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Published in final edited form as: Curr Opin Microbiol. 2015 Feb 1;24:88–95. doi: 10.1016/j.mib.2015.01.006

Diverse Mechanisms Regulate Sporulation Sigma Factor Activity in the Firmicutes

Kelly A Fimlaid 1,2, Aimee Shen 1,*
PMCID: PMC4380625  NIHMSID: NIHMS656251  PMID: 25646759

Abstract

Sporulation allows bacteria to survive adverse conditions and is essential to the lifecycle of some obligate anaerobes. In Bacillus subtilis, the sporulation-specific sigma factors, σF, σE, σG, and σK, activate compartment-specific transcriptional programs that drive sporulation through its morphological stages. The regulation of these sigma factors was predicted to be conserved across the Firmicutes, since the regulatory proteins controlling their activation are largely conserved. However, recent studies in (Pepto)clostridium difficile, Clostridium acetobutylicum, Clostridium perfringens, and Clostridium botulinum have revealed striking differences in the order, activation, and function of sporulation sigma factors. These studies indicate that gene conservation does not necessarily predict gene function and that new mechanisms for controlling cell fate determination remain to be discovered in the anaerobic Clostridia.

Introduction

Of the many species of bacteria known to humans, only a small subset can undergo the defense strategy known as sporulation. During this developmental process, a vegetative bacterial cell differentiates into a metabolically dormant cell-type known as an endospore (referred to as a “spore” hereafter) [1–3]. By forming spores, bacteria can survive adverse environmental conditions by “reawakening” when favorable conditions return [4]. For obligate anaerobes that inhabit or transiently live in the gut, spore formation is essential for survival [5,6].

Sporulation begins with the formation of an asymmetrically placed septum, which creates two morphologically distinct but genetically identical compartments: a larger mother cell and smaller forespore ([1,3,7], Figure 1A). Migration of the mother cell membrane around the forespore during a phagocytic-like event known as engulfment leaves the forespore suspended in the mother cell cytosol as a double membrane-bound protoplast. A thick peptidoglycan layer known as the cortex forms between the double membranes, while a series of protective proteinaceous coat layers form around the forespore [8,9]. Upon completion of forespore maturation, the mother cell undergoes programmed lysis, releasing the mature spore into the environment [10].

Figure 1. Morphological Phenotypes of Sigma Factor Mutants in Bacillus subtilis, (Pepto)Clostridium difficile, Clostridium acetobutylicum, Clostridium perfringens, and Clostridium botulinum.

Figure 1

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utations in sporulation specific sigma (σ) factor genes, sigF, sigE, sigG, and sigK, result in a variety of phenotypes. A. Morphological stages of sporulation (I–VII) defined by transmission electron microscopy (TEM) studies in B. subtilis [7]. Stage I, decision to sporulate (no morphological phenotype); Stage II, completion of asymmetric division; Stage III, completion of engulfment; Stage IV cortex formation (pink) between the inner and outer forespore membrane; Stage V, coat assembly around the outer forespore membrane (blue); Stage VI, spore core maturation, e.g. chromosome condensation; Stage VII, release of fully mature spore following mother cell lysis. Key structures that comprise a mature spore are labeled. B. B. subtilis sigF− and sigE− complete asymmetric division (Stage II), while sigG− and sigK− complete engulfment but fail to make coat or cortex (Stage III). C. difficile sigF− completes asymmetric division; it also makes coat that mislocalizes to the mother cell cytosol (blue); sigE− arrests at asymmetric division (Stage II); sigG− does not complete engulfment in strain JIR8094 (shown, [46]), although it does in strain 630Δerm [48]. Regardless, both sigG− strains fail to make cortex, despite producing coat (blue) that localizes around the forespore; sigK− produces cortex (pink) but no detectable coat. C. acetobutylicum sigF−, sigE−, and sigK− do not complete asymmetric division (Stage I), although a small population (< 2%) of sigE− mutants complete asymmetric division [55]; sigG− completes engulfment and makes some cortex along with coat-like fragments that localize next to the forespore. C. perfringens sigE− is blocked at asymmetric division (Stage II), while sigK− does not initiate asymmetric division (Stage I); sigF− and sigG− phenotypes have yet to be analyzed by TEM. C. botulinum sigF− and sigE− complete asymmetric division (Stage II); sigG− completes engulfment and appears to make coat fragments that localize around the forespore; sigK− TEM has not been performed.

Extensive studies of this developmental process in B. subtilis indicate that sporulation is primarily controlled at the transcriptional level by four sporulation-specific sigma factors, σF, σE, σG, and σK [11]. During sporulation, these sigma factors are post-translationally activated at specific times in specific compartments. This pattern of regulation produces distinct lines of gene expression in the mother cell and forespore that drive sporulation through its morphological stages.

While the morphological changes required to form a spore are generally conserved [2], recent studies in clostridial species have revealed that diverse mechanisms control the activation and function of the sporulation-specific sigma factors. This review summarizes these findings and highlights that there are many paths to producing a spore. While significant differences also exist in the initiation of sporulation among Firmicutes, these have been reviewed elsewhere [12,13].

Regulation of sporulation in Bacillus subtilis

Activation of the master transcriptional regulator Spo0A leads to the sequential activation of σF in the forespore, σE in the mother cell, σG in the forespore, and σK in the mother cell. Multiple transmembrane signaling events are required to produce this “crisscross” pattern of sigma factor activation, which links transcriptional changes to morphological changes ([11], Figure 2, black arrows).

Figure 2. Conserved sporulation-specific sigma factors regulate sporulation in diverse ways in the Firmicutes.

Figure 2

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In B. subtilis, sigma factor activity is regulated in a “crisscross” pattern: σF, σE, σG, and σK are sequentially and alternately activated in the forespore and mother cell compartment, respectively. After Spo0A-mediated activation of sigF and sigE, σF becomes active in the forespore. Expression of the σF regulon leads to the activation of σE in the mother cell. Products of the σF and σE regulon activate σG in the forespore compartment. σG auto-activates its production and inducesσK activation in the mother cell. σF and σE are required for sigG and sigK expression in the forespore and mother cell, respectively. In C. difficile, after Spo0A-mediated transcriptional activation of sigF and sigE, σF activates σG via an unknown post-translational mechanism. σF also activates σE, although the requirement is only partial (dotted black line) due to σF-independent expression of spoIIR. σE activates sigK transcription; σK is active following translation. sigG expression is activated by Spo0A and may be subject to auto-activation. In C. acetobutylicum, σK activates spo0A expression. Spo0A in turn activates sigF and sigE transcription. The mechanisms controlling the activity of σE, σG, and/or σK are unknown (question marks), although initial studies suggest that σG may be dispensable for late σK function. It is hypothesized that σK may be epigenetically inherited so that Spo0A can be activated during vegetative growth ([56], red arrow). Green arrows designate transcriptional activation; black arrows designate post-translational activation; dotted arrow indicates partial requirement for activation; question marks indicate that more work needs to be done to determine the mechanisms controlling sigma factor gene transcription and/or sigma factor activation.

The early sigma factors σF and σE are produced in a Spo0A-dependent manner prior to the formation of a polar septum; however, these factors remain inactive until asymmetric division is complete. In the forespore, σF activation begins when the phosphatase SpoIIE preferentially activates the anti-anti sigma factor SpoIIAA following polar septum formation [14,15]. Activated SpoIIAA antagonizes anti-sigma factor SpoIIAB binding to σF in the forespore [16], freeing σF to activate sigG and spoIIR expression. SpoIIR is secreted into the intermembrane space and activates the SpoIIGA protease to remove an inhibitory pro-peptide from σE in the mother cell [17–19].

In the mother cell, σE activation drives the transcription of genes encoding a hydrolase complex [20,21] SpoIIM/SpoIID/SpoIIP that mediates engulfment [22,23]. By thinning the peptidoglycan layer of the polar septum, the complex permits the assembly of a channel composed of components controlled by both forespore-specific σF and mother cell-specific σE [24,25]. This channel, consisting of SpoIIQ (σF-controlled) and SpoIIIAA-AH (σE-controlled), also known as the ‘feeding tube’, bridges the mother cell and forespore compartments and is necessary for σG activation in the forespore [26–28]. The feeding tube has homology to secretion systems and transports unknown small molecules into the forespore that are required for σG activation [26–28]. σG activity also appears to require completion of engulfment, [29] although recent studies challenge this notion [30]. Regardless, σG activation depends both on the status of the mother cell and forespore. Active σG auto-activates its own production [31] and drives a new transcriptional program encoding determinants important for late sporulation sigma factor σK activation in the mother cell [32].

The mother cell-specific σK, the last sigma factor in the sporulation pathway, is regulated by multiple mechanisms (Figure 3). sigK expression requires both σE and SpoIIID, a σE-regulated transcription factor [33]. σK production depends on σE- and SpoIIID-mediated activation of spoIVCA, which encodes the recombinase required for excising the skin element, a phage DNA-like insertion that disrupts sigK [34]. σK activity requires proteolytic cleavage by the intramembrane protease SpoIVFB [35], which removes an inhibitory pro-peptide from σK similar to σE. Since SpoIVFB activity depends on the σF-/σG-dependent protease, SpoIVB [35] σK activation in the mother cell is coupled to σG activity in the forespore. Active σK induces the expression of genes whose products are required for cortex and coat formation [21].

Figure 3. Regulation of σK activity differs in the Firmicutes.

Figure 3

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σK is regulated by transcriptional and post-translational mechanisms during sporulation. In B. subtilis and C. difficile, a phage DNA-like skin element must excise from genomic DNA prior to sigK transcription. In B. subtilis, after skin element excision, σE and the σE-regulated transcription factor SpoIIID activate sigK expression. σK is produced with an inhibitory pro-peptide that undergoes proteolytic activation via SpoIVFB. In C. difficile, σK regulation is similar to B. subtilis with the exception that σK lacks a pro-peptide and is active upon translation. In C. acetobutylicum, σK acts at two stages during sporulation. Early σK function has been hypothesized to occur via epigenetic inheritance or by early activation of sigK transcription by σA [56]. However, these hypotheses have not been tested. Late sigK transcription is mediated by σE, although the involvement of SpoIIID for transcriptional activation remains to be determined. It is unclear whether σK undergoes proteolytic activation, although SpoIVFB would be predicted to cleave σK. In C. perfringens, early sigK transcription appears to occur via read-through transcription of CPR_1739 [57], while late transcription is mediated by σE. Whether SpoIIID activates sigK transcription has not been determined, although σE does not control spoIIID expression. While SpoIVFB is conserved, it is unclear if it removes the pro-peptide from σK; whether cleaved σK is the active form has also not been determined. In C. botulinum, factors controlling sigK transcription are unknown. WhetherσK undergoes proteolytic activation is also unclear. Based on gene conservation, it is hypothesized that σE and SpoIIID activate sigK transcription, and SpoIVFB activates σK pro-peptide cleavage.

Consistent with their roles in regulating engulfment, both sigF and sigE mutants arrest at asymmetric division ([36], Figure 1B). A sigG mutant completes engulfment but fails to make σK-dependent cortex or coat [27,37]. A sigK mutant phenocopies a sigG mutant [38].

Nested within this regulatory framework are a series of feedback and feedforward loops that allow the late sigma factors (σG and σK) to replace the activity of the early sigma factors (σF and σE) in their respective compartments [39]. σE-regulated gene products activate σK and down-regulate σE-mediated gene expression [40], while a σF-regulated anti-σF sigma factor promotes σG activation [41].

Thus, the B. subtilis sporulation cascade is organized such that the sporulation-specific sigma factors are sequentially activated through a series of intercompartmental signaling events, which in turn are coupled to morphological changes. Events in the forespore are intimately tied in space and time to events in the mother cell, and additional regulatory loops fine-tune the activity of each sporulation-specific sigma factor.

Regulation of sporulation in the Clostridia

Since the sporulation-specific sigma factors are strictly conserved among spore-forming bacteria [39,42,43], and the signaling proteins controlling their activity in B. subtilis are largely conserved, the sigma factors were predicted to be sequentially activated across the Firmicutes. This hypothesis, however, has been overturned by recent studies in multiple clostridial species.

(Pepto)Clostridium difficile

In the Clostridia, sporulation has been most characterized in the nosocomial pathogen PeptoClostridium difficile (still known as C. difficile) [44]. While C. difficile was previously classified as a Clostridium species in the family Clostridiaceae, recent phylogenetic analyses reclassified C. difficile as part of the Peptostreptococcaceae family, genus Peptoclostridium [45].

At the transcriptional level, C. difficile sporulation sigma factor gene regulation resembles that of B. subtilis with the notable exception that sigG transcription does not depend on σF [46,47]. C. difficile sigma factor activity exhibits a similar compartmentalization as B. subtilis, with σF and σG activity being forespore-specific, and σE and σK being mother cell-specific [48]. However, in contrast with B. subtilis, minimal intercompartmental signaling is observed during C. difficile sporulation, and sigma factor activation is not as tightly coupled to morphological changes [49].

Although σF is likely activated by a similar mechanism as B. subtilis based on the conservation of SpoIIE, SpoIIAA, and SpoIIAB [39,42,43], C. difficile σE activation only partially requires σF-regulated transmembrane proteolysis because spoIIR is still expressed in a C. difficile sigF mutant [47]. As a result, partial σE processing and activity is observed in the absence of σF. C. difficile σG activation occurs independent of both engulfment and σE-controlled transmembrane signaling events [46,47], since the σE-controlled feeding tube appears to be dispensable for σG activity. Instead, C. difficile σG requires σF for post-translational activation via an unknown mechanism [46]. σK activity occurs independent of the σG-mediated transmembrane signaling observed in B. subtilis, since C. difficile σK lacks an inhibitory propeptide and is active upon translation ([50], Figure 3). Production of σK nevertheless still requires skin element excision [48,51] similar to B. subtilis, an event that may regulate the timing of σK function [49].

Both C. difficile sigF and sigE mutants are blocked at asymmetric division [46,47], Figure 1B), consistent with their regulation of the peptidoglycan hydrolase genes spoIIP and spoIID [46,47]. Due to the partial activation of σE in a sigF mutant [46,47], mislocalized coat can be detected in the mother cell cytosol in sigF− cells (Figure 1B) [46]. C. difficile sigG mutants have been observed to arrest either during engulfment [46] or following engulfment [47]. The variability in these phenotypes may reflect differences in the strains and growth conditions used. Nevertheless, sigG mutants do not make cortex, and, in contrast with B. subtilis, they polymerize coat around the forespore [46,48]. In further contrast with B. subtilis, a C. difficile sigK mutant produces cortex, indicating that σK is dispensable for cortex development [46,48] and that the forespore regulates cortex synthesis in C. difficile. σK nevertheless controls coat polymerization, similar to B. subtilis, since no polymerized coat is observed in a C. difficile sigK mutant [45,46,48].

Thus, the regulatory architecture of sporulation in C. difficile produces a bifurcated rather than crisscross pathway, with the forespore line of gene expression occurring largely independently of the mother cell line of gene expression (Figure 2, black arrows). Accordingly, the sequence of sigma factor activation is not as tightly controlled as in B. subtilis, since σG can be activated before σE and σK [46–48], coincident production of σE and σK during C. difficile sporulation does not strongly impact spore formation [50] in contrast with B. subtilis [52], and negative feedback loops do not function as prominently in C. difficile [40,50].

Lastly, the sporulation sigma factor regulons are collectively smaller in C. difficile than in B. subtilis, with global transcriptional analyses identifying ~200 genes being activated by these factors in C. difficile [46,47] relative to the ~450 genes in B. subtilis [21,32,53]. Whether this difference is functionally significant, the sigma factors control different cellular processes during C. difficile sporulation relative to B. subtilis (Figure 1B).

Clostridium spp

Studies of sporulation sigma factor function in Clostridium spp. have mainly been limited to C. acetobutylicum, C. perfringens, and C. botulinum. While most sporulation sigma factor mutants have been constructed, the mechanisms controlling sporulation sigma factor activation have not been fully characterized. Based on gene conservation, the minimal machinery for activating σF, σE, σG, and σK are conserved in these species [13,39,42,43], and both σE and σK carry a propeptide predicted to inhibit their function [39]. Despite this gene conservation, C. acetobutylicum SpoIIE is necessary for sigF expression [54] in contrast with B. subtilis [15] and no processing of σE is observed [55] in contrast with B. subtilis and C. difficile, suggesting that divergent mechanisms may control sigma factor activation in Clostridium spp. Indeed, the timing of σK activation in Clostridium spp. exhibits clear differences relative to B. subtilis and C. difficile, with σK being activated early during sporulation ([56–58], Figure 2) and independent of skin excision (Figure 3). In C. acetobutylicum, σK surprisingly functions at two discrete stages of sporulation ([56], Figure 2). Prior to sporulation initiation, σK activates spo0A expression, while at late stages of sporulation, σK activates coat protein gene expression [56]. Since σK carries a pro-peptide and spoIVFB is only expressed late during sporulation [56], it is unclear how early σK activity is achieved; an intriguing mechanism that has been proposed is that active σK is epigenetic inherited by later cell generations [56].

In C. acetobutylicum, differences in sporulation sigma factor gene expression are also observed. While σF induces sigG expression similar to B. subtilis, C. acetobutylicum σF activates sigE expression [59] in contrast with B. subtilis and C. difficile. Late during sporulation, σE induces sigK expression [56] similar to C. difficile and B. subtilis, whether SpoIIID is required for this induction is unclear.

In contrast with B. subitlis and C. difficile, C. acetobutylicum sigK, sigF, and sigE mutants are blocked prior to asymmetric division ([55,56,59], Figure 1B). Since σF activation depends on polar septum formation in B. subtilis [16], and C. acetobutylicum σE is required for asymmetric division [59], C. acetobutylicum σF may require σE for activity. While a C. acetobutylicum sigG mutant completes engulfment similar to B. subtilis, coat fragments are visible around the forespore [55] in contrast with B. subtilis but similar to C. difficile. In contrast with both organisms, a C. acetobutylicum sigG mutant makes cortex (although to a lesser extent than wild type, Figure 1B, [55]). Notably, artificial expression of spo0A in a sigK mutant allows sporulation to proceed past engulfment but prior to coat and cortex formation [56].

The mechanisms controlling C. perfringens have not been characterized, although both σE and σK undergo proteolytic processing [57]. Consistent with the early function of σK during sporulation, C. perfringensσK is required for sigE expression, even though σE induces late stage σK production [57]. Whether C. perfringens σK activates spo0A expression, and whether SpoIIID activates sigK expression, remains to be tested. However, in contrast with B. subtilis and C. difficile, C. perfringens spoIIID expression occurs independent of σE [57]. While Western blot analyses indicate that σF controls σE production [60] similar to C. acetobutylicum, semi-quantitative RT-PCR analyses suggest that σK up-regulates sigF [57], highlighting the need for further studies in C. perfringens.

The morphological stage(s) at which C. perfringens sigF and sigG mutants are arrested are unknown, although a sigK mutant is blocked before asymmetric division [57] similar to C. acetobutylicum. In contrast with C. acetobutylicum, a sigE mutant arrests after asymmetric division ([57], Figure 1B).

In C. botulinum, studies of sporulation sigma factor regulation have been restricted to transcriptional analyses. sigK exhibits a similar two-stage expression profile [61] as C. acetobutylicum and C. perfringens, with sigK being expressed early during sporulation but rising dramatically in a σE- and σF-dependent manner late during sporulation [61]. Similar to C. acetobutylicum, σK activates spo0A and sigF [58]. Whether σK also regulates late stages of sporulation needs to be determined, as do the regulatory mechanisms controlling σF and σG function. Intriguingly, spo0A transcripts are under-expressed in sigF, sigE, and sigG mutants via an unknown mechanism [61].

C. botulinum sigF and sigE mutants arrest at asymmetric division [61] in contrast with C. acetobutylicum, while a C. botulinum sigG mutant completes engulfment and forms coat fragments but no cortex [61]. Thus, σG regulates cortex but not coat formation similar to C. difficile and C. acetobutylicum. Whether a sigK mutant is arrested at an earlier stage of sporulation than sigF and sigE mutants is unclear.

While further analyses are clearly needed to elucidate the mechanisms controlling the activity and compartmentalization of Clostridium spp. sporulation sigma factors, the control of the master transcriptional regulator Spo0A by σK defined in C. acetobutylicum may be broadly conserved in Clostridium spp., since a C. perfringens sigK mutant arrests at an earlier stage of sporulation than a sigE mutant. Nevertheless, since variation in sporulation sigma factor mutant phenotypes are observed between Clostridium spp. (Figure 1B), divergent mechanisms may control sigma factor function in different Clostridium spp. Transcriptional profiling of sigma factor mutants in multiple Clostridium spp. would provide critical insight into these questions. Lastly, it should be noted that σK regulates additional cellular processes beyond sporulation in these species: in C. botulinum it controls stress responses [62], and in C. acetobutylicum it controls solventogenesis [56].

Conclusions

In B. subtilis an intricate dialogue between the forespore and mother cell controls the cellular differentiation program of sporulation. However, this intercompartmental dialogue is not broadly conserved in (Pepto)clostridium difficile and Clostridium spp. despite the conservation of many signaling proteins. Instead, sporulation-specific sigma factors are activated with minimal intercompartmental signaling in C. difficile and in a different sequence in Clostridium spp. The sigma factors also control different morphological processes during sporulation in these clostridia. Accordingly, determining the mechanisms controlling sporulation sigma factor activation in different Firmicutes will undoubtedly lead to new insights into how differential gene expression is coordinated to produce different cellular fates. Even in B. subtilis, open questions remain, such as what is the feeding tube transporting, and why is σG activation coupled to engulfment completion? In C. difficile, what is the mechanism for post-translationally activating σG? To what extent do sporulation studies in C. difficile extend to other Peptoclostridium spp.? In Clostridium spp., how is early σK function achieved? How are the remaining sporulation sigma factors regulated? More broadly, what evolutionary pressures might explain this diversification in regulatory architecture?

Highlights.

  • Conserved sporulation sigma factors are differentially regulated in the Firmicutes.

  • The order in which sporulation sigma factors function is not conserved.

  • The mechanisms controlling σK activation are particularly divergent.

  • C. difficile sigma factor activation is not strictly tied to morphological changes.

Acknowledgments

K.F. is supported by T32 AI055402 from the National Institute of Allergy and Infectious Disease. A.S. is a Pew Scholar in the Biomedical Sciences, supported by The Pew Charitable Trusts, and is supported by Award Number R00GM092934, R01GM108684, and start-up funds from Award Number P20RR021905 from the National Institute of General Medical Sciences. The content is solely the responsibility of the author(s) and does not necessarily reflect the views of the Pew Charitable Trusts, the National Institute of General Medical Sciences, the National Institute of Allergy and Infectious Disease or the National Institute of Health.

Footnotes

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