The process of sporulation is a survival strategy for species belonging to the phylum Firmicutes. Bacterial endospores from this phylum, and more specifically the Bacillales and Clostridiales orders, exhibit remarkable resistance to numerous environmental insults such as heat, dessication, radiation, extremes in pH and pressure, as well as chemical oxidants. Many of clostridial spore-forming species, such as Clostridium difficile, C. perfringens, C. botulinum, C. tetani, C. acetobutylicum, and C. beijerinckii, are medically and industrially important. This group of organisms primarily uses fermentation as a metabolic strategy, but is ubiquitous in the environment and thus can be found in a wide variety of habitats, including mammalian hosts. The mechanism of sporulation is largely conserved amongst these species (Dalla Vecchia et al., 2014). However, there are large variations in the initiation of sporulation as well as in the process of spore coat formation and germination, due to differences in the environmental niches in which these bacteria thrive (Dalla Vecchia et al., 2014; Paredes-Sabja et al., 2014).

The resistance of spores to environmental attacks is attributable to the unique morphology of the spore, which is formed by several layers: the core, the cortex, the coat, the crust, and in some cases, the exosporium. These structures protect sensitive macromolecules such as DNA and crucial proteins, included in the spore core. Dipicolinic acid (DPA), with its ability to bind and chelate Ca²⁺ ions, plays a key role in the dehydration, and mineralization of the spore core. Dehydration of the spore core is the main factor underlying the resistance of spores to wet heat (Paidhungat et al., 2000).

The process of sporulation has been extensively studied for several decades for the model organism Bacillus subtilis. The basic morphological structure of spores is conserved in all the endospore-forming bacteria studied. This unique structure is the basis for the resistance of spores. While some of the resistance determinants of spores have been revealed and it is known that several factors contribute to the development of each type of resistance, the precise mechanism of their action remains to be uncovered (Orsburn et al., 2008). The best-studied mechanism of spore resistance is that to UV, achieved with small-acid soluble proteins (SASPs), which bind to the spore’s DNA and promote its conformational change (Setlow, 1995; Raju et al., 2006; Lee et al., 2008). Recently, it was determined that DNA was packed into crystalline nucleoids through binding by α/β type SASPs, hence protecting DNA from modification (Dittmann et al., 2015). Such protein-DNA structures allow spores to endure multiple types of environmental insults such as non-ionizing radiation, wet and dry heat, and desiccation, probably because the movement of internal molecules is restricted (Dittmann et al., 2015). In B. subtilis, superdormant spores were identified and were defined as spores able to withstand multiple rounds of germination conditions in the dormant state (Ghosh and Setlow, 2009; Ghosh et al., 2009).

The second most studied type of spore resistance is that to wet heat. This particular form of resistance is of major concern in the food industry because of food spoilage post sterilization. The major factor contributing to spore resistance to wet heat is water content in the spore core. In the dehydrated core, aqueous chemical reactions are inhibited, minimizing damage to biomolecules in case of exposure to thermal extremes (Setlow, 2006). It is well established that dehydration of the spore core directly correlates with its mineralization (Beaman et al., 1982). Thus, water content in the spore core is inversely proportional to the amount of DPA and Ca²⁺ (Douki et al., 2005; Setlow, 2006). DPA in the spore likely provides wet heat resistance through (a) lowering the core water content, (b) protecting proteins from denaturation from wet heat exposure, and (c) helping to stabilize DNA and to afford protection from damage (Raju et al., 2006; Setlow, 2006).

In B. subtilis, DPA is synthetized in the mother cell during late sporulation stages and is then taken up together with Ca²⁺ by the engulfed forespore (Tovar-Rojo et al., 2002; Li et al., 2012). The major component responsible for DPA synthesis is the SpoVF protein complex, which consists of two subunits, SpoVFA and SpoVFB (Daniel and Errington, 1993). A B. subtilis spoVF mutant strain forms DPA-less spores that contain a high amount of water and are less heat resistant than spoVF spores formed in the presence of exogenous DPA (Paidhungat et al., 2000). Exposure of such spores to wet heat results in damage to spore proteins, which is directly proportional to the time of exposure to this condition. After accumulation of defects in proteins, spores may still be able to germinate but growth into viable cells is compromised (Coleman et al., 2007). Genome analysis has revealed that although some Clostridia possess a SpoVF homolog within their genome, it is not the case for species of Clostridium sensu stricto (cluster I) (Onyenwoke et al., 2004). In those species, DPA synthesis could be catalyzed by the EtfA protein (electron transfer flavoprotein A), as was shown for Clostridium perfringens (Orsburn et al., 2010).

There is strong evidence of heterogeneity in spore populations with respect to resistance of spores to thermal treatment. A single cell analysis approach, where physiology of individual spores was monitored during the incubation at 80–90°C, revealed a variable response of spores to wet heat. The release of DPA from spores itself only takes a few minutes. However, individual spores differ in the so-called lag phase, which is the period from exposure to germination conditions until the commitment of germination, the rapid release of DPA/Ca. The lag period ranges from minutes to hours for individual spores. It is presumed that the spores with the longest lag phase are the most resistant to wet heat. However, molecular, physiological, or environmental factors underpinning this variability in spore populations are poorly understood (Coleman et al., 2007; Zhang et al., 2010, 2011).

Previous studies of resistance of spores have shown that there are large differences regarding the level of resistance to various environmental insults amongst various species (Brul et al., 2011; Maillard, 2011; Paredes-Sabja et al., 2011; Xiao et al., 2011; Setlow, 2013, 2014). In the present work, we focus on several Clostridium species belonging to Cluster I of genus Clostridium (sensu stricto). This cluster is morphologically and physiologically the best described group of the genus Clostridium, which contains over 160 species divided into multiple sub-groups according to results of 16S rRNA analysis (Gupta and Gao, 2009). Here, we examine the group of industrially important species belonging to this cluster, specifically C. acetobutylicum DSM1731 and DSM792, C. beijerinckii strains DSM791 and NCIMB 8052 and C. collagenovorans 3089, for their level of resistance to wet heat as well as for their DPA/Ca content. The goal of the study is to evaluate the link between heat resistance and DPA/Ca content, as well as to compare DPA/Ca levels among Clostridium species.

Most of the studies of spore resistance strategies have been performed on B. subtilis spores due to the easy acquisition and large availability of existing mutants. Here, we focused on the heat resistance properties of spores of species belonging to the genus Clostridium sensu stricto (Cluster I).

Species analyzed in this study are very closely related phylogenetically (Figure 1), but they also share the same ecological niches as they are ubiquitous in soil, water, and feces; maybe with the exception of C. collagenovorans which was isolated from sewage sludge (Jain and Zeikus, 1989). In fact, many C. beijerinckii strains were considered to be C. acetobutylicum, until DNA fingerprint analyses and sequencing of 16S rRNA proved otherwise (Keis et al., 1995). All of these strains are anaerobic and use a fermentative type of metabolism. C. acetobutylicum and C. beijerinckii strains also share the same physiology and are able to produce solvents in the process of solventogenesis. Solventogenesis together with sporulation belong to strategies to survive extreme acidic environment, which is induced by acid-generating metabolism of Clostridia (Dürre, 2009). During solventogenesis, cells utilize some of the by-product acids to produce neutral solvents, and thus the pH of the environment is restored to favorable levels. In solventogenic Clostridia, solventogenesis and sporulation are coincident, and are both regulated by the stationary phase regulator, Spo0A (Ravagnani et al., 2000).

C. collagenovorans DSM 3089 is also closely related to C. acetobutylicum (Ludwig et al., 2009) (Figure 1). In addition to being acetogenic, it is also able to produce an active collagenase that can hydrolyze collagen and release peptides from animal proteins (Jain and Zeikus, 1989). In contrast to all other Clostridium strains considered, it is not solventogenic (Jain and Zeikus, 1988).

Our results show that despite the close relatedness of analyzed species, they exhibit large variability in calcium and DPA content as well as heat resistance, regardless of phylogeny. The Clostridium species considered here can be divided into several groups.

First, C. collagenovorans DSM 3089 behaves similarly to spore-forming organisms outside of the Clostridia. It exhibits high heat resistance, and uniformally high calcium and DPA concentrations in the core. Thus, this organism represents one end of the spectrum of spore-forming organisms. The Clostridium species with the next lowest wet heat resistance is C. beijerinckii NCIMB 8052 that shows an 81.5% resistance at 10 min of exposure and 0.36% at 60 min. The DPA concentration is comparable to that of D. reducens and B. subtilis and a large percentage of the spores characterized display a high calcium concentration (Table 2). However, in contrast to C. collagenovorans, a few spores contain low calcium concentrations (∼1 atomic %). Thus, there is sufficient intra-population variability to be identified from a relatively small sample size (12 spores).

There is a large decrease in heat resistance between the two aforementioned strains and C. acetobutylicum DSM 1731. Only 37% of the spores survive 10 min of heat treatment and interestingly, the same fraction survives 60 min of the same treatment. Surprisingly, the calcium and DPA concentrations are high (greater than those in B. subtilis and D. reducens). It is unclear why the wet heat resistance is much lower in the case of strain DSM 1731 than the control strains. A possible explanation for this discrepancy is that the DPA synthesis pathway is distinct for Cluster I of the Clostridium genus as compared to organisms outside that cluster. More specifically, the EtfA flavoprotein encoded by etfA, was identified as the DPA synthase in C. perfringens, a Clostridium that doesn’t possess the homolog of SpoVF DPA-synthase from B. subtilis (Orsburn et al., 2010). In fact, homologs of SpoVF are missing throughout the whole Cluster I of the Clostridium genus and it is very likely that in these species EtfA functions as the only DPA synthase. It is unclear whether this difference in pathway may also impact the timing of the production of the DPA and how it is distributed within the spore.

Finally, C. beijerinckii DSM 791 and C. acetobutylicum DSM 792 have very low wet heat resistance (3.9 and 4.6% survival after 10 min, respectively). For C. beijerinckii DSM 791, the low wet heat resistance is combined with low DPA and calcium concentrations (Table 2). Additionally, in the same strain, there is negligible heat resistance at 60 min of heat exposure, confirming the findings at 10 min. Thus, in this case, low calcium and DPA appear to correspond to low wet heat resistance. While the STEM-EDS results indicated relatively uniform Ca concentrations within the spore population, the STXM data (accompanied by the STEM-EDS data for the same spores) evidenced individual variability with a range of DPA concentrations and Ca concentrations ranging from 0.31 to 2.69 atomic %. Thus, the DPA and Ca are low but display some variability.

For C. acetobutylicum DSM 792, we found a wide distribution of calcium concentrations in spores (Table 2). A large fraction (57%) of the spores characterized exhibited an average calcium content of 5.7 atomic % while the rest contained an average of 2.94 atomic % for three spores and 0.4 atomic % for six spores. The average spore DPA concentration was 2.1 pg/μm³, approximately half that of the control strains D. reducens and B. subtilis.

Thus, results obtained from wet heat testing of spores of various clostridial species suggest that DPA/Ca content in spores is correlated to their wet heat resistance level, particularly with respect to short exposure to the treatment. After longer exposure to wet heat (∼ 60 min), spores survival decrease dramatically in all strains, with the exception of C. acetobutylicum DSM 1731. In this strain, the survival of spores is identical after short wet heat treatment, thus, the survivors retain a relatively high ability to germinate and recover even after prolonged exposure to wet heat.

The relatively low DPA/Ca concentrations in C. acetobutylicum DSM 792 and C. beijerinckii DSM 791 spores could potentially be a consequence of environmental adaptation. However, the four strains C. acetobutylicum and C. beijerinckii strains considered in this study have essentially the same type of metabolism (fermentative and solventogenic), and all live in similar environments. Thus, there is little evidence that environmental conditions may lead to this difference.

Another hypothesis was that the strains that produce butanol rather than ethanol may produce more resistant spores. Indeed, C. acetobutylicum DSM 1731 and C. beijerinckii NCIMB 8052 are known to be some of the most efficient at producing butanol amongst the solventogenic Clostridia (Chua et al., 2013). Thus, we propose that difference in solvent yields amongst the strains considered may explain the heat resistance and Ca/DPA content discrepancies.

As discussed above, in all Clostridia belonging to Cluster I, EtfA, an electron transfer flavoprotein, is likely the physiological DPA synthase. Some species of this cluster actually contain more homologs of etfA in the genome, roles of which are not assigned up to date (Orsburn et al., 2010). The function of EtfA may also include the production of butanol (Boynton et al., 1996). Thus, solventogenic strains that produce abundant butanol (DSM 1731 and NCIMB 8052) may also produce abundant DPA through the activity of EtfA. This link between metabolism and sporulation could also explain the heterogeneity in average DPA content of spores amongst strains, which are otherwise closely related phylogenetically. We can speculate that the heterogeneity in DPA/Ca content of spores within a given spore population may be attributable to differences in expression level of etfA prior to sporulation.

Bacillus subtilis, the model organism for the study of physiological and morphological processes in spore formers, also possesses the etf locus (etfA and etfB) in the genome. The function of EtfA flavoprotein in B. subtilis resembles the one in solventogenic Clostridia in several aspects. Not only is this protein in B. subtilis also involved in fatty acid synthesis, but it also plays a role in de-acidification or the internal environment of the cell by oxidizing NADH and consuming excess protons, thus maintaining the proton motive force of cellular respiration and membrane potential of the cell (Barabesi et al., 2007; Marvasi et al., 2010). While in solventogenic Clostridia the etf locus is located in the same operon as solventogenic genes, in B. subtilis the etf locus is part of lcfA operon, which is involved in process of calcite formation during biofilm development (Barabesi et al., 2007). Although it may seem that the function of etfA differs greatly in the two systems, a connection can be found: the transfer of electrons between NADH/NAD+ and consequent regulation of complicated redox reactions. Products of the etf operon are thus involved in processes associated with pH homeostasis in the cell, such as solventogenesis in Clostridia, where repeated oxidizing of NADH to NAD+ favors formation of reaction intermediates which lead to the production of neutral solvents rather than volatile acids. Because of the buffering properties of calcium carbonate, the precipitation of calcite as a result of B. subtilis metabolism is also a way to maintain the intracellular and extracellular pH within the boundaries suitable for neutrophiles.

It is interesting to note that the only non-solventogenic Clostridium strain considered here, C. collagenovorans, exhibits the highest Ca and DPA concentrations and the highest spore survival rate to heat treatment among Clostridia.

Overall, this study shows that there is great variability in the calcium, DPA concentrations in Clostridium Cluster I spores and that those concentrations correlate well with spore heat resistance. In addition, heat resistance also appears to correlate with the type of metabolism supported by specific organisms. Solventogenic strains generally harbor less DPA/Ca than non-solventogenic strains and within solventogenic strains, the ones producing more butanol appear to harbor more Ca/DPA and hence present greater heat resistance. We propose that the link between DPA synthesis and butanol production is through the etfA gene that serves in both pathways. Additional works is required to pinpoint the mechanistic underpinnings of this finding.

JJ carried out the STXM work and initiated the project. ZC and CL carried out most of the experimental work. BB did the electron microscopy. IB and RB-L designed the project and RB-L, ZC, and IB wrote the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.