The chemical composition of spore has been studied extensively since 1950 with peculiar attention to dipicolinic acid and calcium content and distribution. In the last 50 years of studies, many efforts have concerned polymeric components of spore structure and their relation with resistance to physical and chemical exposure.
The capability of spore to strength to environmental stress is certainly related to water content, metal ions and dipicolinic acid (Tipper and Gauthier, 1972; Murrel 1969). In particular, mineralization of spores gives a relevant contribution to heat stability.
Chemical analysis of spore demonstrated a different composition in the diverse integuments and a more elevate mineral content in the protoplast (Scherrer and Gerhardt,1972; Scherrer and Shull, 1987). The minerals in spores seem to be in a crystalline phase forming complexes with spore polymers and low-molecular mass anions (Marquis & Shin, 1994).
The mineral constituents are mostly potassium, calcium, manganese, magnesium, copper, and phosphorus and related to the different phases of spore cycle, the calcium is the most abundant and "participant" (Thomas 1964, Curran et al. 1943; Powell and Strange, 1956; Vintner, 1957.
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The calcium is certainly the most abundant mineral present in the mature spores (2-3% Ca2+) in a molar a ratio of 1:1 with dipicolinic acid (Murrel, 1967). More recently, the calcium complex with DPA has been found to constitute the 10% of the total spore mass (de Vries, 2004; Scully et al., 2002). The Ca2+ content is variable and the amount in the spore depends on the availability of Ca2+ in the environment outside the cell. During germination the Ca2+-DPA chelated is excreted together. In opposite, during spore forming process, the Ca2+ and DPA are transported together from mother cell into the developing spore as complex Ca2+-DPA (Hintze & Nicholson 2010). The accumulation in mature spore of the chelated Ca2+-dipicolinate form is a final product, but calcium ions are transported from outside in unbound form in sporulating cells to be tightly bound with dipicolinic acid in the mature spore. Eisenstadt and Silver in 1971 proposed an active transport system of Ca2+ from outside the mother cell with internal accumulation following saturation kinetics, and it was suggested a membrane-situated calcium transport system. The calcium is chelated by DPA in the mother cell and finally is located inside the cortex or core (tutti e 2?) of the mature spore. It has been shown in Bacillus subtilis and Bacillus megaterium a carrier-mediated transporter of Ca2+ suggested as Ca2+/H+ exchanger driven by the electrical gradient over the plasma membrane (Seto-Young 1981) and according with these results, an antiporter Ca2+/H+ has been characterised from B. subtilis (Matsushita, 1986). In B. subtilis has been also suggested a contribution for calcium transport of P-type transport ATPase that provides the formation of resistant spores (Raeymaekers, 2002).
Murrel in 1969 reported accurate studies on mineral composition of spores in Bacillus and Clostridium and in table 1 are listed the inorganic elements as he showed:
Table 1 (Questa tabella l'ho riportata tale e quale per cui non so se si può mettere da rivedere)
Major inorganic elements in spores % dry wt
Average of 13 species and 14 unidentified strains (from Murrel W.G. 1969).
Table 2: Levels of monovalent and divalent ions in spores of various species. Values are reported in micromoles per gram of dry spores.
Table and data from Loshon 1993 and value from aStweart et al 1980, b,e,f,g,Loshon et al. 1993, cWarth 1978, dMurrel 1969.
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The elements present in spores in higher amount are Ca, K, P, Mg and Mn and sometimes are reported Cu and Si. Copper and manganese have been also detected in B. subtilis and B. megaterium in higher amount then in vegetative cells (Windle and Sacks 1963) with a role in inducing sporulation and increasing heat resistance (Charney et al., 1951; Weinberg, 1964; Roberts & Hitchins, 1969). A study performed by x-ray microanalysis in B. coagulans showed greater extent of calcium, manganese, and phosphorus detected in the core and the coat (Amaha et al., 1956?). This distribution of the elements was consistent with the results obtained in B. cereus with x-ray microanalysis where clearly almost all of the calcium, magnesium, manganese were confined in the core and most of the phosphorous were located both in the core and a part among cortex/coat interface (Stewart et al.1980); magnesium, seems to be essential for sporulation but not for heat resistance, interfering with Ca-DPA complex formation (Murrel 1969; Roberts & Hitchins, 1969). The sulphur was detected in all structures (REF..). Potassium is also implied in spore formation in some Bacillus species (Wakisaka et al., 1982). In B. coagulans spores, iron was revealed in the coat but not in the core and in C. botulinum is necessary for heat resistance (Sugiyama, 1951). Sulphur and phosphorus were located in the coat with larger distribution in the outer coat layers. Particularly, phosphorous is present in mature spore in relevant amount as 3-phosphoglyceric acid (Loshon & Setlow, 1993). Silicon was not detected in the coat, in opposition to what observed in B. cereus and B. megaterium but this was considered to be due entirely to contamination (Stewart et al. 1981).
The ions spore composition and the distribution in the layers is considered of fundamental relevance on spore formation, resistance, dormancy and germination. The localization of calcium in the core as Ca2+-DPA chelate has long been recognized where the molecule is largely accumulated (Eisenstadt and Silver. 1972, Stewart et al. 1980, Stewart et al. 1981, Gerhardt and Marquis 1989, Murrell and Warth 1965, Murrell 1967, Setlow 1994).
The metal content of spores depends on the cultural conditions and on the metal availability in the cultural medium during growth and sporulation (Slepecky and Foster, 1959). The higher is the concentration of metal in the sporulation medium, the larger is the incorporation in the spores of the relative divalent cations, more specifically Ca2+, Ni2+, Zn2+, Cu2+, Co2+, Mn2+. At high concentrations Zn2+, Mn2+ and Ni2+ competed for Ca2+ sites in the spores, but the content of the alternate metal was incorporated less than the decrease of incorporated Ca2+ (Murrell 1967). The spores grown at higher temperatures, showed higher calcium content. Furthermore, the spores containing less Ca2+ content were more susceptible to heat stress. A similar behaviour was observed when high concentrations in the growth medium of divalent ions, such as Zn2+, Ni2+ and Mn2+, results in substitution of Ca2+ in the spore. Thus, these spores the heat resistance is not re-established did not replaced it with the same efficiency (REFâ€¦). Studies on Sr2+ and Ba2+ as substitutes of Ca2+ divalent metal gave different response (Foerster & Foerster, 1966?). The spores obtained in presence of Sr2+ had less DPA and were less heat resistant whenever there was a quite relevant accumulation. The barium replacement of Ca2+ as the others divalent metals were less effective quantitatively and qualitatively. Moreover, many of the spores with Ba2+ content were not viable. These results showed the predominance of Ca2+ efficiency for DPA chelated complex although other divalent cations as Mg2+ and Mn2+ are present in significant amount (Murrell 1967).
Native dormant spore of B. megaterium studied in detail for dielectric properties, showed ions tightly bound and immobile in the core, conferring a small conductivity. The inner membrane, that in dormant spore forms a tight dielectrically homogeneous complex with the core, was considered a good electrical insulator that begins to affect dispersions when germination starts and ions become mobile. In decoated spores, it has been observed a raise of the conductivity levels in response to environmental ion flux from outside the spore. This attributes to the coat a role of insulating layer able to maintain its own fixed charge concentration with an order of 10 to 20 milliequivalents per liter and a corresponding number of mobile counterions. The presence in spores of two dielectrically effective membranes, plasma membrane and outer membrane, other than the presence in the core of dormant spores of ions tightly bound has been found both in B. cereus and in B. megaterium (Carstensen, 1979).
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Some metal ions, such as Cu, Mn, Fe, Co, Ni and Zn, required for the growth and survival of microrganisms, could behave, at the same time, as potentially toxic if their intracellular concentrations become too high (REFâ€¦). Among these, Mn plays a relevant role in spore cycle because its homeostasis is fundamental to initiate sporeforming activity in vegetative cells. The Mn2+ is necessary for the septum formation in the process regulated from pre-spore transcription factor ÏƒF and accumulated in the new spore. The mature spore has a storage reservoir of 3-phosphoglyceric acid (3-PGA) that is metabolized when the germination process starts. The accumulation of (3-PGA) is achieved during sporeforming activity by an important drop of pH reaching a value of 1 in the intracellular compartment by a concomitant dissociation of the Mn2+ cofactor from 3-phosphoglycerate mutase (Kuhn et al. 1995, Jakubovics and Jenkinson 2001).
Furthermore, it was shown the relevance of Mn2+ and superoxidismutase (SOD) during sporulation in B. subtilis. It is well known the role of the enzyme (SOD) in bacteria to defend the cell from reactive oxygen molecules (ROS). By using SOD negative mutant and medium depleted of Mn2+ it was observed the sensitivity of sporulating cells and premature spores to O2- . The Mn-superoxidismutase has been shown to increase the resistance of vegetative cells, sporulating cells and spores against oxidative stress (Inaoka at al. 1999). Recently Granger et al. (2011) has studied the role of Mn2+ and other divalent cations to understand the mechanisms that allow the increase of spore resistance to a variety of treatments including dry heat, gamma irradiation and UV. Actually these agents can damage the spore by generation of ROS, including H2O2 . It was demonstrated that high content of Mn2+ alone was not sufficient to protect the spore from external damaging conditions in particular from ionizing radiation and H2O2. The DNA-binding proteins the Î±/Î²-type small, acid soluble proteins (SASP) are very relevant to protect DNA from damage desiccation, wet heat, dry heat, H2O2 and gamma radiation (Moeller et al. 2008) together the high content of mineral ions in the core chelated with DPA, the effect of protection of the thick coat and the low level of water content (Magge et al. 2008, Settlow 2006). Mn2+ complexed with DPA and with or without ortophosphate was demonstrated to protect the restriction enzymes in particular from ionizing radiation. The most effective protection against ROS was purchased with mixture Mn2+, DPA, Ca2+ and orthophosphate protecting the ROS-sensitive enzyme BamHI (Granger et al. 2011). Studied performed in B. subtilis and B. megaterium to establish the hierarchy of mineralization in spore for protection against moist heat damage shown the most efficacious to be Ca2+ followed by Mn2+, Mg2+ (and K+). The Na+ and K+ resulted give not reliable protection. Ineffective protection resulted when the spores were remineralizated with mineral usually present in trace as Li, Sr, Cu and La (Marquis and Shin 1994). It has been suggested the efficacy against specifically ROS damages of Mg2+ and Cu2+, in particular the accumulation of Mn2+ replacing Fe2+, to protect the active sites of enzymes from oxidative damage (Anjem et al 2009).
Cations and their role in spore structure
The amount of monovalent and divalent cations were in some extent similar in spores of Sporosarcina, Clostridium and Bacillus species, suggesting that the levels of these small molecules in spores are comparable in all Gram positive organisms. This assumption reflects important and conserved characteristics of the sporulation process and dormant spores (Loshon & Setlow, 1993). The importance and significance of ions for the spore life cycle has been underlined with many studies in the years but many mechanisms are not yet clear (xxxx). The calcium is the cation in most relevant amount in the spores because is chelated with dipicolinic acid in the core.
The spore allows ions exchange through its different layers, membranes and the structures core, cortex, coat. In the spore there is a ions flux by vapour water permeation and ions diffusion trough channels in the structure of hydrated ions. By using nuclear magnetic resonance spectroscopy it has been shown in mature spore of B. subtilis that water affects the phosphorous mobility. An increase of the hydratation state has resulted in increase of the phosphorous and carbon mobility by a migration between the inner compartments of spore to outside environment. The experiment was performed in freeze dried spores where the phosphorous was detected as non mobile molecular component. Rehydration of dormant spores increased the molecular mobility of phosphorous and carbon. In opposite DPA remained immobilized in the core, probably in a water-insoluble network (Leuschner and Lillford, 2000).
The permeability to water vapour and solutes depends on the molecular weight, the charge of ions and lipophilicity (Gerhardt P.and S. H. Black, 1961). The germination is a complex process and is characterize by the release of H+ and monovalent cations such as K+, Na+ and Zn2+. When the germination starts, the ions flux moves across the spore membrane (teguments), dipicolinate and calcium are excreted and, at the same time, monovalent cations as potassium, sodium, xxxx permeate into the core through the exosporium, coat and cortex.
The spore has been for long time considered impermeable, since many studies and the use of different techniques and experiments have demonstrated as the entire spore, including the core is permeable to water and other molecules (Black and Gerhardt, 1961; Black and Gerhardt, 1962; Black et al., 1960; Gerhardt and Black, 1961; Lewis et al., 1960). Recently Ghosal et al. (2010) applied successfully high resolution secondary ion mass spectrometry (NMR) to analyze the spatial distribution of permeating water vapour and ions in B. thuringiensis subsp. israeliensis. The experiments were performed by exposure of the spores to cations and anions and in dehydrated and aqueous conditions. To study the cations flux in the spore, Li+ and Cs+ were selected because these cations are usually not present in the spores. The absorption of LiF did not affect the percentage of phase-bright spores therefore did not induce germination process. The Li+ permeated the entire layer of the spore, showing that the core of dormant spore was subjected to flux of water and small ions as Li+. The amount of Li+ after three days of exposure increased of 104 compared with not exposed spores. The NMR analysis allowed to show the distributions of Li+ concentration in the spore structures, that was higher in exosporium, coat, and cortex and relative into the core (Ghosal et al. 2010). E il Cs?
The efficiency of certain bivalent cations to permeate and form a complex with dipicolinic acid was studied in B. megaterium subsp. texas by using the technique of converting spore in hydrogen form (H-spores) by titration to pH 4 with HNO3. Calcium, strontium and barium were the bivalent cations interchangeable in the formation of endotrophic spores and the efficiency of spore germination rate were closely related. In opposite magnesium and sodium were less efficient in the formation of spore with capability of germination while nickel, cobalt, cadmium, zinc were not effective. The importance of strong electrolytes as sodium and potassium phosphate buffers used for germination test was confirmed in the experiments underlying the role played by the uptake of exogenous strong electrolytes; in fact in acidic conditions without the presence of sodium phosphate buffer, spore failed germination (Rode and Foster 1962, Rode and Foster 1966).
The release of Ca pyridine-2,6-dicarboxylic acid and Zn2+ commences immediately on addition of germinants (Johnstone 1982)
Anions and their role in spore structure
Anions uptake studies were carried out by NMR analysis when the Li+ and Cs+ cations were used to analyze the fluxes in B. thuringenisis subsp. israelensis (Ghosal et al. 2010). The spore were exposed to LiF or CsCl to study the uptake and localization of anions and cations. Chloride (Cl-) were localized more abundantly in the outer structure of spores, exosporium, coat and cortex while most fluoride (F-) was localized in the core. In effect Cl- and F- were detected in abundance in the spore without previous exposition because these elements are ubiquitous in the natural environment and in the medium used for sample preparation for the experiment. The fluoride concentration was shown to be in equilibrium with the LiF concentration of solution outside of spores and the fluxes and redistributions of F- in the spores was strictly related to the exposure treatments. Furthermore the Cl- content of spore changed during the experiments in relation to exposure of different concentrations of CsCl and Cl- was distributed in the outer part of spores; it was shown as washing treatments can remove accumulation of anions or contaminants in the spore outer structures. The different distribution of F- and Cl- has been suggested due to the different affinity of F- to calcium (Ghosal et al. 2010). Moreover the anion Cl- has been demonstrated to be a germinating agent in B. megaterium after exposure of spores to CsCl (Rode and Foster 1962).
Ions fluxes during sporulation, dormancy and germination
Roles of ions in sporulation and in the developmental cycle of spore formers bacteria
Several experiments have shown that there is a significant variability in ions incorporated into spores depending on the mineral composition of the medium (Amaha & Ordal 1957; Aoki & Slepecky 1973; El-Bisi & Ordal 1956; Krueger & Kolodziej, 1978; Levinson & Hyatt, 1964; Mallidis & Scholefield, 1987; Slepecky & Foster, 1959; Marquis 1989; Murrell, 1967). Sporulating cultures have a strong tendency to accumulate metals, including Ca, Zn, Mn, Ni, and Cu, and that the ion content of the spores affected heat resistance (Levinson and Hyatt, 1964). The very first hypotheses about the specific functions of metal ions in sporulation described their probable role as catalytic activators of the various enzyme systems necessary for sporulation which involves protein degradation and resynthesis (Kolodziey 1962). Kolodziey and Slepecky, in 1964, analysed the sporulation cycle in Bacillus megaterium finding traces of metal cations connected to the phenomenon. Using a purified medium supplemented with copper for sporulation they found in spores traces of the following metals: iron, 0.5,ug/ml of ferrous ion; zinc, 1.1 ,Ag/ml of zinc ion; manganese, 0.037 pg/ml of manganese ion; and calcium, 0.9 ,g/ml of calcium ion. Determining the approximate time required by the cell to use these ions, they postulated that copper could be one possible requirement for sporulation in B. Megaterium and then in B. cereus var. mycoides and var. albolactis; this role could be highlighted by the previous demonstration of its presence in vegetative cells and spores by spectrochemical analysis (Curran, Brunstetter, and Myers, 1943), colorimetric means (Slepecky and Foster, 1959), diphenylcarbazone staining (Troger, 1959), and electroparanmagnetic resonance studies (Windle and Sacks, 1963). Moreover Powell and Strange (1956) demonstrated with their data a strong chelation between DPA and copper. Copper behaves, in fact, as a cofactor or a metal ion activator in many animal, plant, and bacterial systems (McElroy and Glass, 1950; Underwood, 1959; Stiles, 1961). They observed that a concentration within 0.025-0.10 Âµgm/ml range of CuSO4âˆ™5H2O stimulated maximum sporulation. Lower concentrations limited sporulation while greater resulted in incomplete sporulation within 62 hours.
Contrary to what observed until that time, Kihm et al. in 1990 conducted the first studies on Clostridium botulinum sporulation influenced by transition metals, particularly Cu and Zn. During metabolism, clostridia produce organic acids that assist in solubilizing metals and making them available for growth and incorporation into spores. It was found that Cu incorporation into spores during sporulation resulted in increased thermal destruction. Cu inside Clostridium spores can react with essential macromolecules catalyzing hydrolytic reactions (Williams 1981) and spontaneously producing free radicals, greatly toxic to DNA and cellular components (Loeb, 1988). As a matter of fact, the possible use of copper to limit Clostridium sporulation, germination and growth of vegetative cell is now at studying (Rodriguez & Alatossava, 2010); the model supposes the interaction between copper ions and spore-specific molecules involved in sporulation and germination processes, such as spore membrane and coat proteins rich in cysteine residues which are object of disulphide bridge formation (Henriques and Moran, 2000; Chada et al., 2003). Cu2+ targets consequently sulphydryl groups of spore proteins with the formation of non-functional metal-protein complexes resulting in a reduction of the sporulation process.
Molybdenum was considered in some extents stimulatory but was not so required for sporulation (Kolodziej, 1964).
At the same extent, iron and zinc requirements were established for the sporulation of B. megaterium; but their role for sporulation has been considered vague and not so certain.
Manganese is the most firmly established metal requirement for sporulation (Charney, Fisher, and Hegarty, 1951; Grelet, 1952a, b; Curran and Evans, 1954; Amaha, Ordal, and Touba, 1956; Powell, 1957). Recent genetic works in B. subtilis have demonstrated that Mn2+ homeostasis is fundamental not only for an efficient sporulation (Que & Helmann, 2000), but also in several steps of its developmental cycle (Jakubovics & Jenkinson, 2001). During vegetative exponential growth initial Mn, together with Mg, uptake was rapid and ended by mid-log phase. Secondary uptake followed immediately and continued through stage V (Krueger & Kolodziej, 1978). The manganese accumulated during growth and early sporulation is exchangeable and therefore relatively "free"; intracellular manganese is converted later during sporulation into a bound form that cannot be released (Eisenstadt, 1973). In sporulation Mn2+ seems to be a cofactor of SpoIIE serine phosphatase (Schroeter et al., 1999) implied in the formation of the spore polar septum through interactions with FtsZ (King et al., 1999) and, at the same time, involved in the activation of ï³F in the forespore by dephosphorylation of SpoIIAA (Kroos et al., 1999).
Magnesium active transport is of particular interest since this cation is involved in the functioning of the nucleic acid and protein synthesis apparatus, as well as being necessary for the functioning of numerous individual enzymes. Magnesium was considered, therefore, essential for sporulation (Murrell, 1969), but not for heat resistance (Roberts and Hitchins, 1969). Moreover, magnesium decreases heat resistance of Bacillus species, interfering with formation of the Ca-DPA complex (Murrell, 1969). Mg2+, phosphate, and an energy source in minimal concentrations were required in order that early stationary-phase cells become committed (stage II) (Greene, 1972). Commitment to sporulation at stage IV in B. cereus can be explained on the basis of permeability effects, since the forespore at stage IV is surrounded by the inner and outer forespore membranes, and the plasma membrane of the mother cell. While during exponential growth, the intake of Mg was rapid, its excretion continued to t1 with release of 33% accumulated. Secondary uptake began by t5 (stage IV) and continued slowly through sporulation (Krueger & Kolodziej, 1978). In Clostridium perfringens Mg seemed to be important for growth of vegetative cells and maintenance of normal cellular morphology (Lee, 1978). Considering the magnesium uptake and transport system, Scribner et al. (1973) affirmed that active transport of magnesium in B. subtilis is indicated by the energy dependence of the transport system and by the substrate saturation kinetics of magnesium uptake. They noted that, at the very first beginning of sporulation, there was a sudden decline in the Mg accumulation, and they observed that 4.5 or 8 h after the end of log-phase growth, the rate of magnesium uptake had decreased almost 80% and seemed to be independent of substrate concentration. In B. subtilis, two magnesium carriers have been postulated, so that the Mg decline during sporulation could be associated to the fact that one of two magnesium transport systems could no longer function late in sporulation. Such specificity of regulation is not surprising since it has been already shown that potassium transport in sporulating B. subtilis continued at essentially the same rate as with log-phase cells (Eisenstadt, 1972) and that manganese transport increased in rate during sporulation in response to the external growth conditions. This Mg++ kinetics could be explained by the fact that it is implied in the four components phospho-relay system. As already known, the first two steps of this system involve induced auto-phosphorylation of either of two kinases, KinA or KinB, and transfer of the phosphoryl group to the response regulator, SpoOF. A phosphotransferase, SpoOB, then catalyzes the transfer of the phosphoryl group from the SpoOF protein to the transcriptional activator/repressor, SpoOA. SpoOA-P plays a dual role; it represses expression of proteins that regulate the vegetative phase and activates gene expression important for sporulation (Hoch, 1993). During sporulation KinA-mediated Spo0F-P formation also displayed both activation and inhibition by Mg++. The concentration dependence was such that Mg++ ions stechiometry with ATP produced full activity, while levels higher gave inhibition (Grimshaw et al., 1998).
Few are hypothesis regarding the role of potassium in sporulation. In normal growth, B. subtilis accumulates potassium against a concentration gradient. Eisenstadt (1972) first, studied the K+ implication in B. subtilis; he found that sporulating cells had a potassium content similar to that of vegetative cells, cause this mineral is fundamental for protein synthesis, its activity is enhanced not only in normal growth but also in sporulating cells (Eisenstadt, 1972). Moreover, it was observed that addition of manganese to stationary phase cells before sporulation produced a rapid intake of potassium ion. This relation was quite unusual but it made suppose that during the sporulation phase it happens a global activation of different ions transport systems. This concept could explain also the role of manganese in stimulating magnesium accumulation in stationary cells (Eisenstadt et al., 1973). Differently from calcium, potassium in sporulating cells is not firmly bound (Eisenstadt & Silver, 1972).
Calcium is considered to play a fundamental role in bacterial cells controlling chemotaxis, stress response, gene transcription and cell division. In spore formers bacteria was shown to be a requisite for the formation of refractile and heat-resistant spores, and it was necessary at the time of sporangium formation (Kolodziej, 1964; Lee, 1978). During the onset of sporulation, in fact, a specific-Ca2+ transport system seems to develop. This is in close agreement with the demonstration that the calcium content of cells increased during the formation of the spores within the sporangium (Vinter, 1956). As the spores matured, they progressively accumulated calcium until they contained several times more calcium than their respective vegetative cells. Its proper mechanism of transport has not yet been elucidated. Usually in the cell, microrganisms move calcium through secondary transport mechanisms such as Ca2+/H+ exchangers which use the energy accumulated in the gradient of other ions (Smith, 1995; Norris et al., 1996). A similar apparatus could be thought also for sporulating cells where a transporter accumulates Ca2+ in the cytosol of the mother cell, followed by passive diffusion of Ca2+ into the prespore; here the levels of calcium are low due to the complex formation with DPA (Seto-Young, 1981). Recently, some works have hypothesised about the presence of a putative P-type Ca2+ transport ATPases in B. subtilis codified by the yloB gene (Raeymaekers et al., 2002). This protein is observed to be express only during the induction of sporulation and it's possible that it catalyzes also the transport of Mn2+ and other cations.
Ions transport and germination
The uptake of spore ions in bacteria, the transport system and more specifically the release of anions is still poorly understood. The most important event is the accumulation of Ca2+ in the spore mature thus the transport from the mother cell to the forespore. This process occurs concomitant with the cell synthesis of DPA. During germination the spore releases in the first minutes monovalent ions, probably with concomitant release of anions (Swerdlow et al.1981).
The first step of germination in natural environment probably is triggered as response to nutrients named germinants (Setlow 2003). The germinants may be single nutrients or a combination of nutrients and they are functionally different depending on the species of sporeforming bacteria. The germinants (Table XX) have to encounter the specific receptor and to permeate the outer coat and cortex layers of the spore. The germination is triggered when nutrients or germinants reach their cognate receptor located in the spore inner membrane (Paidhungat and Setlow 2001). The second event is the release from spore core of monovalent ions, more frequently Na+ and K+ followed by the release of the large depot of Calcium-DPA. The role of Ca-DPA in germination is different among the studied species. In B.subtilis the release of Ca-DPA is the signal molecule for the activation of cortex-lytic enzyme CwlJ (Paidhungat et al 2001). In C. perfringens Ca-DPA has a different role, it acts as signal molecule in GerKA and GerKC to trigger germination but has not effect on hydrolytic enzymes (Paredes-Sabja et al 2008, Paredes-Sabja et al 2009). The result of interaction from germinants and cognate receptors triggers the release of monovalent (H+, Na+, K+) and divalent ions (Ca2+, Mg2+, Mn2+). The ions uptake and the hydrolysis of cortex allow the hydration of spore and to expand and resume water content to the level found in growing cells with resumption of enzymatic activity and energy metabolism (Settlow 2003, Paredes-Sabja et al 2009).
It was suggested that in the early stages of germination the release of K+ and Na+ occurred due to an increased permeability of spore layers and at the same time by low levels of K+ and Na+ in germination medium therefore the release and balance of K+ and Na+ was the consequence of gradient between the inside and the outside of the spore to rise pH. The release of Na+ and K+ has been shown to be one of the first step of germination coming before the DPA release causing a rise of the spore core's pH; at least 80% of the spore's Na+ and K+ is released in the early germination steps and this efflux is followed later by the reuptake of K+ by an energy dependent system (Swerdlow et al.1981).
It was suggested that the release of calcium and DPA in germinating spore is possible for the presence in the spore of one or more channels for the ions to across the layers of spore structure; furthermore the channels are opened to allow germinants to reach and bind germinants with appropriate receptors (Setlow 2003).
Different Na+/H+-K+ antiporter has been identified in sporeforming bacteria, GerN in B. cereus and B. megaterium, GerA B. subtilis , GerO in Clostridium perfringens (Thackray et al. 2001, Southworth et al. 2001, Paredes et al. 2009). In B. cereus has been studied the GerN, homologus with GrmA of B. megaterium, this antiporter is important in germination and by using a gerN null mutant was perturbed the normal order of germination events or drastically reduced with alanine by germinants or blocked with inosine (Thackray et al. 2001). Differently recent study of GerO and GerQ putative Na+/H+-K+ antiporters in C. perfringens has shown to be involved in the transport of monovalent cations the results indicated a role during sporulation and germination (Paredes-Sabja et al. 2009a) and the involvement of GerO for germination in presence of Na+ and inorganic phosphate as germinants (Paredes-Sabja et al. 2009c). The role of Na+ and sodium phosphate (100 mM, pH 6) has been demonstrated by using null mutant of germinant receptors. Germination test was performed with C. perfringens spores mutant of gerKA-KC, gerAA and gerO and was shown their function of receptor of the sodium phosphate germinant and more precisely that the Na+ acts as cogerminant with inorganic phosphate and the presence of the putative Na+/H+-K+ antiporter is necessary (Paredes-Sabja et al. 2009c). Previously the authors (Paredes-Sabja et al. 2008) studied the the role of KCl as germinat in C. perfringens. Different germinants were assayed and the most effective resulted the KCl. The K+ ions was an efficient germinant in strains of C. perfringens responsible of food poisoning. The response was different related to the localization of the chromosomal enterotoxin cpe gene or plasmid-borne cpe gene. It has been hypothesized that the spore of food poisoning strains isolates from meat during ripening have a system to sense the ions that trigger the germination. This mechanism it will be not present in spores from strains not food poisoning. Differently from other germinant ions the sodium phosphate require all the four Ger receptors (GerKA-KC, GerAA, GerKB) and also the putative antiporters GerO and GerQ are required to have a normal germination. The receptor GerKA-KC is also the involved to trigger germination in presence of KCl and L-asparagine(Paredes-Sabja et al. 2009c, Paredes-Sabja et al. 2008).
The release of rapid Calcium Dipicolinate was studied in Clostridium tyrobutyricum by using ray X microanalysis. The molecular mechanisms that regulate the germination of this clostridial species is yet unknown. Similarly to the data and observation in C. perfringens the combinations of ions and aminoacids was more effective to trigger germination than the germinant in single. The most efficient combination resulted to be the germinant L-alanine, L-lactate pH4,6 in NaHCO3 buffer. Moreover significant differences were obtained in germination measurements when phosphate, Na+ or aminoacid like alanine, lactate were assayed in single to trigger germination. Most release of Ca-DPA was reached when germinants as alanine or lactate were used in solution with ions, NaHCO3 (Bassi et al. 2009).