1 Introduction

Endospore-forming bacilli constitute a prominent group of bacteria, not only for the pathogenic species it includes (i.e., Clostridium botulinum, Clostridium difficile, Bacillus anthracis), but also for its saprophytic (i.e., Bacillus subtilis) and industrially important microorganisms (i.e., C. acetobutylicum). Additional significant spore-forming species include the genera Desulfotomaculum, Paenibacillus, and Alicyclobacillus. Other recently described Gram-positive bacteria such as Caldalkalibacillus thermarum TA2.A1 (Peddie et al. 1999; Xue et al. 2006), which is a member of alkaliphilic bacteria but otherwise related to the Bacillales order, has been recently shown to contain at least three annotated operons involved in spore germination (de Jong et al. 2020), including the genes gerABC and yndE. As this alkalophilic bacterium is old in terms of evolution, it has to be assumed that the ability of endospore-forming emerged soon in the evolution of Gram-positive bacteria.

The bacteria exhibiting this exclusive ability, when encounter unappropriate physicochemical conditions initiate the formation of important small molecules, that are collectively known as “alarmones” which are part of the heat shock response in B. subtilis (Schäfer et al. 2020); two of them classic and well known such as pppGpp, ppGpp, and the newest one pGpp, with at last recognized effect as alarmone (Yang et al. 2020), all of them involved in a classical bacterial response known as “stringent response” and recently also found in metazoan (Ito et al. 2020). These elements (highly conserved in Nature and known for more than five decades; Cashel and Gallant 1969) show a variety of pleiotropic effects and are involved in a number of metabolic pathways in bacteria, including the development of endospores. Therefore the alarmones represent a new way for bacterial survival (Fernández-Coll and Cashel 2020). In addition, it has been shown recently that the ComX quorum sensing peptide of B. subtilis positively affects the sporulation process (Špacapan et al. 2020). Differentiation processes in B. subtilis, such as endospore formation, involve multiple paralog Rap-Phr systems that are highly redundant, and that according to Gastélum and colleagues in 2020, interconnect this first-order morphogenetic event with others such as the development of competence.

B. subtilis is, therefore, and without a doubt, the best-known Gram-positive bacterial rod, and contains three subspecies [i.e., subtilis (Nakamura et al. 1999), spizizenii (Nakamura et al. 1999), and inaquosorum (Rooney et al. 2009)]. These three subspecies are so similar (they share ca 3300 ORFs) that they can only be differentiated by phylogenetic analysis of multiple proteins, as their 16S rRNAs exhibit an extremely high sequence identity (for a genomic insight into the taxonomic status of the three B. subtilis subspecies, see Yi et al. 2014).

Although endospore-forming bacteria can exhibit different metabolic and genetic abilities, they all belong to the phylum Firmicutes and share the capacity to survive harsh environmental conditions via the production of highly resistant endospores; this is a superior biological development, normally subjected to catabolic regulation (Schaeffer et al. 1965). These highly resistant structures have been recently reviewed from the point of view of the different technologies usable today that cause endospore death (Cho and Chung 2020). Espores from B. subtilis have been used recently in chickens with positive results as adjuvants in vaccines against the avian influenza H9N2 orthomyxovirus (Lee et al. 2020)

Endospore formation follows the same genetic program in all bacteria, with little variation from species to species; this fact led Hutchison and coworkers to suggest in 2014 that “a robust and sophisticated developmental framework was already in place in the last common ancestor of all extant Firmicutes.” Nearly 90 different bacterial genera can form endospores and, although Gram-positive microorganisms are predominant among them, this endospore-forming group also includes many Gram-negative species.

This survival structure was originally described by Ferdinand Julius Cohn, in the nineteenth century (1875). The author, although a botanist, became one of the founding fathers of modern bacteriology and microbiology by demonstrating the ability of Bacillus to form endospores and describing the basic steps in spore formation (Drews 2000). Cohn, due to his background in algal taxonomy, also made a significant contribution to bacterial taxonomy, although his bacterial classification was not accepted by many of his colleagues, who still believed that bacteria could spontaneously arise from decaying biological matter (Cohn 1875; Drews 2000).

The number of endospores produced by bacteria can vary from one (monosporic species), two (bisporic), or many (polysporic), and they always are genetically identical copies of the vegetative cells. The morphogenetic process resulting in endospore production is usually initiated by a lack of nutrients essential for vegetative growth (mainly nitrogen source depletion). This process is tightly regulated by SPO genes and different σ factors, that define the sporulation stages, and terminates with the formation of a multilayer, refractive, highly resistant structure that can withstand the challenges posed by factors such as extreme temperatures and DNA-damaging agents (Errington 2003). This survival structure is what microbiologists call “endospore,” characterized by its metabolically inactive “dormant state.” In some bacterial groups, however, the sporulation process gives rise to multiple intracellular offsprings, some of which do not undergo a dormancy period; many of these spore-forming bacteria, although hard to grow axenically, were identified as Clostridia, one of the endospore-forming bacterial group (Hutchison et al. 2014).

Unraveling the mechanism of endospore formation, triggered by starvation, resulted not only in the understanding of this basic bacterial morphogenetic process and in obtaining a variety of mutants with different metabolic and genetic abilities, but also in the discovery of novel non-Firmicutes and remote bacterial strains displaying certain characteristics of the Firmicutes. One of these traits is the resistance to soil-dwelling predatory microorganisms, such as the delta proteobacterium Myxococcus xanthus. It is well known that nondomesticated strains of B. subtilis, capable of producing bacillaene (a polyene antibiotic), can resist the attack of the predatory bacteria, eventually forming spores and hence becoming fully resistant to the predator. On the other hand, laboratory strains of B. subtilis, usually unable to produce the antibiotic, are easily predated by M. xanthus (Müller et al. 2014).

Starvation-induced sporulation is the last survival resort for some bacteria. The sporulation process involves a cellular decision-making stage (commitment point), that can last several hours depending on the bacterium, accompanied by the development of actinomycin resistance (Sterlini and Mandelstam 1969). During this time, the bacterium explores other possibilities of survival, such as the secretion of enzymes to use alternative food sources, production of antibiotics to eliminate competing microorganisms, and the induction of cell competence to uptake exogenous DNA. Sporulation is suppressed until all other possibilities are shown inviable and, once the commitment point is reached, sporulation is irreversible. The sporulation process is spatially and temporally orchestrated and represents one of the most thoroughly investigated cellular processes. Some of the genes involved were mapped on the B. subtilis chromosome (Piggot and Coote 1976; Piggot and Hoch 1985) by means of either transformation or transduction. Sporulation studies in Bacillus and Clostridium determined that, although the process is continuous, it can be structured into several stages. Sporulation starts with Stage 0: the decision to sporulate and ends with Stage VI/VII: spore release (Fig. 1), as proposed by Ryter in 1965. Already in 1974, Hranueli et al. proposed that spore formation in Bacillus involves at least 37 operons. For a recent review, see Setlow and Johnson (2019).

Fig. 1
figure 1

Sporulation stages in the Gram-positive bacteria Paenibacillus favisporus. (a, b) Fore-spore formation; (c) spore maturation, displaying the typical surface of the spores from this species; d, e, and f) lysis of sporangium and spore release (modified from Velázquez et al. 2004). Scale bar is 0.7 mm (a, b, c, and d) or 0.2 mm (e and f)

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The years 1996 and 1997 saw the publication, in Microbiology and Nature respectively, of first the computerized genetic map of B. subtilis (Biaudet et al. 1996) and then the complete sequence of B. subtilis genome (Kunst et al. 1997). B. subtilis genome spans 4,214,810 base pairs, encompassing 4100 protein-coding genes, as well as at least ten prophages or their remnants and a large number of genes for using a variety of nutrients, many of plant origin. More recently, the publication of complete genome sequences, such as that of Clostridium perfringens (Shimizu et al. 2002), has permitted to carry out comparative genomics with other important Gram-positive anaerobic sporulating rods.

Unraveling this complex genetic and biochemical pathway not only contributes to a better knowledge of the biology of sporulating Gram-positive bacteria, but could also result in the discovery of novel antibiotics, or even contribute to the knowledge of associated flavors in certain beverages, such as the Chinese Maotai Liquor (Wang et al. 2018).

Endospore formation is a major morphological feature used in bacterial taxonomy and the characteristics of the spore, such as location within the sporangium (mother cell), sporangium distension and number of spore per sporangium, are also important for the classification of both aerobic and anaerobic spore-forming Gram-positive bacilli.

Starvation is not the only trigger for sporulation, in fact, siderophore production is another factor affecting endospore formation. Grandchamp and coworkers demonstrated in 2017 that the production of bacillibactin facilitates sporulation, and even enterobactin (a siderophore from E. coli) induces B. subtilis sporulation. However, while the uptake of either siderophore involves binding to just one protein (FeuA), the onset of sporulation in the presence of the siderophores requires a different protein for each siderophore, such as the esterase BesA for bacillibactin and the esterase YbbA for enterobactin (Grandchamp et al. 2017).

B. subtilis spores have recently found quite unusual applications (Sun et al. 2020). The authors used spore coat proteins CotB and CotC as anchors for the heterogenous antigen in a system grass carp reovirus combined with the genes cwlJ and sleB able to control the pore germination. Heterologous antigens using this method were able to elicit a strong humoral as well as cellular response in Ctenopharyngodon idella.

One tends to consider the SPO proteins (all those so far related to the sporulation process) as exclusive of those bacteria able to carry on with the formation of endospores, but the truth of the matter is that there is a large variety of bacterial species (including Escherichia coli) that contain sporulation-related repeated domains, known to bind peptidoglycan and also to enhance the activity of the penicillin-binding proteins and hence of the transpeptidase activity (Pazos et al. 2020).

The study of endospore formation in B. subtilis has been an important increase of our knowledge in terms of genetics, biochemistry, and developmental biology, but indeed it has resulted in practical applications. One of these has been the development of a new strain of B. subtilis that harboring the β-lactam-induced regulatory system BlaR1/BlaI from Staphylococcus aureus, which can be used as an efficient biosensor to evaluate the presence of β-lactams in solution (Lautenschläger et al. 2020). Another interesting application involving the spores of Bacillus subtilis is related to the use of these spores to prepare vaccines against B. anthracis. So, Oh, and colleagues reported in 2020 the obtention of a new B. subtilis strain that originates spores with the anthrax protective antigen on the surface. All in all, and as Errington and van der Aart have recently proposed B. subtilis has been and still is a workhorse as a model for studying cellular development, including the generation of asymmetry, cell fate, and prokaryotic morphogenesis in general (Errington and van der Aart 2020).

2 Genes and Factors Affecting Endospore Formation

The initiation of sporulation is a prime example of developmental biology in Gram-positive bacteria that strongly involves biochemical and genetic factors. It occurs in Nature constantly in this group of bacteria, when encountering inappropriate physicochemical conditions, and the picture of the whole process may be altogether blurred by the continuous growing of B. subtilis under laboratory conditions in what has been denoted as “loss of social traits during domestication process of Bacillus subtilis” (Barreto et al. 2020).

Endospore formation depends on a major signal transduction system known as “the sporulation phosphorelay” that controls phosphorylation of the key Spo0A transcription factor (Burbulys et al. 1991; Ohlsen et al. 1994; Wang et al. 2001), as well as the synthesis of sporulation-specific sigma factors (Fimlaid et al. 2015) involved in the subsequent sporulation stages. Most of the biochemical changes during sporulation appear to occur during the first two “sporulation stages” mentioned above (0 and II); during this period, a new cell differentiates within the mother cell and isolates itself, although it maintains a specialized connection system to the mother cell, to receive from her a variety of nurturing compounds, such as activators and sigma factors.

Initiation of sporulation in B. subtilis stops normal growth (stage 0; Fig. 2), this is followed by the synthesis of a septum (stage II, see below). The Spo0A protein is activated through phosphorylation (Sonenshein 2000) in stage 0 and is responsible for the regulation, either directly or indirectly, of more than 500 genes (Fawcett et al. 2000). When studying the σ factors involved in the sporulation process, it soon became clear that a single vegetative σ factor could not be responsible for the RNA transcription carried out from a variety of promoters which, in addition, are different from those responsible for vegetative growth and primary metabolism. Further proof of this was provided by Linn and coworkers that, already in 1973, demonstrated that the activity of the vegetative sigma subunit of B. subtilis RNA polymerase dramatically decreases once sporulation starts, and its levels remained low throughout the sporulation process. These findings were confirmed by Brevet the following year (Brevet 1974).

Fig. 2
figure 2

Key stages of the sporulation cycle in Bacillus subtilis. Sporulation phases 0 to VI are indicated in the diagram, and the main genes involved in the process are summarized. Stage VI represents the final events leading to spore maturation inside the mother cell, while stage VII requires mother cell lysis for the spore to be released

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An example of activation of sporulation-specific genes/regulons is the cascade reaction initiated by the arbB gene, which encodes a protein (ArbB) that acts as a repressor of spo0H expression. The gene spo0H encodes the σH protein (Weir et al. 1991), which regulates the expression of σF protein, responsible for entering sporulation stage II (Wu et al. 1992; Sonoda et al. 2015). Figure 3 summarizes the different σ factors involved in the main sporulation stages.

Fig. 3
figure 3

Summary of sigma factors involved in Bacillus subtilis sporulation. SpoIIT is involved in the activation of σE in the mother cell, whereas SpoIIIL is required for σG activity in the forespore (Meeske et al. 2016)

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Briefly, the spore formation pathway mainly depends on two pivotal kinases integrated into the phosphorelay process of sporulation. The main activators and repressors systems required for sporulation initiation are depicted in Tables 1, 2, 3, 4, and 5.

Table 1 Main activator proteins involved in Bacillus (mainly subtilis) initiation of sporulation
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Table 2 Main repressors involved in Bacillus (subtilis) initiation of sporulation
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Table 3 Main genes and proteins involved in sporulation stage II
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Table 4 Main genes and proteins involved in sporulation stage III
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Table 5 Main genes and proteins involved in sporulation stage IV
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Bacteria rely on histidine kinases to react to a variety of external signals, and this also applies to sporulation. KinA is perhaps the main histidine kinase involved in the initiation of endospore formation in the family Bacillaceae. Winnen and collaborators described in 2013 that this kinase had an N-terminal region (residues 1–382) spanning three tandem Per-ARNT-Sim (PAS) domains, believed to constitute the sensor sporulation module. Upon nutrient starvation in endospore-forming bacteria, KinA inhibits the antikinase activity of KipI (gene homologues of kipI are found almost throughout all bacterial kingdom; Jacques et al. 2011a), hence allowing sporulation. KipI and KipA are the fourth and fifth genes, respectively, of a seven-cistron operon that is upregulated by high glucose concentrations and down-regulated in the presence of nitrogen. The combined actions of KinA and Kipl trigger the regulatory pathway known as the sporulation phosphorelay, which in turn activates Spo0A (the main component of the sporulation cascade). The protein Sda (Fig. 4) is also involved in KinA phosphorylation, as well as in replication and sporulation coordination (Veening et al. 2009). The gene products involved in stage 0 are depicted in Tables 1 and 2.

Fig. 4
figure 4

Early events in endospore formation, including sporulation stages 0 and II

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Sporulating stages II and III involve a differentiation program that lasts 5 hours, and, according to Eichenberger and coworkers in 2004, it involves 383 genes epistatically controlled by transcription factors σE, σF, σG (they activate 81 genes), and σK. This stage is characterized by an asymmetric division that gives rise to a sporangium, formed by the mother cell and separated from the future forespore by a closing Z-ring that leaves a narrow tunnel, also known as the “feeding tube” (Mastny et al. 2013) that links both compartments. The tunnel also contains a DNA filament that extends from the mother cell. As it is, and before the beginning of the asymmetric division to form the prospore, an axial DNA filament is formed containing two chromosomes along the longest axis of the cell, and firmly attached to each pole thanks to proteins such as RacA, Soj, Spo0J, and MinD (Wu and Errington 2003; Willis et al. 2020). In this way, when the prespore is finally formed, it tapes ca. 30% of one chromosome and the remaining 70% of the chromosome relays on the feeding tube, and particularly on the translocase SpoIIIE (Bath et al. 2000; Willis et al. 2020), which is a hexameric protein that embraces the double-stranded DNA, and translocates each arm into the prespore, presumably through the formation of small pores. It is known that the terminus chromosomal region in B. subtilis is comprised between 152 and 187°, and that this region is the last one to be translocated into the prospore (Willis et al. 2020). The feeding tube, therefore, is crucial for spore formation and maturation, as this process requires many gene products expressed by the mother cell genes that are transferred to the forespore through this tunnel. The genes involved in this stage and their function are summarized in Table 3.

Sporulating stage III is characterized by the engulfment of the forespore by the mother cell; this results in the forespore being covered by a double-membrane, inner and outer membranes (McKenney et al. 2013), within the mother cell cytosol. This phase is accompanied by a simultaneous synthesis of modified peptidoglycan, which contains the modified sugar muramic-δ-lactam and a low level of peptide cross-links between the glycan strands (Popham 2002), located between the inner and outer membranes. Deposition of a proteinaceous layer takes place mainly externally, thus completing the formation of the spore “cortex,” that constitutes the characteristic structure of Stage IV (see Tables 4 and 5 for its main components and functions).

A peculiarity of B. subtilis sporulation is that there is a temporal dissociation between the occurrence of late events and the expression of genes that determine them (Jenkinson et al. 1980), as the proteins responsible for the changes during stages V and VI are already synthesized by the end of stage IV. Stage V is characterized by the formation of the spore coat, which contains approximately 70 proteins, originated from the mother cell, many of which started migrating to the spore surface at the time of engulfment (Popham 2002). Stage VI (maturation and sporangium lysis; summarized in Table 6) starts with the synthesis of dicarboxylic dipicolinic acid (derived from l-aspartate, see Fig. 5), that chelates high amounts of Ca++ and transforms the spore into a refractile structure containing the coat proteins (Fig. 6), selectively stainable by malachite green at high temperature, and thus forming a spore crust (which is the outer most layer of spores lacking sporangium). The crust structure is composed of several coat proteins such as CotV, Cot W, CotX, CotY, CotZ, and CgeA (Bartels et al. 2019), being CotY the most important in the crust structure in terms of scaffolding and morphogenetic functions (Shuster et al. 2019; Dubois et al. 2020) along with CoX and CotZ. In addition to these coat proteins, the crust contains a variety of glycans with functions largely unknown, although at least two different glycans have been proposed: one linked to the outer coat proteins and another strictly linked to the crust (Shuster et al. 2019; Dubois et al. 2020). In this sense, the genes spsM, spsABCDEFGIJKL, yfnHGFED, ytdA-ytcABC, and cgeAB-cgeCDE have been involved in the synthesis of the surface proteins (Cangiano et al. 2014). Lately, it has been demonstrated (Dubois et al. 2020) that these sps genes encode the legionaminic acid pathway that is required for crust assembly. The legionaminic acid is a 9 carbon, beta-neuraminic acid derivative (5,7-diamino-3,5,7,9-tetradeoxy-d-glycero-beta-d-galacto-non-2-ulopyranosonic acid), also found on the flagellin of Helicobacter pylori and Campylobacter jejuni.

Table 6 Main genes and proteins involved in sporulation stages V and VI/VII
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Fig. 5
figure 5

Mechanism of synthesis of dipicolinic acid by Bacillus subtilis

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Fig. 6
figure 6

Summary of the different cot genes involved in coat formation in Bacillus subtilis spores

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Finally, the mature spore is normally released, as a dormant resistant cell, by lysis of the sporangium wall (old mother cell’s). The spore can remain dormant for a long period of time, many years until reactivation (germination) takes place when environmental conditions, such as food and temperature, permit it. During the first phases of germination, efflux of ions occurs and step by step also disassembly of the coat proteins and the cortex; most of the previously captured calcium is released. During all the time the spore was a dormant structure, well within the spore core there were a variety of unaltered mRNAs. The question is, are these mRNAs functional during germination?. This question and several others have been recently proposed by Setlow and Christie in their recent review of 2020. The existence of fully functional mRNAs in spores would indeed speed up the germination processes, since the germinating spores would pass directly to translation as the ribosomes became, in turn, functional.

Futuristically, it would be interesting if B. subtilis had receptors for 4,5-dihydroxy-2,3-pentanedione derivatives, collectively known as “autoinducers AI-2” and involved in quorum sensing responses. This, without a doubt, would facilitate the coordination of sporulation in an otherwise asynchronous culture (a recent communication on the role of autoinducer AI-2 may be found in Zhang and colleagues in 2020).

3 Secondary Metabolites Produced During Endospore Formation

3.1 Antibiotics

Spore-forming bacteria are excellent secondary metabolite producers, including antibiotics. Bu’Lock already described in 1961 the relationship between intermediary metabolism and antibiotic synthesis (Bu’Lock 1961), while Weinberg summarized the main characteristics of secondary metabolites (Weinberg 1964). According to this author, a secondary metabolite has a restricted distribution (best if species-specific), does not play an obvious role in general metabolism, and is rapidly synthesized even when bacterial growth is minimal or non-existent. Sermonti concluded in 1980 that secondary metabolism is a primitive type of metabolism. Kalenova et al. (2017) recently reported that secondary metabolites produced by Bacillus sp., isolated from late Neogene permafrost, have a very potent effect on cytokine production by human peripheral blood mononuclear cells. These metabolites induced the production of both proinflammatory (TNF-α, IL-1β, IL-8, IL-2, and IFNγ) and anti-inflammatory (IL-4 and IL-10) cytokines, and the secretion levels of cytokines were far higher than those induced by B. cereus, medicinal strain IP5832, metabolites. These results propound a putative role for these secondary metabolites in the development of immunomodulating drugs.

Manganese and copper are two transition metals that appear to be important both in endospore formation (Weinberg 1964; Krueger and Kolodziej 1976) and in secondary metabolite synthesis (i.e., iron for mycobacillin or cobalt for d-glutamyl peptide; Jansen and Hirschmann 1944; Foster and Woodruff 1946). Manganese, in particular, appears to be essential as, according to Weinberg, no other biologically active element can substitute it. Apart from transition metals, starvation (depletion of a usable nitrogen source) triggers both sporulation and secondary metabolism (including synthesis of antibiotics), originating a metabolic state known as the “stringent response” that involves GTP and active ribosomes (Lukin et al. 1983; Ochi and Ohsawa 1984).

We envisage that research into novel sources of antibiotics and secondary metabolites (as well as other pharmaceutically relevant compounds) in the near future will involve the study of yet unknown microorganisms isolated from insects, plants, or animals. Indeed, insects represent the most diverse group of animals and should constitute an excellent source of microorganisms capable of producing bioactive molecules as secondary metabolites. In his review, Bode provides prime examples of entomopathogenic bacteria as sources of secondary metabolites, these include Bacillus thuringiensis, Pseudomonas entomophila, Xenorhabdus, and Photorhabdus (Bode 2009).

The genus Bacillus is an eminent antibiotic producer (mostly polypeptidic), with already 167 peptides described by Berdy in 1974 and a number of new ones characterized since (see review by Katz and Demain 1977). The classical antibiotics produced by B. subtilis include mycobacillin, subtilin, bacilysin, bacillomycin, fungistatin, bulbiformin, bacillin, bacillaene, subsporin, bacillocin, mycosubtilin, fungocin, iturin, neocidin, and eumycin. B. brevis secretes gramicidin S, tyrocidine, linear gramicidin, brevin, edeine, eseine, bresseine, and brevistin. B. pumilus synthesizes micrococcin P, pumilin, and tetain, while B. mesentericus produces esperin, and B. licheniformis generates bacitracin, licheniformin, and proticin. Antibiotic production in B. polymyxa includes polymyxin, colistin, gatavalin, and jolipeptin, while B. circulans secretes butirosin, circulin, polypeptin, EM-49, and xylostatin. B. cereus makes biocerin, cerexin and thiocillin, and B. laterosporus synthesizes laterosporamine and laterosporin.

Those described above are all peptide antibiotics, listed by Katz and Demain in 1977, and all share the following basic properties: (1) their size is much smaller than “normal” antibiotics; (2) they are usually produced as a close family of peptides; (3) they can be constituted by either amino acids only, or be complexed with other compounds, such as polymyxins, that contain either 6-methyloctanoic acid or 6-ethylheptanoic acid as a fatty acid residue; (4) frequently contain d-amino acids not found in proteins, and (5) they are mainly resistant to hydrolysis by peptidases and proteases.

The peptide antibiotic synthesis requirements are the same for all of them; they all require amino acids, ATP, the appropriate synthase (that can be purified from cell-free extracts), Mg2+ ion, and a reducing agent. The antibiotic extends from the N-terminal to the C-terminal end, as is the case in protein synthesis, and only enzyme-bound intermediates are involved (Katz and Demain 1977). Lipmann and collaborators proposed a mechanism for the synthesis of cyclic peptide antibiotics, such as gramicidin S, which involves peptidyl transfers from enzyme-bound thioester intermediates (Gevers et al. 1969; Lipmann 1973). When the peptide antibiotic is linear (i.e., gramicidins) the pentadecapeptide remains thioester-linked, and formylation occurs after completion of the polypeptide synthesis (Bauer et al. 1972). Despite all the advances in our knowledge of the genetics, biochemistry, and synthesis of sporulation-related antibiotics, little is known about the role (or roles) that these compounds play in the producing organism. The suggested function as a biochemical sink has its merit, although, as indicated by Katz and Demain (1977), these antibiotics are produced specifically when the cell detects harsh conditions and could either be packaged in the Bacillus spore to provide a favorable environment (by eliminating competitors) during germination or inhibit spore germination until environmental conditions are favorable.

Antifungal antibiotics produced by Bacillus are somehow linked to sporulation, as they are secondary metabolites. They are not common in these bacteria, although there are some lipopeptides (Hamley 2015) with antifungal action, such as fengycin, surfactin, and iturin family compounds (Dunlap et al. 2013); and more recently, Knight and coworkers described one secreted by B. subtilis subsp inaquosorum (Knight et al. 2018). All these antibiotics are synthesized by synthetases not linked to ribosomes, they exhibit different types of cyclization and varied length of the fatty acid chain. Fengycin was the first antifungal identified (Vanittanakom et al. 1986), although surfactin is perhaps the most powerful biosurfactant and the iturin family displays a broad-spectrum antifungal activity (Knight et al. 2018).

Iturins are a group of lipopeptide antifungal amphiphilic antibiotics that act on the cytoplasmic membrane altering K+ permeability. Iturins increase membrane permeability by forming ion-conducting pores, due to their interaction with sterols and phospholipids present in the membrane. The antifungal activity of these compounds increases with the number of aggregates formed and depends on the type of amino acids contained by the lipopeptide, as well as the type of sterols present in the cytoplasmic membrane.

Iturin A (Fig. 7) is the archetype for B. subtilis lipopeptide (Besson et al. 1976). It is encoded by the iturin A operon, which spans over 38 kb and contains four open reading frames, ituD, ituA, ituB, and ituC (Tsuge et al. 2001). Recently (Zhou et al. 2020) have reported on the isolation from deep sea, of a new bacterial strain, tentatively classified within the Bacillus genus, that synthesizes two new iturin-like lipopeptides, designated as C14 iturin W, and C15 iturin W, with fungicidal activity by introducing damage into the fungal plasmalemma. Mycosubtilin, also produced by some B. subtilis strains, is similar to iturin, although there are minor differences between the two antibiotics, both in the conformation of serine and asparagine and in the order the two amino acids are found on the lipopeptides.

Fig. 7
figure 7

Comparison of the structures of Iturin A and mycosubtilin. Although the two compounds are very similar, the amino acids at positions 6 and 7 in the mycosubtilin sequence are D-Ser→L-Asn, while in iturin A these amino acids are inverted

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Iturins may have additional roles as biocontrol agents. It has been reported lately (Wang et al. 2020), that iturin A directly extracted from B. subtilis strain WL-2 readily exerts a controlling role on the fungus Phytophthora infestans (potato late blight disease that shapes a threat worldwide for Solanum tuberosum culture) through disruption of the cellular membrane and oxidative stress.

Plipastatin (A and B) are potent Bacillus antimicrobial lipopeptides (inhibitors of phospholipase A2; Volpon et al. 2000), thought to replace shortly conventional treatments in plant–fungal infections. B. subtilis synthesizes this antibiotic directed by the operon ppsABCDE operon (Vahidinasab et al. 2020); the authors accomplished the construction of a new strain able to produce in a constitutive manner, increased amounts of plipastatin.

Interestingly, recently it has been reported that some fungal–bacterial interactions are able to select mutants able to synthesize increased levels of compounds with antifungal activity (Albarracín-Orio et al. 2020). Surprisingly, the authors found that interactions of B. subtilis with the fungus Setophoma terrestris, originated bacterial variants which had lost the ability to form lipopeptides and instead had gained the capability to synthesize compounds with antifungal activity.

Genome mining applied to B. subtilis NCD-2 is giving positive results as far as unraveling the potential to find antimicrobial compounds in this strain, and also to determine the specificity of respective gene clusters (Su et al. 2020). The strain is a good one to fight soil-borne plant pathogenic fungi, since it has been described as producer of broad-spectrum antifungal compounds. Additional species of the Bacillus genus, such as B. velezensis have been described as bein g good sources of L ipopeptides and polyketides (Ruiz-García et al. 2005; Rabbee and Baek 2020), that allows the bacterium to exert quite positive antagonistic effects against plant pathogens, such as Verticillium dahlia that causes wilt in olive trees (Castro et al. 2020), or to promote the growth of Malus hupehensis Rehd (Wang et al. 2019) while related to B. subtilis, is different in that it contains nine gene clusters (namely, srf, bmy, fen, dhb, bac, mln, bae, dfn, and nrs) by which the bacterium produces a large variety of antimicrobial compounds (Rabbee and Baek 2020).

Table 7 summarizes the most relevant antibiotics produced by Bacillus.

Table 7 Main antibiotics produced by Bacillus species
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3.2 Alkanes

Alkanes, with a general molecular formula of CnH2n+2, represent the simplest organic molecules that are widely distributed in nature; they are stable due to their backbone carbon atoms, having attained their octet of electrons through the formation of four covalent bonds.

Alkanes can be used as an advanced biofuel because of their high-energy content, which is 30% higher than ethanol. Although it has been reported that recombinant E. coli strains can produce a different range of alkanes, such as pentadecane and heptadecane (Choi and Lee 2013), the use of these compounds is far from being industrially exploited, and this includes the alkanes produced as secondary metabolites in Gram-positive sporulating bacteria. However, most sporulating bacteria appear to be good alkane degraders. Efficient microbial biosynthesis of alkanes with long carbon chains is difficult to achieve in a single organism (Lehtinen et al. 2018), as this process requires a two-step pathway. Hence, the first step of CO2 reduction to acetate should be carried out by a homoacetogenic bacterium following the Wood–Ljungdahl pathway. Transformation into long-chain hydrocarbons, on the other hand, would be best achieved by a second engineered microorganism expressing the enzymes acyl-ACP reductase (AAR) and aldehyde deformylating oxygenase (ADO); ADO is regarded as the bottleneck for the alkane biosynthesis, due to the low activity of the enzyme.

The available data indicate that aerobic Gram-positive sporulating bacteria do not naturally exhibit the ability to generate alkanes, at least not in enough quantities to be industrially relevant. In fact, some results suggest that these microorganisms are totally unable to do so unless they are genetically engineered. However, this appears not to be the case for anaerobic clostridia; Bagaeva and Zinurova reported in 2004 that Clostridium pasteurianum could in fact synthesize alkanes (C25-C35 intracellular and C11-C24 extracellular) at the end of its logarithmic growth phase, in an atmosphere formed by a mixture of CO2+H2/ argon. A particularity of this bacterial species, not present in Gram-negative bacteria, is its ability to produce branched alkanes. Despite the aforementioned, there have been recent papers describing the ability of certain strains of B. subtilis to form a “volatilome” formed by secondary metabolites that include hydrocarbons, ketones, alcohols, aldehydes, ester, acids, among many others (up to 231), and some having the property to control the fungal population in the rhizosphere (Kai 2020).

3.3 Parasporal Crystals

Parasporal crystals constitute one of the few examples in Biology in which a cell contains a crystallized structure with biological activity. The archetypes for these structures are the bipyramidal parasporal crystals of B. thuringiensis, a Gram-positive, endospore-forming bacterium closely related to both B. cereus and B. anthracis, the causative agent of anthrax. The crystals are synthesized during endospore formation and are hence associated with the secondary metabolism of Bacillaceae. This microorganism was initially described by Ishiwatari Shigetane (1901) in the silkworm and named Bacillus sotto. It was later renamed as B. thuringiensis after Berliner (1915) isolated it from the gut of the flour moth caterpillar in Thuringia, Germany (Milner 1994). There are currently several known B. thuringiensis subspecies (all producing parasporal crystals) that display different toxicity towards insects, such as Lepidoptera, Coleoptera, Diptera, Hymenoptera, and Nematoda (Schnepf et al. 1998; Wei et al. 2003; Soberón et al. 2013). The proteinaceous nature (δ-endotoxin or cry proteins) of the parasporal crystal was described by Hannay and Fitz-James in 1955, while the crystal-specific toxicity towards caterpillars of the lepidopteran species Pieris brassicae was known since 1965 (Lecadet and Martouret 1965). This research defined the type subspecies, Berliner, while further subspecies, such as kurstaki, israelensis, and aizawa, were later described. In 1968 de Barjac and Bonnefoi carried out the first attempt to rationalize the taxonomy of B. thuringiensis subspecies and varieties. Cry proteins are encoded by cry genes, which are located on a plasmid in most B. thuringiensis strains. In 1979 both Robert A. Zakharian and coworkers and Miteva independently reported the plasmid location of the cry genes, suggesting a role for the plasmid in both endospore and crystal formation (Zakharian et al. 1979; Miteva 1979)

There are multiple studies on the mode of action of B. thuringiensis toxins (i.e., Koch et al. 2015) which, unlike chemical pesticides, are effective only after being ingested by the insect. The parasporal Cry proteins are approximately 70–140 kDa and, once within the gastrointestinal tract of insects, they become activated by proteases and specifically bind to epithelial cells receptors (mostly cadherin-like glycoproteins); they create pores, formed by oligomers of six Cry molecules (this is essential for lethality), that cause a dramatic cellular osmotic imbalance which eventually leads to the death of the insect.

Since the cry genes were cloned in 1981 (Schnepf and Whiteley 1981) there have been many successful attempts to express them in transgenic crop plants, such as corn, some of which involved biotechnological companies such as Monsanto. The initial concerns about the possible negative effects of the thuringiensis toxins, either released into the environment through the roots of the transgenic plants, or present in the foodstuffs, resulted in the experiments being concealed from the public, such as the work by Saxena and Stotzky in 2000. In fact, there was no need for such concern, as indicated by Koch and coworkers in 2015: “Cry proteins are very limited in their duration of effectiveness because they can be washed off the plant (e.g., by rain) or inactivated by sunlight within days after application, and they require considerable water, heat, and feedstock to produce, and must be manually applied, either by hand sprayer on small plots or by machine if applied to large tracts.” Because of their safety of use, a variety of Cry proteins have been approved for use in at least one country to protect against lepidopteran pests, and these include: Cry1Ab inserted into maize by Monsanto; Cry1Ac expressed in cotton, corn, brinjal, and soy by Monsanto; Cry1A.105 + Cry2Ab2 and Cry1Ac + Cry2Ab2 were introduced in maize varieties by Monsanto; Cry1Ac + Cry1F in cotton and soy by Dow; Cry1Fa2 in maize by Dow; Cry1Ac + Cry1F in cotton and soy by Dow; Cry1Ab + Cry2Ae in cotton by Bayer. In addition, Cry34Ab1 + Cry35Ab1 were expressed in maize by Dow and DuPont to protect from Coleoptera (Koch et al. 2015). The economic importance of Cry proteins in crop protection was reviewed by Marques and coworkers in 2019. As for the price for the production of these proteins, it passes through the obtention of Cry protein-overproducing strains. An easy way of doing this was recently reported by Quan and coworkers in 2020. The authors, by simply deleting the leu B gene (encodes for the 3-isopropylmalate dehydrogenase in the leucine synthesis pathway) in a conditionally asporogenous B. thuringiensis, were able to overexpress such a protein.

The isolation of new and natural strains of B. thuringiensis must proceed at whatever pace, since Nature has always provided new useful mutations for human industrial applications. In this sense, Liu and colleagues reported in 2020 the isolation of a new strain B. thuringiensis, X023, which exhibits enhanced insecticidal (against Plutella xylostella) activity by copper ions. This ion promoted the expression of cry1Ac and vip3Aa, the synthesis of aminoacids, the glyoxalate pathway, as well as the poly-β-hydroxybutyrate accumulation; all these compounds are necessary for the synthesis of parasporal crystals (Liu et al. 2020).

Concerning the safety of use of these biocides, they are generally considered as safe, as they are quite specific in their mode of action against lepidopteran or Diptera insects; however, their use may disturb the general metabolism of other insects initially thought not to be susceptible to the cry toxins. In this sense, Nawrot-Esposito and colleagues reported in 2020 that these bioinsecticides cause defects in the larval development of Drosophila melanogaster, by reducing the protein digestion. Differential side-effects of thuringiensis biocides have also been reported on this fly by Babin and coworkers in 2020 non-target Drosophila flies.

Late reports (Ursino et al. 2020) have shown that B. subtilis may be directly used to produce mosquitocidal toxins against species of Aedes, known to transmit some arbovirus-caused diseases. Some of these diseases include Dengue fever and Yellow fever (transmitted by Aedes aegypti), Japanese Encephalitis and Rift Valley fever (transmitted by Culex tritaeniorhynchus), among others. It is clear that the genetic background of B. subtilis is by far better known than that of B. thuringiensis; so any genetic manipulation with projection in the industry (i.e., increase production of lepidopteran or dipteral toxins, or obtention of altogether different toxins) should have a better outcome if developed in B. subtilis. The deepest study on this topic follows in the next chapter.

3.4 Lanthipeptides

Lanthipeptides constitute “natural products,” ribosomally synthesized by bacilli as secondary metabolites, and are posttranslationally modified peptides (RiPPs) (Nolan and Walsh 2009; Dias et al. 2015). These modifications include the formation of meso-lanthionine and 3-methyllanthionine, as well as dehydrated amino acids. Xin and coworkers classified lanthipeptides into four groups in 2015, depending on the enzymes involved in post-translational processing. In group I, amino acid dehydration is carried out by a dedicated lanthipeptide dehydratase, and cyclization is catalyzed by a lanthipeptide cyclase; in group II, the lanthipeptide is modified by specific proteins; whereas in groups III and IV, lanthipeptide dehydration and cyclization reactions are carried out by multifunctional enzymes. B. thuringiensis and B. cereus are able to produce more than 20 bacteriocins, many with potential usage both in the Food Science industry and in the clinical control of pathogenic bacteria (Rea et al. 2010). Cerecidins merit a special citation among the lanthipeptides produced by the cereus group, for their prospective usefulness in controlling pathogenic bacteria (Wang et al. 2014). In fact, cerecidins A1 and A7 are known to be active against Gram-positive bacteria, displaying remarkable efficacy against both multidrug-resistant S. aureus (MRSA strains) and vancomycin-resistant Enterococcus faecalis.

As a general rule, lanthipeptides are encoded by structural genes (lanA), normally synthesized as non-active precursors that are later hydrolyzed into an N-terminal peptide and a C-terminal peptide; the N-terminal leader peptide is important for post-translational modifications (Yang and van der Donk 2013; Dias et al. 2015). The structural genes for these peptides (lanA) frequently cluster with genomic islands, this is the case for lanthipeptides synthesized by Bacillus methylotrophicus (Dias et al. 2015), and this supports the notion that their production might be the result of evolutionary adaptation to best achieve their in vivo function, either as controllers of other microorganisms (Wang et al. 2014) or as plant growth promoters (Hao et al. 2012). It appears that Gram-positive spore-forming bacteria require antimicrobial lanthipeptides to conquer harsh environments, as the strains and bacterial species isolated from harder habitats seem to produce novel lanthipeptides with new characteristics (Othoum et al. 2018). The structural lanthipeptide genes have been cloned (Ongey et al. 2018) and are in the process of being genetically modified in order to both increase production of these compounds, that are normally produced in low amounts by their “natural” bacterial species, and broaden their application. Lanthipeptides are very promising bioactive compounds with a great potential use not only in human and veterinary medicine but also in the control of bacteria that cause food spoilage.

4 Secondary Metabolites in the Environment

Microbiologists are still blatantly ignorant concerning the number of bacterial species on earth and can only hypothesize to estimate the enormous number (perhaps up to 80%) of bacteria that cannot yet be grown in axenic conditions in the laboratory. This is either due to the lack of appropriate culture media or because microorganisms are rarely found in nature in pure culture (only pathogenic microorganisms constitute a monoculture when causing a disease), and to flourish, they need to be in contact with other microorganisms, often through “quorum sensing” mechanisms, or may require secondary metabolites such as antibiotics or lanthipeptides. Zengler and coworkers researched this topic in their interesting publication entitled “Cultivating the uncultured” (2002), putting forward a proposal for a universal method to detect, or at least estimate, the numerous unculturable microorganisms present in the environment. According to Nai and Meyer (2018) “Only a paradigm shift in cultivation techniques—from axenic to mixed cultures—can allow a full comprehension of the (chemical) communication of microorganisms, with profound consequences for natural product discovery, microbial ecology, symbiosis, and pathogenesis.” This means that it is essential to develop the microbial co-culture technology, as well as understand the effects of secondary metabolites produced by a given microbial specimen on the biological development of neighboring organisms. Despite our lack of knowledge in these basic research areas, some advances are slowly taking place, among them are the early reports by Johnson and colleagues and Patel and Roth, both in 1978. More recently, Shank (2013) studied bacterial co-cultures to examine the influence of secondary metabolites on microbial interspecies interactions in the natural environment. In addition, Nai and Meyer (2018) reported that the three technical approaches currently used (3D-bioprinting, single-cell metabolomics, and microfluidics) can allow systematic co-culture of three or more microorganisms. Hopefully, the next few decades would bring a much better understanding of the complex microbial relationships that occur in “natural” environments.

This knowledge and understanding could revitalize the search for novel natural compounds with antimicrobial activity, such as antibiotics, a task currently practically abandoned by pharmaceutical companies throughout the world. Some authors estimate that there are still up to 1000 novel antimicrobials awaiting discovery, as well as a great number of yet unknown enzybiotics (Veiga-Crespo et al. 2007). Production of novel drugs could be attained by microbial co-cultures in which the secondary metabolites secreted by one species induce expression of antibiotics or antimicrobials in another species (Bertrand et al. 2014). Gram-positive organisms and spore-forming bacteria, together with members of the Pseudomonadaceae family, are prime candidates to use in co-culture experiments, as they are among the best secondary metabolite producers. Although the number of combinations for laboratory co-culture experiments is high, the family Bacillaceae (B. subtilis, B. cereus, B. licheniformis, B. thuringiensis, or B. brevis) can be anticipated as good candidates for co-culture with antibiotic-producing fungi, such as Penicillium, Aspergillus, or Acremonium. These co-cultures could result in the production of novel, improved β-lactams. Other good contenders for co-culture experiments are members of the Streptococcaceae and Myxococcaceae families, as they constitute well known antibiotic producers. This opens up the exciting possibility of obtaining new and improved antibacterials in the near future, as long as both governments and private companies are willing to invest in this new venture. This research is essential for the future of antibiotic development and must be done now to find new antimicrobials to counteract the threat of poly-resistant bacterial strains. Antibiotic resistance was described by the World Health Organization in 2018 as “one of the biggest threats to global health, food security, and development” facing humanity today.

5 Toxins

The ability of spore-forming Gram-positive bacilli (such as Bacillus or Clostridium) to produce toxins is very high and, in most bacteria, it is linked to secondary metabolism. These compounds include some of the most potent neurotoxins known in nature (i.e., C. botulinum, C. perfringens, C. sordellii, or Cl. tetani). Although the toxigenic phenotype has mainly been assigned to the strict anaerobic Clostridium genus, this ability is also displayed by some species of the mostly aerobic Bacillus genus, such as B. cereus and B. anthracis. Clostridium botulinum was named Bacillus botulinus by Emile van Ermengem, who originally isolated it from spoiled ham (1897). The American bacteriologist Ida Albertina Bengtson (1881–1952), the first woman hired to work at the National Institutes of Health (Lindenmann 2005), renamed it as Clostridium in 1924, as it is an anaerobic organism, hence restricting the genus Bacillus to aerobic spore-forming rods. Despite this, the bacterium was still referred to as Bacillus in publications well into the 1950s, such as in the article by Bulatova and Matveev (1957) concerning clostridial species. Finally, Collins et al. (1994) reorganized and redefined the species included in the genus Clostridium.

These neurotoxins produced by these bacteria are proteinaceous in nature and composed of two subunits (α and β). Botulism toxin was originally purified and crystallized by Lamanna et al. (1946), and is classified into eight types, referred to as A to H (Dover et al. 2014); A and B are the most important to humans. This toxin prevents the release of the neurotransmitter acetylcholine from axon endings at the neuromuscular junction and causes flaccid paralysis. The botulinic toxin is currently used in a number of medical applications, ranging from wrinkle reduction to the treatment of limb spasticity after a stroke (Sun et al. 2019); it is also applied in esthetic plastic surgery to treat facial sagging (Zhou et al. 2019), as well as in the treatment of Parkinson’s disease (Cardoso 2018), bruxism (Tinastepe et al. 2015) and strabism (Scott 1981).

Eklund et al. demonstrated in 1971 that, when C. botulinum type C is cured of its prophage, the bacteriophage Ceβ, it ceases to produce toxin and becomes nontoxigenic C. novyi type A. This discovery could open the possibility of toxin gene movilization among different clostridial species (Eklund et al. 1974). In the late twentieth century, a neurotoxigenic Clostridium butyricum strain, isolated from food, was found to be involved in an outbreak of food-borne type E botulism (Aureli et al. 1986; Meng et al. 1997). In addition, Cassir and coworkers recently demonstrated (2016) that Clostridium butyricum, normally used as a probiotic, could become a new emerging pathogen. Enterococcus faecium has also been reported as a potential producer of botulinum toxin, presumably due to horizontal transmission of the toxic gene from a clostridial strain (Zhang et al. 2018)