Bacterial Spores: Current Research and Applications

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1.Water in the bacterial spore

In both prokaryotic and eukaryotic biological systems, decrease in water content is fundamental in the shift from active cell life and dormancy and contributes to resistance towards adverse conditions. Thus, dehydration is one of the key factor responsible of the extraordinary resistance of bacterial spores. This process is reversible and rehydration is fundamental to restore normal cell life (Powell and Hunter, 1955).

At the very first attempts to study bacterial spores, numerous doubts were raised about spore permeability and its dryness to front heat insults. Spores were, in fact, nonstainable, highly refractive and, often, nonwetable. The preliminary hypothesis was that spores were dry and impermeable to water (Fischer, 1877). During the end of 1880's, on the basis of water vapour sorption by bacterial spores, researchers attributed their heat resistance to the fact that spores had a lower moisture content than vegetative cells (Drymont, 1886). Subsequently, different methods have been used to measure water content in spores and in vegetative cells. Henry and Friedman (1938), in their pioneering work using cryoscopic methods, affirmed that vegetative cells and spores contained the same amount of water. Based on hygroscopic properties, Virtanen and Pulkky (1933), affirmed there were no differences between spores and vegetative cells. It was first across 1950-1960, that numerous scientists marked the difference between the hydration state of spores and cells (Stewart and Halvorson, 1953; Waldham and Halvorson, 1954) demonstrating that the vegetative cells were more hygroscopic than spores. The same theory was supported by Murrell and Scott (1958) that, using D2O, measured the 96% of freely isotope exchange with spore water and demonstrating that almost all the water in spores was exchangeable with external water in the environment. They added also that the dry space in the spore could be approximately the 30-40% of the volume of the spore. It was particularly studying the heat resistance of spores of Bacillus and Clostridium that some hypothesis were made; i) spores, differently from vegetative cells, contain thermoresistant enzymes (Green and Sadoff, 1965); ii) spores have a high refractive index, suggesting that they contain a large amount of refractile material or that the water content of spores is low (Leman, 1973; Ross and Billing , 1957); iii) due to spore apparent gravity, it was postulated that these dormant cells present a condensed structure with an atypical low water content, compared to the vegetative cell (Lewis et al., 1965). While the first hypothesis wasn't confirmed by experimental data, most of the pioneering studies of 60' and 70' were aimed to address three main questions: first, the water content of spores and its role in dormancy and resistance, second the mechanisms which lead to spore dehydration and third, the permeability of spore envelope to water and solutes.

Water content of spores

Precise determination of the total water content of the fully hydrated spores was accomplished at the time by using different methods. Black & Gerhardt (1962) demonstrated in Bacillus that the water content of spores was lower than in vegetative cells, being respectively 65% and 76% of the total cell weight. Other studies in Bacillus indicated that the water content in total was depending on species and varied among 0.45 and 0.65 g of water per g of wet spore (Marshall & Murrell, 1970; Watt 1981). At first, such a water content appeared to be in contrast with the high refractive index of spores and the strong gravity observed. However, this was justified if water would be very unequally distributed within spores, with more in an exterior compartment and less in an interior compartment. Using laser difractometry, Ulanowski et al. (1987) and De Pieri et al.(1993) measured water content and size of two layers of Bacillus sphaericus spores, which they recognized as the cortex and the core respectively on a total spore crops. They found a water content of ≈0.6 g/ml in the cortex and only ≈0.3 g/ml in the core, with only a few modifications due to growing temperature.

These observations contributed to assume that spores are partially dry and this feature is prerogative to dormancy and resistance (Gould, 1977). Most of the investigations about water properties and content in spores were made on members of Bacillus genus (Black 1961; Leuschner 2000; Lewis 1960; Neihof 1967; Plomp 2005; Rubel 1997; Setlow 1980; Sunde 2009; Westphal 2003). Ishihara and colleagues (1994) studied the free and bound water in vegetative cells and spores of B. subtilis using time-domain reflectometry demonstrating that spores contained only two thirds of the two types of water compared to vegetative cells.

The mechanism which leads to low water content of spore was firstly investigated by Lewis et al. (1960), that proposed the mechanical pressure created by spore cortex contraction during sporulation as responsible of forcing out water from the tight core. A different mechanism was hypothesised by Alderton & Schnell in 1963 which pointed out that the core dehydration is the result of mechanical force exerted by cortex expansion. At the same time, these studies raised the problem about the presence of specific envelopes highly impermeable to water (Lewis et al., 1960) and on the mechanism which leads dehydration. Both these hypothesis were confuted when different authors demonstrated that dormant spores are permeable to water and small solutes, and that this is influenced by their molecular weight, charge and lipophilicity (Black and Gerhardt, 1961; Black and Gerhardt, 1962; Black et al., 1960; Gerhardt and Black, 1961; Lewis et al., 1960). Later, Marshall and Murrell (1970) in experiments of H2O/D2O exchange demonstrated that almost 97% of spore water is exchangeable with the environment. Therefore, the impermeability theory was completely excluded, but the nature of water distribution in the spore structure became complicated and remained a wide and interesting area of study. In the following paragraphs these themes are discussed in detail.

1.1 Core water content

The unusual capacity of spores to remain dormant for years and to resist to physical and chemical insults, was mostly attributed to the dehydration of the spore protoplast which appeared to be encased within a more robust integument even when spores are exposed to excess water. This could help to understand why spores can stay dormant for long times. The driving evidence for this idea was the historical light-microscopic image of the bacterial spore with a refractive core and a non/refractive surroundings which presumptively corresponded to the protoplast and the exterior integument respectively (Beaman et al., 1984). A great difficulty was initially represented by the effort to measure the distribution of water content because the protoplast could not be isolated without loss of its intrinsic in situ properties. Moreover, with the finding that the spore possessed not only an inner membrane surrounding the cytoplasm but also an outer membrane protecting the cortex (Crafts-Lighty & Ellar, 1980), the question became more complex.

The high resistance of spores to environmental insults, if compared to vegetative cells, was traditionally ascribed to a sort of molecular stabilization in the core caused by the presence of specific compounds such as dipicolinic acid (Beaman, 1986) and to a low water content of spore core. Thus the core, also in fully hydrated spores, has a low water content (Beaman & Gerhardt, 1986). It has been estimated that the water content of the spore protoplast varies among Bacillus species between 26 and 55% of the total wet weight basis, depending on the species (Nakashio & Gerhardt, 1985; Lindsday et al., 1985; Beaman et al., 1984; Beaman & Gerhardt, 1986; Gerhardt & Marquis 1989; Leuschner et al., 2000). The quantity of free water in the spore core is also particularly low and, for this, the consequent movement of macromolecules is strongly inhibited (Cowan et al., 2003). The low water content is essential for spore's metabolism dormancy (Gerhardt & Marquis 1989), but little is known about the cell's behaviour during sporulation that allows the core rapid reduction of water. As seen by Cowan and Setlow (2003), during the germination process the water uptake happens in the very first minutes, so that macromolecular dynamic and enzymatic activity are restored.

Other than water, the physical space in the core is quite completely occupied by the genome, about half of the spore's proteins, ribosomes, tRNAs and a large depot of calcium and other divalent ions bound by DPA (Gerhardt et al., 1989; Setlow 2006). Dipicolinic acid has been considered to be one of the major constituent of the spore core, with a quantity of 5-20% of the dry weight of spores of both Bacillus and Clostridium species, where it is most likely chelated 1:1 with divalent cations, largely Ca2+ (Gerhardt and Marquis 1989; Murrell, 1969; Lenz & Gilvarg, 1973; Kozuka et al., 1985; Setlow B. et al., 2006). DPA is produced only in the mother cell during sporulation, it is captured by the forespore and excreted during the first steps of germination. At now, the precise state of the DPA in the core is not known. What it is more clear is that the accumulation of high amount of DPA in the spore core is responsible for some of the reduction in core water content during sporulation, and DPA also plays a significant role in the UV photochemistry of spore DNA (Setlow, 2006).

Gould in 1986 proposed that the moisture content of core is so low to be comparable to a vitrified solid state (glassy), responsible of the high resistance of spores. This hypothesis was supported by differential scanning calorimetric (DSC) and NMR studies (Ablett et al., 1999) which provided evidence that the core region of spore is in a low moisture glassy state. This theory was consistent with the mechanism developed by anhydrobiotes, such as plant, seeds, pollen and fungal spores. In these organisms the dormancy is achieved by increasing the cellular viscosity and transforming the cytoplasm in a glassy state, conferring long term survival in a dry condition. The formation of intracellular glasses is the result of the accumulation of disaccharides and other compatible osmolytes (Crowe et al., 1998; Buitink and Leprince, 2008). If so, spore dormancy could be explained as a consequent suppression of chemical reactions and extreme slowness in denaturation and metabolic processes.

Other studies supported the glassy state hypothesis: experiments determining the dielectric permittivity (Carstensen et al., 1979) and the electron paramagnetic resonance spectra (Johnstone et al., 1982) demonstrated that many ions in the core are immobilized. Using NMR spectroscopy Leuschner et al. (2000) pointed out that core's storage of pyridine-2,6-dicarboxylic acid (DPA) is in a glassy solid state. Moreover, experiments done in B. subtilis expressing green fluorescent protein, demonstrated that in the spore core the proteins are four orders of magnitude less mobile than in the cytoplasm of the vegetative cell (Cowan et al., 2003; Gould, 2006; Newsome, 2003). More recently, Ghosal et al. (2010), accordingly with Black and Gerhardt (1962) and Leuschner and Lillford (2000), showed clearly that the core is permeable and behaves as an insoluble heat-stable gel which exchanges water with the external environment.

Water mobility in the dense spore core of B. subtilis has been recently studied using NMR technique that has allowed to determine water dynamics. This has been done quantifying water mobility, in the different spore compartments, by the dynamic perturbation factor (DPF) that is the rotational correlation time averaged over all water molecules in a spore's region considered divided by the same quantity in bulk water (Sunde et al., 2009). The DPF value permitted the evaluation of the water molecules external to proteins and other solutes but not of water inside polar cavities of proteins or at the interfaces of associated macromolecules (Mattea et al., 2008). in addition, a symmetric flip motion of water molecules bounded to Ca2+ bivalent ions in the core has to be considered to obtain a proper water mobility evaluation (Denisov and Halle, 1995).

According to the "core glassy state" hypothesis (Gould, 1986; Sapru, 1993; Ablett, 1999), the core water has been defined as a "rigid lattice" (Sunde et al., 2009). In effect, it was found that the core water was only 30-fold less mobile than bulk water, observation that made conclude that the core is not in a glassy state (Sunde et al., 2009). As a matter of fact, water in biological systems is often evaluated as "bound" and "free" water, presuming the fact that if water is bound, it should be strongly rigid and immobile. This misguiding concept could be confuted if we think at the fact that water molecules prefer to be in a bound state rather than in a chaotic state without interactions (vapour); so that, hydrogen-bounded condition doesn't immobilize water molecules but, on the opposite, core water could be defined together bound and mobile at the same time.

From these recent advances, it has been concluded that water in the core hydrates preferably non protein molecules. As a consequence, core proteins are surrounded by at most one water layer and this dehydrated state is probably sufficient to alter their active folding and stop their metabolism (Gregory, 1995; Setlow, 1994). Bacterial spores could be resistant stopping their metabolism without immobilizing the solvent (glassy state), but simply stabilizing proteins against their thermal denaturation and maintaining their unfolded state (Gregory, 1995; Rupley 1991). However, minor changes in the water content could affect this protection (Eijlander 2010; Mafart 2010). As a consequence, if denaturation should be reversible, there was not possibility for the spore to be killed. Only when the level of hydration is high enough to allow intermolecular disulfide exchanges, proteins unfolding translates to irreversible aggregation that affects the spore viability (Klibanov 2004). This last position agrees also with Raman spectroscopy experiments made on B. subtilis spores (Zhang 2009), in which a heat activation at 70°C has been followed by partial but reversible protein denaturation.

Some points remain to be elucidated relevant to core water content; we know that the low water content in the core is responsible of heat resistance but we don't know the exact quantity of free water in the core; we ignore how much core proteins are hydrated and following which via, wet heat is able to kill spores.

Water mobility across the inner membrane (IM)

Numerous hypothesis point out that the spore's inner membrane (IM) is the major permeability barrier selecting the flux of chemicals and small molecules into and outside the core (Swerdlow 1981; Paidhungat 2002; Gerhardt 1971; Westphal 2003; Setlow 1980; Setlow 2000). The rare permeability of the spore IM to hydrophilic and hydrophobic molecules is, in this way, one of the factor that influences spore chemical resistance (Gerhardt 1972). It appears to be in a gel state (Cowan, 2004) and could have a protective role towards the core DNA, due to its low permeability to ionic species and even to small non ionic solutes (Setlow, 2006).

A very small molecule such as methylamine has been used to study the IM permeability, observing that its passage through the membrane was very slow; at the same way water crossed the IM very slowly (Setlow and Setlow 1980; Swerdlow 1981; Westphal 2003; Cortezzo 2004; Cortezzo and Setlow 2005).

The IM in the dormant spore is very tightly packed and it is composed by lipid molecules which are largely immobile and create a crystalline structure (Cowan et al., 2004). Although, the great part of lipids in the IM are immobile, there is an important ratio that remain mobile. These authors proposed that the low diffusion value (D) of spore inner membrane is strictly related to the organisation of mobile and immobile lipids. The first hypothesis recognizes that the two leaflets of the IM present a different mobility. This differential lipids mobility could be due to the low water content in the core compared to the cortex area (Gerhardt 1989). This leads to lower mobility of lipids in the inner part of the membrane towards the core. When germination occurs, the core water grows, restoring the normal lipids mobility (Gerhardt 1989; Popham 1996; Setlow 2001). A second theory is that the IM is organised in different domains which can contain respectively mobile and immobile phospholipids. This view is supported by studies on lipid and protein composition of B. subtilis and B. megaterium IM. Ellar et al. (1978) reported that the IM has a strongly higher protein content than the growing cell and this might contribute to establish large areas of ordered domains under the special dehydrated core state. These domains have been distinguished in growing cells of B. subtilis (Vanounou 2003), while in B. megaterium the IM has been observed as composed by two fractions of similar lipids but with different protein composition (Swerdlow 1984).

The unusual low permeability of IM was initially attributed to some abnormalities in the lipid composition, but deeper studies demonstrated that the IM lipid structure was very similar to that of the plasma membrane of growing cells (Cortezzo 2004; Cortezzo 2005). Additionally, the spore's IM has the particular behaviour to vary its volume according to the different steps of spore-formers cell cycle; in fact, it decreases as much as two-fold late in sporulation and increases up to two-fold in the first minutes of spore germination, when the spore's peptidoglycan cortex is degraded and the outgrowing cell expands (Warth 1978; Popham 1996; Gould 1969). This increment in volume happens without new membrane synthesis and no ATP production, restoring the permeability to the germinating spore's plasma membrane (Swerdlow 1981; Gerhardt 1971; Setlow 2001; Setlow 1970).

Considering that the spore's IM is: (i) the major barrier avoiding the passage of small molecules such as DPA that are essential for spore resistance; (ii) the position where are found a number of proteins involved in the triggering of spore germination; and (iii) where membrane active agents such as cationic surfactants act to trigger spore germination (Setlow 2003). It seems probable that a deeper knowledge of the structure of the inner membrane will give us new insight regarding not only spore resistance, but also spore dormancy and spore germination.

The cortex and its osmoregulatory function

The cortex, a layer composed by a cross-linked peptidoglycan matrix, is the most hydrated region of the spore. Gould and Dring (1974, 1975a) underlined that the polymer in this spore layer is water-filled and highly expanded if compared to the dense water-depleted core. Such a structure was in line with the high refractive index and peculiar gravity of the bacterial spores, but also with the total high water content of these dormant cells, compensating the water-deprived state of the core.

The spore core dehydration state is in part dependent on the inner forespore membrane but greatly also on the spore cortex (Popham et al., 2002). Which is the process that allows to maintain a perfect and equal distribution inside the spore's integuments? The first studies postulated that the leading force responsible of this behaviour was mainly osmosis and the cortex was designated as an osmoregulatory organelle (Gould & Dring 1975a). Since the cortex is mainly composed by electronegative peptidoglycan, it is supposed to contain also positive counterions conferring therefore an osmotic potential calculated appreciatively around 2 MPa or more. In this way, the expanded cortex should be in osmotic equilibrium with the dehydrated enclosed core through the inner membrane in such a system that regulates the passage of water from the core to the cortex or vice versa until the total osmotic pressure is balanced. The equilibrium between the two compartments is dependent also on the osmotic activity of the core molecules. Since in the cortex usually reside molecules of high molecular mass or salts as calcium dipicolinate, calcium salts of other weak acids like glutamic, sulpholactic or phosphoglyceric, the osmotic balance is in the direction of a high water content cortex surrounded by a lower water protoplast content. If spores are suspended in water, they are stimulated to absorb it into the cortex; successively, the hydrostatic pressure inside the spore will balance the osmotic pressure to set the system in perfect equilibrium. Different experiments have been conducted to validate this new hypothesis. Spores with modified coats have been produced using chemical treatments so that they were permeable to multivalent cations; all these spores were heat sensitive if in the medium were present high concentrations of salts of calcium and of other multivalent cations (Gould & Dring 1975a). The effect caused by the bivalent cations that succeed in passing through the spore coat was a strong interaction with the electronegative peptidoglycan of the cortex giving rise to its collapse, the replacement of mobile counterions and a consequent rehydration of the core that becomes heat sensitive. The theory validation was confirmed by the opposite reaction of full heat resistance when spores are suspended in solutions of sugars enough concentrated to establish anew, by osmosis, the protoplast dehydration (Gould 1977). Anyway, this mechanism should be in part at the basis of the germination process. In fact, spores that have entered germination become more and more tender to heat than the ungerminated ones, because of core hydration. This water flux can occur within very short periods of time (in less than 1 minute) and results in a wide expansion of the core due obviously to water. As seen before, at this time, the inner membrane has to increase almost four-times in area because the spore doesn't synthesise in this step new plasma membrane (Dawes and Halvorson, 1972).

Water dynamics through spore coat

Proceeding towards the interior, the spore's first line of defence is the coat, the membrane composed of multiple protein layers which behaves as a barrier able to select which molecules could pass through the spore. In the coat of B. subtilis at least 50 different individual proteins have been found into two major layers (Driks, 1999, 2002; Lai et al., 2003). Only a small subset of coat proteins play essential roles in the determination of coat morphology (Driks and Setlow, 2000; Driks, 2002; Henriques et al., 2004). Several studies have assigned to coat proteins an important role in defending spores against a number of chemicals, including sodium hypochlorite, chlorine dioxide, ozone, organic solvents, and lytic enzymes that can damage the spore cortex. It was observed that chemically decoated wild-type spores were much more sensitive to these agents than intact wild-type spores (Setlow 2000; Setlow 2006; Young & Setlow, 2003;Young & Setlow, 2004). Differently, the coat is not responsible for protection against heat, radiations and some other chemicals (Driks, 1999; Nicholson et al., 2000; Setlow 2000; Klobutcher et al., 2006). No specific coat protein has been discovered to be responsible of spore resistance to these chemicals and the role of the coat seemed to be that of a 'reactive armour' (Setlow 2005) acting to filter these chemicals when they enter into the spore.

Studies conducted on functions of Bacillus spore coat proteins revealed an unexpected grade of disorder in this structure (Kim et al., 2006). This opens the discussion on how a discrete biological disorder in the network of coat proteins not only can be tolerate, but also may help to maintain an important function such as the ability of the spore to flex and move. This means that every coat protein could be able to bind to each other, presumably through relatively low-affinity, low-specificity interactions and this may contribute directly to function, by permitting structural flexibility.

If compared to cortex and core compartments, few water is present in the spore coat, as demonstrated in B. stearothermophilus by calculating the dry mass, dry volume, wet mass, and wet volume of the spore and its components (Algie & Watt, 1984). This view was confirmed by similar results achieved in B. subtilis and B. cereus T (Algie, 1984). More recently the water content of coat has been investigated by means of a NMR approach called magnetic relaxation dispersion (MRD) profile (Sunde et al., 2009), a method which determines the water 2H and 17O spin relaxation rates in D2O-exchanged B. subtilis spores. This study, aimed to calculate the water distribution in the different spore compartments, demonstrated that the internal water fraction of coat is limited, accounting for the 0.14% of spore water trapped inside proteins. This slightly lower value may evidence a lower internal-water content of coat proteins or a larger fraction of internal water molecules with residence times >10 µs in the extensively cross-linked coat proteins.

For what concerns permeability, the spore coat is a semi-permeable layer allowing to molecules with a molecular mass less than 5 kDa, including water, and allows the passage of molecules to deeper layers, particularly through the inner membrane that contains specific receptors (Driks, 1999; Henriques & Moran, 2007; Setlow, 2003). Westphal et al. (2003) measured as the humidity influences spore response of B. thuringensis in terms of water flux and swelling of dried spores exposed to humidity. Using an automated scanning microscope and observing the kinetics of size changes at different values of relative humidity, they demonstrated a rapid diffusion of water into the spore coat and cortex layers with a time scale close to 50 seconds. In the same experiments the time for diffusion of water into the spore core was found to be much longer, being approximately 8 minutes. Taking in account the time needed for water diffusion and the estimated thickness of coat, cortex and core, these authors proposed a simplified model where water diffusion time through the spore layers is proportional to square root of the summed radial distance of the different spore's layers.

Clear explanation of the precise function of the spore coat has been prevented by the difficulty to obtain spores without most coat layer. Ghosh et al. (2008) studied a B. subtilis mutant strain lacking of CotE, a protein essential for assembly of many coat proteins as well as the outer coat layer, and GerE, a DNA binding protein acting in the mother cell compartment late in sporulation and which positively regulates the expression of genes coding for a number of proteins in the spore coat. These spores, which present a modified coat, had a normal core water content supporting the hypothesis of the limited action of coat in reducing core water content during spore development, which is the primary cause of spore resistance to wet heat.

Unexpectedly, in fact it was found that the water permeability of the IM is greatly enhanced in these coat-deficient spores.

The coat seems only to act as a detoxifying filter for chemicals with its large amount of proteins but it has no involvement in resistance to heat and in water flux through the spore.

1.5 Exosporium: a semi-permeable barrier

As the outermost layer of the spore, the exosporium is the primary site of contact with the environment, including host defences in case of pathogenic spore-formers (Guidi-Rontani, 2001). This envelope acts as the source of surface antigens (Gerhardt, 1967; Steichen et al., 2003) and as a semi-permeable barrier that excludes large, potentially harmful molecules such as antibodies and hydrolytic enzymes (Gerhardt, 1967; Gerhardt & Black, 1961). It has a wide surface and, in some cases, a hirsute appearance, and it occupies a great part of the spore's volume.

Some strains of Bacillus lack of this outermost layer (Setlow, 2006). The exosporium was firstly studied in Bacillus cereus T, a strain frequently used in basic studies of spore resistance (Gerhardt & Ribi, 1964; Gould, 1977; Beaman et al., 1982). This strain has a superficial, loose-fitting, bilayered exosporium which, when spores are suspended in solution, is expanded and when spores are packed, tends to collapse. This is explained by the presence in the space between exosporium and spore coat of liquid, macromolecules and in some cases protein crystals, suggesting a free flow of water through the exosporium.

An additional factor which may influence water permeability is surface hydrophobicity of spores, which depends mainly on its exosporium and its components. It's well described that hydrophobic features of spores are strictly related to the presence or not of the exosporium (Takubo et al., 1988; Koshikawa et al., 1989). However, both dimensions and hydrophobicity of the exosporium were shown to be strain dependent (Tauveron et al., 2006). For example, spores of Bacillus subtilis, B. licheniformis, and B. macerans do not have distinct exosporium layers and are less hydrophobic than are exosporium-containing B. cereus, B. brevis, and B. thuringiensis spores (Koshikawa et al., 1989). Specific information on structural component(s) responsible for the hydrophobic behaviour of spores is limited. In B. anthracis the collagen-like protein (BclA) present in the exosporium markedly influenced the spore hydrophobicity, probably affecting the access of molecules to the internal spore compartments (Brahmbhatt et al., 2007). Moreover, the same authors observed that deletion mutant in bclA gene were less water repellent than wild-type. Differently, recent studies in B. thuringiensis subsp. israelensis spores, analysing the uptake of water by exposing dry spores to D2O vapour, demonstrate that exosporium is highly permeable to water (Ghosal, 2010).

Data available up to date indicate that the exosporium, due to its composition, elasticity and to the fact that it loosely surrounds the spore periphery often dissociating from the spore itself (Zaman et al., 2005), have a limited role in water and ions fluxes.