, J.-Y. Maillard Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, UKSearch for more papers by this author First published: 10 May 2012 https://doi.org/10.1111/j.1365-2672.2012.05336.xCitations: 169 Jean-Yves Maillard, Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff CF10 3NB, UK. E-mail: [email protected] AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Summary The structure and chemical composition of bacterial spores differ considerably from those of vegetative cells. These differences largely account for the unique resistance properties of the spore to environmental stresses, including disinfectants and sterilants, resulting in the emergence of spore-forming bacteria such as Clostridium difficile as major hospital pathogens. Although there has been considerable work investigating the mechanisms of action of many sporicidal biocides against Bacillus subtilis spores, there is far less information available for other species and particularly for various Clostridia. This paucity of information represents a major gap in our knowledge given the importance of Clostridia as human pathogens. This review considers the main spore structures, highlighting their relevance to spore resistance properties and detailing their chemical composition, with a particular emphasis on the differences between various spore formers. Such information will be vital for the rational design and development of novel sporicidal chemistries with enhanced activity in the future. Introduction When cells of certain Gram-positive bacteria, for example Bacillus and Clostridium spp., encounter environmental stresses such as nutrient starvation, they form a dormant structure termed an endospore (simply referred to as a spore in this review). Bacterial spores can survive in this dormant state for many years (Kennedy 1994), with some studies suggesting that they may even persist for millions of years (Cano and Borucki 1995). Faced with the challenge of surviving prolonged periods of dormancy, spores have evolved many mechanisms to protect themselves from damage, which also serve to protect them from modern disinfection/sterilization procedures (Setlow 2006). It is this highly resistant characteristic that makes them such a problem in the food industry, where Bacillus cereus is commonly responsible for food-borne diseases (Bottone 2010), and in healthcare settings where the spore-forming Clostridium difficile is a major cause of hospital-acquired diarrhoea (Lyerly et al. 1988; Wilcox and Fawley 2000). It is therefore of interest to investigate how bacterial spores withstand environmental stress, including their ability to resist disinfectants and sterilants. Much of the work on spore resistance to date has centred on spores of Bacillus subtilis, owing principally to the ease with which this organism may be genetically manipulated (Nicholson et al. 2000; Setlow 2006), as well as the relatively early availability of its complete genome sequence (Kunst et al. 1997). This review provides an update on what is known about spore structures, highlighting their detailed composition where known and noting any similarities/differences between Bacillus and Clostridium spores in particular. Consideration will also be given to any known resistance factors in the spore structure itself. Spore-former life cycle The process of sporulation is classically divided into seven stages (Hitchins and Slepecky, 1969; Piggot and Coote 1976; Errington 1993; McDonnell 2007; Fig. 1) and is basically identical for Bacilli and Clostridia, except that Clostridia undergo a considerable cell lengthening during sporulation and visible clubbing on development of the forespore (Fitz-James and Young 1969). Normal vegetative cell growth can be defined as stage 0 with regard to sporulation, and is followed by stage I/II, where the vegetative cell undergoes asymmetric cell division, forming two compartments, the smaller of which is termed the prespore, separated by a septum; stage I – presentation of the cell DNA as an axial filament – was originally defined by Ryter (1965), but is generally no longer recognized as a defined stage (Piggot and Coote 1976; Errington 1993). During stage III, the prespore is engulfed by the mother cell to form a distinct cell termed the forespore bound by the inner and outer forespore membranes. Stage IV sees the synthesis of the spore cortex, composed of peptidoglycan (PG), between the inner and outer forespore membranes, which is followed by stage V, spore coat formation. During stages IV and V, the mother cell also synthesizes a very abundant spore-specific molecule, pyridine-2,6-dicarboxylic acid [dipicolinic acid (DPA)]. This accumulates in the forespore and is accompanied by a reduction in the forespore water content. Spore maturation takes place during stage VI, where the coat material becomes denser in appearance. The final stage (VII) sees the lysis of the mother cell and release of the mature spore structure (1, 2). The mature spore structure protects the dormant micro-organism from external influences until the conditions once more become favourable for vegetative cell growth. The dormant spore is then re-activated and undergoes germination and outgrowth. Figure 1Open in figure viewerPowerPoint Key morphological changes that take place during sporulation. Modified from McDonnell (2007). Figure 2Open in figure viewerPowerPoint Spore structure. A representation of a ‘typical’ bacterial spore (structures are not drawn to scale). Modified from Setlow (2006). The transition from dormant spore to vegetative cell involves three separate phases: activation, germination and outgrowth. Activation can be triggered by appropriate conditions of heat, pH or chemical exposure and renders the dormant spore poised to enter germination, thus breaking its dormant state (Keynan and Evenchik 1969). Activation is a reversible process that does not necessarily commit the spore to germination and outgrowth, and activated spores retain most properties of the dormant spore (Keynan and Evenchik 1969). In contrast, once a spore is committed to germinate, the spore can no longer return to its dormant state (Gould 1969). Germination can be initiated in response to various stimuli, often varying depending on the species. These include, but are not limited to, metabolizable nutrient germinants, such as specific amino acids and sugars (although these germinants’ metabolism is not required for their triggering of germination), nonmetabolizable germinants such as some ionic species, cationic surfactants and chelates (in particular, a Ca:DPA chelate), and some physical treatments such as high pressures (Gould 1969; Setlow 2003). ‘Outgrowth’ is defined as all developmental events taking place after germination, including initiation of metabolism and macromolecular synthesis, swelling of the spore, emergence (where the outer spore layers are shed) and growth of the new cell, and represents a return of the spore to vegetative cell growth (Strange and Hunter 1969). Spore structure The structure (Fig. 2) and chemical composition of the spore differ considerably from those of the vegetative cell. These differences largely account for the unique spore resistance to environmental stresses, including disinfectants and sterilants (Setlow 2006). They are considered in further detail below and, unless stated otherwise, the discussion refers to spores of B. subtilis. Exosporium The exosporium is the outermost structure of many bacterial spores, in particular those of the B. cereus group, which also includes Bacillus anthracis and Bacillus thuringiensis (Todd et al. 2003; Redmond et al. 2004), but is also found in some other Bacilli and Clostridia, including the pathogenic Cl. difficile (Lawley et al. 2009; Permpoonpattana et al. 2011). The presence of an exosporium is by no means universal, and this structure may be either absent or greatly reduced in many species, including B. subtilis (Waller et al. 2004); this has resulted in a lack of information regarding its composition (Todd et al. 2003). Based on studies with B. cereus, the exosporium is composed principally of protein (43–52% of dry weight), but also contains lipids (15–18% of dry weight) and carbohydrates (20–22% of dry weight), as well as a minor (around 4%) component described as ash, which contained both calcium and magnesium as well as some undetermined components (Matz et al. 1970; Beaman et al. 1971). The exosporium protein component is notable for its low level, or lack, of the sulfur-containing amino acids cysteine (a prominent component of the spore coat) and methionine, as well as histidine and tyrosine. Of the lipid component, diphosphatidylglycerol (cardiolipin) represented the only detectable phospholipid (∼30% of total lipids); the majority were neutral lipids, and there were at least 19 fatty acids, 40% of which were normal C16 and C18 fatty acids. Of the remaining fatty acids, nine were straight chained (seven saturated and two unsaturated), seven were branch chained (four iso- and three anteiso-), and one was unidentified (Matz et al. 1970; Beaman et al. 1971). The exosporium polysaccharide component was made up of glucose, glucosamine and rhamnose, with a very small amount of ribose (which was attributed to RNA contamination of exosporium preparations). Whilst the exosporia of clostridial spores have been described (Hodgkiss et al. 1967; Mackey and Morris 1972), there is no detailed breakdown of the chemical composition of these structures. Although a number of major proteins have been identified as components of the B. cereus and B. anthracis spores’ exosporia (Lai et al. 2003; Todd et al. 2003; Henriques and Moran 2007; Fazzini et al. 2010; McPherson et al. 2010), their exact function in the spore is unknown. It has been suggested that the adherent, hydrophobic properties of the exosporium may be involved in the pathogenicity of some spores (Koshikawa et al. 1989; Bowen et al. 2002). However, to the best of our knowledge, the exosporium has not in itself been shown to provide the spore with any significant protection from biocide attack. Spore coat The spore coat sits within the exosporium (if present) and generally comprises a series of thin, concentric layers, the numbers of which differ depending on the organism under investigation (Driks 1999). Indeed, the structure as visualized by electron microscopy and the biochemical composition of the spore coat vary between species and even within different strains of the same species (Fitz-James and Young 1959; Kondo and Foster 1967; Kornberg et al. 1968). Bacillus subtilis spores have two prominent coat layers, the inner and outer spore coats, plus a basement layer between the inner coat and the cortex and an outermost crust (Aronson et al. 1992; Driks 1999; Henriques and Moran 2007; McKenney et al. 2010). The inner coat is often described as having a lamellar appearance and is less dense when viewed by electron microscopy, whereas the thicker outer coat lacks the clear lamellar structure of the inner coat and appears darker under electron microscopy (Warth et al. 1963; Kay and Warren 1968; Aronson and Fitz-James 1976). There are many excellent reviews dealing specifically with the structure and molecular genetic control of coat assembly (Driks 1999; Henriques and Moran 2000, 2007; McKenney and Eichenberger 2012); in this review, we will briefly summarize some of the details of the composition of the spore coat. The coat is made up predominantly of protein, but also contains minor (6%) carbohydrate components, most likely owing to the glycosylation of two low-molecular-weight coat polypeptides of approximately 8–9 kDa (Pandey and Aronson 1979; Jenkinson et al. 1981). The protein fraction of the coat represents 50–80% of the total spore protein (Aronson and Fitz-James 1976; Pandey and Aronson 1979) and can itself be divided into two separate fractions, soluble and insoluble. The soluble fraction accounts for approximately 70% of the total coat protein and may be isolated by treatment with a combination of reducing and denaturing agents at alkaline pH (Goldman and Tipper 1978; Pandey and Aronson 1979). The soluble fraction contains upwards of 40 proteins, as viewed on polyacrylamide gels, ranging from 6 to >70 kDa in size (Driks 1999; Henriques and Moran 2000). Particularly abundant within the soluble fraction is CotG, a hydrophilic protein of 24 kDa that is thought to be morphogenetic and when deleted prevents the incorporation of another protein, CotB, into the mature spore coat (Sacco et al. 1995). Molecular genetic manipulation in the B. subtilis model organism has allowed the identification and investigation of other coat proteins from the soluble fraction, including CotA, CotB, CotC and CotD (Donovan et al. 1987). Unlike CotG, these proteins can be deleted without any major detrimental effects to the mature spore. However, spores lacking CotD germinated more slowly than wild-type spores, and loss of CotA resulted in the loss of the wild-type brown colour (Donovan et al. 1987). Approximately 30% of isolated coat proteins resisted solubilization and define the coat insoluble fraction (Pandey and Aronson 1979). This fraction is characterized by a high cysteine content, which likely contributes to its insoluble nature because of the formation of disulfide cross-links (Goldman and Tipper 1978; Pandey and Aronson 1979). Evidence for the presence and function of such disulfide cross-links in the spore coat is given by Gould and Hitchins (1963) and Gould et al. (1970), who showed that spores became sensitive to hydrogen peroxide and lysozyme following treatment with various chemical disruptors of disulfide bonds. Other types of cross-linking, including dityrosine (Pandey and Aronson 1979) and ε-(γ-glutamyl)lysine (Kobayashi et al. 1996) cross-links, have also been detected in the spore coat. The presence of heavily cross-linked material in the spore coat is likely responsible for some of the spore’s chemical and mechanical resistance (Wold 1981), as is the case in other biological structures such as the sea urchin egg, which is surrounded shortly after fertilization by a rigid envelope containing dityrosine cross-links to protect the developing embryo (Shapiro 1991). Of the 70 or so coat proteins identified in B. subtilis spores (Kim et al. 2006; Henriques and Moran 2007), at least 50 are also shared with its close relatives, B. cereus and B. anthracis (Kuwana et al. 2002; Lai et al. 2003; Liu et al. 2004; Giorno et al. 2007). Whilst little is known about the structure and composition of the clostridial spore coat, only about 20 of the B. subtilis coat proteins have orthologs in the clostridial genomes currently available (Henriques and Moran 2007). It would be interesting to discover whether this discrepancy in the number of known coat proteins is indicative of a simpler coat in these Clostridia, or perhaps the presence of unique coat proteins in the clostridial coat. In a recent study, it was demonstrated that there was no cross-reactivity between antispore serum from Cl. difficile 630 and B. subtilis spore coat proteins, suggesting significant differences between the coats of these two species (Permpoonpattana et al. 2011) and adding weight to the bioinformatic observations of Henriques and Moran (2007) outlined earlier. Such species-specific differences in coat composition could impact upon the biocidal formulation required to rapidly overcome the defence provided by the spore coat. Functionally, the spore coat serves as an initial barrier to large molecules, such as the PG-lytic enzyme lysozyme, which would otherwise have access to the spore cortex (Nicholson et al. 2000). Probably for this reason, the coat is essential for spore resistance to predation by bacteriovores (Klobutcher et al. 2006; Laaberki and Dworkin 2008). In contrast to the coat’s impermeability to lysozyme, smaller molecules such as spore germinants must presumably pass through this barrier (Driks 1999). The spore coat has also been identified as a critical resistance mechanism against many chemicals, especially oxidizing agents such as hydrogen peroxide (Riesenman and Nicholson 2000; Young and Setlow 2004b), ozone (Young and Setlow 2004a), peroxynitrite (Genest et al. 2002), chlorine dioxide and hypochlorite (Young and Setlow 2003; Ghosh et al. 2008), all of which kill spores more rapidly when the coat layer is absent. This protective role was perhaps most clearly illustrated by Ghosh et al. (2008), who showed that B. subtilis spores lacking most coat layers owing to mutations in the cotE and gerE genes (coding for a morphogenetic protein essential for formation of the outer coat, and a DNA-binding protein that itself regulates several genes coding for coat proteins (Driks 1999), respectively) became sensitive to hypochlorite to a level similar to that of vegetative cells. Despite the clear protective role of the spore coat, and an increasingly detailed understanding of the mechanisms, components and genetic controls involved in spore coat assembly (Driks 1999; Takamatsu and Watabe 2002; Henriques and Moran 2007; McKenney and Eichenberger 2012), no individual coat proteins have been identified as an essential protective component. The coat may simply be serving to detoxify these chemicals before they penetrate the inner regions of the spore structure, such as the inner membrane and the core (Nicholson et al. 2000; Riesenman and Nicholson 2000; Setlow 2006). It has also been suggested that the spore coat is unable to protect spores from some toxic chemicals, for example low-molecular-weight alkylating agents (Setlow et al. 1998), which apparently are small enough to bypass the coat’s molecular sieving effect and gain access to their target site in the spore core. It has been suggested that superoxide dismutase (SOD), an enzyme associated with the exosporium or spore coat of B. subtilis and B. anthracis and thought to be involved in the formation of the spore coat (Henriques et al. 1998), may also serve to detoxify potentially damaging chemicals at the spore surface, as is the case for some vegetative cells (Nicholson et al. 2000; Setlow 2006). Whilst such a protective role was not found in B. subtilis (Casillas-Martinez and Setlow 1997), it has been shown in B. anthracis (Cybulski et al. 2009), where SODs present on the surface of the spore protect against oxidative stress and increase spore pathogenicity within the host lung. Other coat proteins have been shown to possess enzymatic activity, such as CotE of Cl. difficile 630. This bifunctional protein shows both peroxiredoxin and chitinase activity, which may be associated with the characteristic inflammation associated with infection by this organism (Permpoonpattana et al. 2011). Outer membrane Under the spore coat lies the outer spore membrane. Whilst this structure is essential for spore formation (Piggot and Hilbert 2004), its precise function remains unclear, reportedly having no great effect on resistance to radiation, heat or some chemicals (Nicholson et al. 2000; Setlow et al. 2000). There is some confusion as to whether the outer membrane, which is morphologically distinct during sporulation, actually serves as an intact membrane in the mature spore (Racine and Vary 1980), and there are no reports of the isolation of a purified outer membrane in the literature. It is difficult to identify the outer membrane in electron micrographs following synthesis and maturation of the coat and cortex, and dormant spores from several species are reported to have poorly defined or indistinguishable outer membranes (Freer and Levinson 1967; Fitz-James 1971; Holt et al. 1975; Aronson and Fitz-James 1976). Despite the lack of conclusive morphological evidence for the presence of an outer spore membrane, there is functional and biochemical evidence to support the presence of such a structure in the mature spore. For instance, there is evidence that 11 spore coat proteins are related antigenically to membrane proteins from vegetative cells (Fujita et al. 1989; as cited in Henriques and Moran 2000). Crafts-Lighty and Ellar (1980) identified cytochromes and enzymes of the electron transport chain in extracts of spore outer integuments (cortex, coats and any outer membrane structure), both of which imply the presence of a membranous element, and which were not because of contamination by inner-membrane fractions. Further evidence in support of an outer spore membrane was presented by Rode et al. (1962), who identified a sharply delineated permeability barrier between the cortex and coats of Bacillus megaterium spores that prevented the uptake of methacrylate. This barrier was disrupted following spore fixation using potassium permanganate (KMnO4). It has also been shown that glucose will only permeate as far as the cortex of dormant spores (Gerhardt et al. 1982), again suggesting the presence of a functioning membrane at this point in the spore. If present, this membrane does not prevent the uptake of the small uncharged, lipophilic molecule, methylamine (Setlow and Setlow 1980; Swerdlow et al. 1981), and presumably does not hinder the passage of germinants, which must penetrate as far as their receptors in the inner membrane. Further study regarding the presence, functionality and in particular the permeability properties of the outer spore membrane would therefore be of interest with regard to spore resistance and susceptibility to biocides and also with regard to permeability to germinants. Cortex and germ cell wall The spore cortex is composed of PG that, whilst broadly similar to vegetative cell PG, has some notable spore-specific modifications, notably the complete absence of teichoic acids from the N-acetylmuramic acid (NAM) residues in spore PG (Atrih et al. 1996). Vegetative cell PG from B. subtilis cell walls consists of glycan chains of alternating N-acetlyglucosamine (NAG) and NAM residues (Warth and Strominger 1971). Approximately 40% of the NAM residues in vegetative cell PG are cross-linked to other glycan strands via their peptide side chains (Warth and Strominger 1971; Popham and Setlow 1993), whilst around 2% are complexed with teichoic acids (Atrih et al. 1999a). In spore cortex PG, approximately 50% of the NAM residues present have no peptide side chains and instead are cyclized to form the spore-specific residue, muramic-δ-lactam (M-L; Fig. 3), whilst a further 25% of NAM residues have only an l-alanine side chain (Warth and Strominger 1969, 1972). Both of these NAM modifications preclude the formation of peptide cross-links between glycan strands (Fig. 3); indeed, only around 3% of spore NAM residues contain peptide side chains that are cross-linked (Popham et al. 1996). Figure 3Open in figure viewerPowerPoint Schematic representations of spore peptidoglycan structure. G, N-acetylglucosamine; M, N-acetyl-muramic acid; M-L, muramic-δ-lactam; Ala, l-alanine; peptide, tri- or tetrapeptide side chains that can form cross-links between glycan strands. Modified from Popham (2002). It has been speculated that M-L, being a structure unique to spore cortex PG, was in some way important in attaining spore dormancy and/or resistance properties (Popham 2002). However, mutants lacking a functional cwlD gene that encodes an autolysin of the N-acetylmuramoyl-l-alanine amidase class (Sekiguchi et al. 1995; Popham et al. 1996) produce cortex PG that lacks M-L, and yet cwlD spores maintain full spore dormancy and have normal heat resistance (Atrih et al. 1996; Popham et al. 1996), although they cannot complete germination and outgrowth. The low level of cross-linking in spore PG has also been identified as a possible mechanism responsible for attaining and maintaining maximum core dehydration, a hypothesis referred to as the contractile cortex concept (Lewis et al. 1960). More recent studies have demonstrated that the level of cross-linking in spore PG does not alter spore dehydration (Popham 2002). The PG from spores of other organisms, including B. megaterium, B. cereus, Bacillus sphaericus (now Lysinibacillus sphaericus), Bacillus stearothermophilus (now Geobacillus stearothermophilus), Clostridium botulinum and Clostridium sporogenes (Warth and Strominger 1969; Atrih et al. 1999b; Atrih and Foster 2001), has also been analysed in some detail and was in all cases very similar to that of B. subtilis. The only subtle difference noted was the de-N-acetylation of an amino sugar, most likely the glucosamine, in B. cereus, B. sphaericus and Cl. botulinum, which was not present in B. subtilis (Atrih and Foster 2001). Bacterial spores contain another PG structure, the germ cell wall (GCW), which becomes the cell wall as the spore undergoes germination and outgrowth. Structural differences between GCW and cortex PG, in particular the absence of M-L, allow the selective degradation of the spore cortex, but not the GCW during spore germination; specifically, the M-L in cortex PG is a key substrate specificity determinant for recognition by cortex-lytic enzymes during spore germination (Atrih et al. 1998). There is currently no indication that the GCW plays any great part in spore resistance properties. Cortex-less mutants of spore-forming bacteria have been produced, for example spoVD and spoVE mutants in B. subtilis, which apparently lack any/most of the cortex (Piggot and Coote 1976; Daniel et al. 1994). However, the resistance properties of these mutant spores have not been studied. Imae and Strominger (1976) used a conditional cortexless mutant of B. sphaericus in which the amount of cortex present was alterable by changing the level of meso-diaminopimelic acid in the growth medium to show that a critical mass of cortex was required for resistance to xylene, octanol and heat. However, owing to the complex nature of spore development, they were unable to attribute resistance specifically to the cortex itself. Inner membrane Several studies have demonstrated that the dormant spore is remarkably impermeable, as small molecules such as the uncharged lipophilic molecule methylamine and even water permeate into the spore core only slowly (Setlow and Setlow 1980; Swerdlow et al. 1981; Sunde et al. 2009). This characteristic has led to the suggestion that the spore inner membrane must differ significantly from the vegetative cell plasma membrane, and this may be responsible for the low spore inner-membrane permeability. However, the lipid composition of the spore’s inner membrane appears very similar to that of the vegetative cell plasma membrane in both B. megaterium where both membranes contain principally phosphatidylglycerol, diphosphatidylglycerol (cardiolipin), phosphatidylethanolamine and glucosaminylphosphatidylglycerol (Bertsch et al. 1969; Scandella and Kornberg 1969; Racine and Vary 1980), and B. subtilis where both membranes contain primarily phosphatidylglycerol, cardiolipin and phosphatidylethanolamine, although vegetative cell membranes contain much more diglucosyl diacylglycerol (Griffiths and Setlow 2009). In contrast, the vegetative cell membrane and spore inner membrane have very different protein compositions, in particular as the spore’s inner membrane contains germinant receptors and SpoVA proteins not found in vegetative cells (Setlow 2003). However, the precise composition of the spore inner membrane does not provide any obvious reason for this membrane’s low permeability. Biophysical analysis of the inner membrane in intact spores (Cowan et al. 2004) has further suggested that it is not the lipid content of the inner membrane that confers its remarkable impermeability, but the state of these lipids in the membrane. By incorporating fluorescent lipid probes into the membranes of dormant spores of B. megaterium and B. subtilis, it was demonstrated that lipids located in the inner spore membra