MINIREVIEW
The regulatory network controlling spore formation in Clostridium difficile tima C. Pereira3, Adriano O. Henriques3 & Isabelle Martin-Verstraete1,2 Laure Saujet1,2, Fa Laboratoire Pathogenese des Bacteries Anaerobies, Institut Pasteur, Paris, France; 2University Paris Diderot, Sorbonne Paris Cit e, Cellule Pasteur, gica, Universidade Nova de Lisboa, Oeiras, Portugal Paris, France; and 3Microbial Development Laboratory, Instituto de Tecnologia Quımica e Biolo
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Correspondence: Isabelle Martin-Verstraete, Laboratoire Pathogen ese des Bacteries Ana erobies, Institut Pasteur, 28 rue du Dr Roux, 75724, Paris Cedex 15, France. Tel.: +33 1 40613561; fax: +33 1 40613123; e-mail: [email protected] Received 19 May 2014; revised 9 July 2014; accepted 13 July 2014. Final version published online 12 August 2014. DOI: 10.1111/1574-6968.12540 Editor: Andre Klier
MICROBIOLOGY LETTERS
Keywords sigma factors; developmental sporulation program; communication; forespore; mother cell; pathogenic Clostridia.
Abstract Clostridium difficile, a Gram-positive, anaerobic, spore-forming bacterium, is a major cause of nosocomial infections such as antibiotic-associated diarrhea. Spores are the vector of its transmission and persistence in the environment. Despite the importance of spores in the infectious cycle of C. difficile, little was known until recently about the control of spore development in this enteropathogen. In this review, we describe recent advances in our understanding of the regulatory network controlling C. difficile sporulation. The comparison with the model organism Bacillus subtilis highlights major differences in the signaling pathways between the forespore and the mother cell and a weaker connection between morphogenesis and gene expression. Indeed, the activation of the SigE regulon in the mother cell is partially independent of SigF although the forespore protein SpoIIR, itself partially independent of SigF, is essential for pro-SigE processing. Furthermore, SigG activity is not strictly dependent on SigE. Finally, SigG is dispensable for SigK activation in agreement with the absence of a pro-SigK sequence. The excision of the C. difficile skin element is also involved in the regulation of SigK activity. The C. difficile sporulation process might be a simpler, more ancestral version of the program characterized for B. subtilis.
Introduction Endosporulation is an ancient bacterial cell differentiation process allowing the conversion of a vegetative cell in a mature spore through a series of several morphological steps (Stragier & Losick, 1996). Bacterial endospores (or spores) are formed by many Bacilli, Clostridia, and related organisms. The spores have the ability to withstand extreme physical and chemical conditions, and their resistance properties allow them to survive in a variety of environments, often for very long periods of time. Clostridium difficile, a Gram-positive, anaerobic, sporeforming bacterium, is the major cause of antibioticassociated diarrhea. The two main risk factors for C. difficile infection (CDI) are antibiotic treatment and hospitalization of elderly and immunocompromised patients. Disruption of the intestinal flora caused by antibiotherapy increases the risk of CDI. Indeed, after ingestion of spores, C. difficile germinates in the intestine in the presence of bile salts (Sorg & Sonenshein, 2008). Vegetative FEMS Microbiol Lett 358 (2014) 1–10
forms multiply and produce two toxins, which are the major virulence factors (Carroll & Bartlett, 2011) and cause enterocyte lysis and inflammation leading to diarrhea, colitis, pseudomembranous colitis, or more severe symptoms including bowel perforation, sepsis, and death. Clostridium difficile also forms spores in the gut that will be released into the environment (Fig. 1) (Lawley et al., 2009; Janoir et al., 2013). The spore, which is the vector of transmission and persistence of C. difficile in the environment or in the host, is therefore central to the infectious cycle (Deakin et al., 2012). Sporulation shares morphological and molecular similarities among the Bacilli and Clostridia (Paredes et al., 2005). All the morphological steps of the sporulation process observed in B. subtilis (Fig. 2) are conserved in C. difficile (Fimlaid et al., 2013; Pereira et al., 2013). The developmental program of sporulation is mainly governed by the sequential appearance of four sporulation sigma factors, SigF and SigG in the forespore and SigE and SigK in the mother cell. The main periods of their activity in ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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Fig. 1. The role of spores in the infectious cycle of C. difficile. Red arrows indicate events of the infectious cycle that are linked to spores or their germination. CA means cholate and CDCA chenodeoxycholate.
the initiation of transcription follow the B. subtilis model. SigF and SigE control early stages of sporulation, mainly prior to engulfment completion, and are replaced by SigG and SigK following engulfment completion. The SigF, SigE, SigG, and SigK regulons of C. difficile have recently been characterized by genome-wide analysis (Fimlaid et al., 2013; Saujet et al., 2013). SigF is involved in the transcription of genes encoding proteins involved in communication between the mother cell and the forespore (spoIIR, spoIIQ, spoIVB). SigE plays a crucial role in forespore engulfment, in the synthesis of spore protective layers (cortex and coat proteins), in the production of the late sigma factor SigK and of the metabolites and energy needed to nurture the forespore. sigF and sigE mutants are blocked at the asymmetric septation stage. The SigG form of RNA polymerase transcribes genes encoding proteins mainly involved in stress resistance (SASP proteins, dipicolinic acid uptake, cortex modification). A sigG mutant produces spore coat proteins. This mutant does not complete engulfment in 90% of the cells in one study (Fimlaid et al., 2013) or is blocked just after engulfment completion in another (Pereira et al., 2013), but different strains and growth conditions (medium, liquid/plate) were used. Finally, SigK plays a crucial role in the synthesis of the spore coat and exosporium, thought to contain many SigK-dependent proteins that are specific to C. difficile. SigK is also essential for spore release into the environment. Unlike in B. subtilis, a sigK mutant assembles the cortex layer. Thus, the main functions of the cell type-specific SigF, SigE, SigG, and SigK conform to some extent to those described for B. subtilis. ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
However, the sporulation initiation pathway of C. difficile and B. subtilis shows major differences. Spo0A and SigH are present and required for sporulation in C. difficile (Underwood et al., 2009; Saujet et al., 2011), but AbrB and the phosphorelay proteins, Spo0F and Spo0B, are absent. The sporulation initiation pathway remains in C. difficile and other Clostridia as a two-component system with Spo0A and associated kinases (Paredes et al., 2005; Underwood et al., 2009; Pettit et al., 2014). SigH is involved in the transcription of spo0A as observed in B. subtilis and of CD2492, encoding one of its associated kinases (Saujet et al., 2011; Edwards & McBride, 2014). Here, we will describe recent advances in the study of the C. difficile sporulation regulatory cascade highlighting major differences from the B. subtilis model.
The forespore regulatory network In B. subtilis and C. difficile, the spoIIAA–spoIIAB–sigF operon is positively controlled by SigH and Spo0A (Britton et al., 2002; Molle et al., 2003; Saujet et al., 2011). In C. difficile, sigF is expressed in the predivisional cell and following asymmetric division in the forespore and in the mother cell (Pereira et al., 2013). However, the expression of gpr (a SigF target) is detected only in the forespore soon after asymmetric division (Fig. 3). The confinement of SigF activity to the forespore indicates the existence of a post-transcriptional control of SigF activity. In B. subtilis, SigF is held inactive by the antisigma factor SpoIIAB until the phosphatase SpoIIE dephosphorylates the antianti-sigma factor SpoIIAA, leading to the release of active FEMS Microbiol Lett 358 (2014) 1–10
The sporulation regulatory network in C. difficile
Fig. 2. Morphological stages and compartmentalized gene expression of B. subtilis sporulation. The process begins with an asymmetric cell division (a). The mother cell membrane then migrates around the forespore (b), engulfing it. At the end of this process, the forespore becomes a free protoplast in the mother cell cytoplasm (c). The cortex peptidoglycan (blue) and coat (red) layers are then synthesized and deposited around the developing spore (d). Upon mother cell lysis, a mature spore is released to the surrounding environment, where it remains in a dormant state (e). The spore can then germinate (f). The compartment and main periods of activity of the sporulation SigF (F), SigE (E), SigG (G), and SigK (K) sigma factors are indicated. Intercompartmental signaling pathways that operate at critical stages of morphogenesis are shown by solid or broken arrows. The broken arrow indicates that E is likely to be required to maintain late forespore gene expression, rather than for the specific activation of G. E and K are activated by proteolysis of an inactive pro-protein. These pathways link the forespore and mother cell lines of gene expression and also result in the tight coupling between gene expression and morphogenesis. PD, predivisional cell; MC, mother cell; FS, forespore.
SigF following asymmetric division (Errington, 2003). The control of SigF activity is also mediated by a transient genetic asymmetry with the origin-proximal region of the chromosome that enters the forespore first and is only followed by the remainder of the chromosome containing the FEMS Microbiol Lett 358 (2014) 1–10
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spoIIAA operon after a delay (Errington, 2003). The SpoIIAA, SpoIIAB, and SpoIIE proteins are present in C. difficile. It seems likely that these proteins modulate SigF activity in C. difficile although an experimental demonstration is still lacking. The spoIIAA operon is not close to the origin of replication, but the possible existence of a transient genetic asymmetry remains to be established in C. difficile. In C. difficile, the expression of sigG and of its target sspA is confined to the forespore (Fig. 3) (Pereira et al., 2013). In B. subtilis, the sigG gene is part of an operon (spoIIGA–sigE–sigG) transcribed from two promoters: a SigA promoter located upstream of spoIIGA and a second promoter located upstream of sigG, recognized first by SigF and later by SigG (Hilbert & Piggot, 2004). In C. difficile, a SigA-dependent promoter upstream of spoIIGA and a second promoter upstream of sigG, corresponding to a forespore sigma factor consensus sequence, have been mapped (Saujet et al., 2011, 2013). Several results strongly suggest that sigG is transcribed from at least two promoters: one in front of spoIIGA recognized by SigA, and one upstream of sigG recognized by SigF and maybe also later by SigG (Fimlaid et al., 2013; Pereira et al., 2013; Saujet et al., 2013). Further studies are necessary to elucidate the regulation of sigG transcription in C. difficile. SigF regulates many SigG-controlled genes in genomewide analyses (Fimlaid et al., 2013; Saujet et al., 2013). In the sigF and sigG mutants, the sspA gene (a SigG target) is not detected using a reporter SNAP fusion (Pereira et al., 2013) and the SspA protein is not produced (Fimlaid et al., 2013). Then, SigF directly or indirectly controls the expression of the SigG regulon. Both SigF and SigG might dually control some SigG targets as observed in B. subtilis (Steil et al., 2005; Wang et al., 2006). This control might also be due to the reduced accumulation of SigG in the sigF mutant (Fimlaid et al., 2013). However, the reduced SigG synthesis in this mutant is probably not sufficient to explain the strict requirement of SigF for sigG target expression, suggesting the existence of another level of control (Fimlaid et al., 2013; Saujet et al., 2013). The positive regulation of SigG target genes by SigF could be exerted at the level of SigG activity (Fimlaid et al., 2013; Saujet et al., 2013) (Fig. 3). Further studies will be necessary to elucidate the molecular mechanism involved in the control of SigG targets by SigF in C. difficile. In B. subtilis, SigG-containing RNA polymerase transcribes spoVT and SpoVT regulates the expression of about half of the genes in the SigG regulon, either as an activator or as a repressor (Wang et al., 2006). In C. difficile, SpoVT is absent in a sigF mutant and is detected in a sigG mutant at a reduced level, suggesting that both SigF and SigG are involved in spoVT expression (Fimlaid et al., 2013). The C. difficile spoVT mutant completes engulfment and forms phase dark immature spores, ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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Fig. 3. The forespore regulatory network in C. difficile. The upper part of the figure represents the cell before asymmetric division. The parallel vertical lines represent the two membranes separating the forespore (right) and the mother cell (left). IFM and OFM mean outer-forespore membrane and inner-forespore membrane, respectively. The sigma factors correspond to oval boxes. Diamond-shaped yellow box corresponds to the inactive form of SigF. The figure also includes a schematic representation of the deduced spatial pattern of transcription of a Pgpr–SNAP or PsspA–SNAP fusions. The Pgpr–SNAP and PsspA–SNAP fusions are specifically controlled by SigF and SigG, respectively. Solid arrows indicate activation at the transcriptional level, and crossed arrows correspond to inactivation processes. White arrows indicate activation at the level of protein activity. Question marks represent mechanisms that are not yet fully understood or demonstrated.
suggesting that the cortex is reduced or absent; accordingly, the mutant fails to produce heat-resistant spores (Saujet et al., 2013). The phenotype of the spoVT mutant of C. difficile differs from that of the spoVT B. subtilis mutant, which forms phase bright spores while showing a reduced ability to sporulate (Bagyan et al., 1996). Finally, SpoVT activates the expression of two SigG-controlled genes, sspA and sspB, and represses the expression of spoIIR and gpr, two members of the SigF regulon (Saujet et al., 2013). In B. subtilis, RsfA represses spoIIR transcription, but this regulator is absent in C. difficile. While we cannot exclude an indirect effect caused by the block in sporulation in the C. difficile spoVT mutant, we hypothesize that the negative effect seen on spoIIR and gpr indicates that SpoVT might play the role of RsfA in the negative control of SigF targets (Saujet et al., 2013). The positive effect of SpoVT on sspA and sspB expression is not sufficient to explain the phase gray spore phenotype of the spoVT mutant. However, no differential expression of other tested genes (pdaA, spoVAC, sigG) is observed under similar conditions. A transcriptome analysis of the spoVT mutant compared with the wildtype strain as well as the visualization of the cortex structure of this mutant by electron transmission microscopy will help to pinpoint its role. ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
In B. subtilis, besides RsfA and SpoVT, YlyA plays a role in the forespore line of gene expression; YlyA binds to RNA polymerase and activates a set of genes required for proper spore germination (Traag et al., 2013). CD2599, an ortholog of ylyA, is a member of the SigG regulon as in B. subtilis (Fimlaid et al., 2013; Traag et al., 2013). Thus, and despite the low degree of similarity with its B. subtilis counterpart, CD2599 may act as an additional forespore-specific regulator in C. difficile. Its role remains to be elucidated.
The mother cell regulatory network In B. subtilis, sigE transcription is activated by Spo0A and initiated at the SigA-dependent promoter located upstream of spoIIGA (Baldus et al., 1994; Buckner et al., 1998; Hilbert & Piggot, 2004). In turn, SigE is involved in the transcription of numerous genes including spoIIID and gerR encoding two mother cell regulators. SpoIIID and SigE jointly activate the expression of sigK (Kroos et al., 1989; Kunkel et al., 1989). Then, the SigK form of RNA polymerase transcribes gerE, encoding the GerE regulator controlling the expression of SigK-dependent genes (Eichenberger et al., 2004). In C. difficile, sigE expression is dependent on Spo0A and is detected in predivisional FEMS Microbiol Lett 358 (2014) 1–10
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The sporulation regulatory network in C. difficile
cells (Rosenbusch et al., 2012; Fimlaid et al., 2013; Pereira et al., 2013; Saujet et al., 2013; Pettit et al., 2014). However, the expression of spoIIIAA and sigK (SigE targets) is detected only in the mother cell just after asymmetric division (Fig. 4), indicating that SigE activity is confined to the mother cell (Pereira et al., 2013). sigK is transcribed from two distinct promoters recognized by SigE and SigK (Pereira et al., 2013; Saujet et al., 2013), suggesting that sigK is first transcribed by the SigE-RNA polymerase and then by SigK at later stages of sporulation (Fig. 4). Disruption of sigE prevents expression of a PsigK–SNAP transcriptional fusion (Pereira et al., 2013) and SigK production (Fimlaid et al., 2013). However, disruption of sigK did not seem to significantly reduce expression of a PsigK–SNAP in the condition tested. In C. difficile, SpoIIID is present but GerR and GerE are absent. SigE is involved in the transcription of spoIIID (Saujet et al., 2013) (Fig. 4). The C. difficile spoIIID mutant is blocked after engulfment completion and produces about 103 fewer heat-resistant spores than the wild-type strain (Saujet et al., 2013). SpoIIID negatively controls the expression of members of the SigE regulon (including spoIVA, sipL, cotB and the spoIIIA and spm operons) and positively controls the expression of sigK (Fig. 4) and of 11 members of the SigK regulon (Saujet et al., 2013). The positive regulation of SigK-controlled genes by SpoIIID involves either a direct binding of SpoIIID to their promoter regions or an indirect effect via the positive control of sigK transcription by SpoIIID. So, SpoIIID plays a major role in the control of gene expression in the mother cell in C. difficile by repressing the
expression of SigE targets and activating sigK and some of the SigK-controlled genes. Finally, four additional SigE-controlled genes code for transcription factors (Fimlaid et al., 2013). Further analysis will be necessary to determine their possible involvement in the mother cell line of gene expression.
Intercompartmental communication between the forespore and the mother cell One of the most interesting features of the B. subtilis sporulation network is the existence of signaling pathways allowing communication between the forespore and the mother cell (Fig. 2). This communication takes place at critical steps of spore morphogenesis and allows the coordinated deployment of the two genetic programs and their coupling to the course of morphogenesis. In B. subtilis, SigF activity in the forespore is necessary for pro-SigE cleavage in the mother cell. Then, the activity of SigG in the forespore requires the function of SigE in the mother cell. Finally, SigG is necessary for the cleavage and activation of SigK in the mother cell (Higgins & Dworkin, 2012). Partial SigF-independent activation of SigE in C. difficile: role of SpoIIR
In B. subtilis, SigF, which is required for the activation of pro-SigE, indirectly controls the SigE regulon. Indeed, SigF allows the production of the signaling protein SpoIIR, which is secreted across the forespore inner-membrane
Fig. 4. The mother cell regulatory network in C. difficile. The upper part of the figure represents the cell before asymmetric division. The parallel vertical lines represent the two membranes separating the forespore (right) and the mother cell (left). IFM and OFM mean outer-forespore membrane and innerforespore membrane, respectively. The sigma factors correspond to oval boxes. Diamondshaped green box corresponds to pro-SigE, the inactive form of SigE. The figure also includes a schematic representation of the deduced spatial pattern of transcription of a PspoIIIAA– SNAP or PcotE–SNAP fusions. The PspoIIIAA– SNAP and PcotE–SNAP fusions are specifically controlled by SigE and SigK, respectively. Black solid arrows and white arrows indicate activation at the transcriptional level or at the level of protein activity, respectively. Crossed arrow corresponds to transcriptional repression. Question marks represent mechanisms that are not yet fully understood.
FEMS Microbiol Lett 358 (2014) 1–10
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into the intermembrane space, where it triggers the SpoIIGA-dependent pro-SigE processing in the mother cell (Karow et al., 1995; Londono-Vallejo & Stragier, 1995). Surprisingly, the expression of the SigE regulon in the mother cell is not strictly under SigF control in C. difficile despite the fact that SpoIIR and SpoIIGA are present (Fimlaid et al., 2013; Pereira et al., 2013; Saujet et al., 2013). Indeed, SigE target genes are either not regulated (Saujet et al., 2013) or only slightly downregulated in a sigF mutant compared with the wild-type strain (Fimlaid et al., 2013). In addition, the PspoIIIAA–SNAP fusion is still expressed in the mother cell compartment of a sigF mutant, indicating that SigE is active in the absence of SigF, although in only a fraction of the cells (Pereira et al., 2013). In agreement with these results, both pro-SigE and processed SigE are detected in the sigF mutant, even if SigE is present in a reduced amount compared with the wild-type strain (Fimlaid et al., 2013; Saujet et al., 2013). As shown in B. subtilis, SpoIIR is required for pro-SigE processing likely through a control of SpoIIGA activity (Saujet et al., 2013). However, spoIIR expression is not strictly dependent on SigF (Fimlaid et al., 2013; Saujet et al., 2013). Indeed, in the sigF mutant, a residual expression of spoIIR that is Spo0Adependent is sufficient to maintain a SigE-dependent activity in the mother cell (Fig. 5) (Saujet et al., 2013). Nevertheless, no Spo0A box is identified in the spoIIR promoter region suggesting an indirect effect of Spo0A. A SigF promoter is found upstream of spoIIR, and its transcription is confined to the forespore and detected only after asymmetric division (Saujet et al., 2013). Presumably, another sigma factor can activate foresporespecific transcription of spoIIR in a fraction of the sporulating cells. Furthermore, expression of a PspoIIIAA– SNAP fusion is detected in only about 20% of the sigF cells (Pereira et al., 2013) in agreement with the idea that spoIIR is expressed independently of SigF in a fraction of cells. To conclude, these results indicate that SigF is not strictly required for SigE functionality in C. difficile, highlighting a major difference relative to the B. subtilis sporulation. Regulation of SigG activity by SigE
In B. subtilis, the main period of sigG transcription and activity takes place after engulfment completion (Partridge & Errington, 1993) and the expression of SigG targets in the forespore depends upon SigE activity in the mother cell. The requirement for SigE is at least in part dependent on the expression of the spoIIIA operon and the formation of the SpoIIIAH–SpoIIQ channel that links the cytoplasm of the two cells and is required for late, postengulfment completion, gene expression in the foreª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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spore (Hilbert & Piggot, 2004; Higgins & Dworkin, 2012). Surprisingly, sigG expression is detected just after asymmetric division and SigG activity begins prior to engulfment completion in C. difficile (Pereira et al., 2013). Furthermore, the SigG protein is detected and several SigG targets are expressed at normal levels in a sigE mutant (Fimlaid et al., 2013; Pereira et al., 2013; Saujet et al., 2013). Indeed, a PsspA–SNAP fusion is expressed in the forespore of this mutant (Pereira et al., 2013). Then, a strict requirement of SigE is not observed for the expression of the large majority of the SigG-controlled genes (Fig. 5). However, some SigG target genes are downregulated in the C. difficile sigE mutant (Saujet et al., 2013). This suggests that SigE participates in the control of the SigG regulon in C. difficile, probably through the control of the expression of the spoIIIAA operon as observed in B. subtilis (Higgins & Dworkin, 2012). Moreover, fluorescence from a PsspA–SNAP fusion enhances after engulfment completion, an increase that is prevented by disruption of sigE (Pereira et al., 2013). To conclude, SigG is active in the absence of SigE in C. difficile, but the increase in SigG activity after engulfment completion probably requires SigE and may rely on formation of the SpoIIIAH–SpoIIQ channel (Fig. 5). The SigK regulon is not controlled by SigG
In B. subtilis, SigG regulates the expression of the SigK regulon in the mother cell through the control of proSigK processing involving the SpoIVFB protease and SpoIVFA and BofA, two membrane proteins that form a complex with and control SpoIVFB activity (Higgins & Dworkin, 2012). SigK lacks a pro-sequence, and SpoIVFB, SpoIVFA, and BofA are absent in C. difficile (Stragier, 2002; Haraldsen & Sonenshein, 2003; Abecasis et al., 2013). Interestingly, the SigK regulon is not controlled by SigG using global approaches or a more targeted strategy coupling morphology and expression using a PcotE–SNAP fusion (Fimlaid et al., 2013; Pereira et al., 2013; Saujet et al., 2013). Moreover, accumulation of SigK as well as of three other SigK-controlled proteins (CotE, CdeC, and SleC) is detected in a sigG mutant (Fimlaid et al., 2013). In addition, the coat protein, CotE, is produced and assembled around the developing spore in the sigG mutant as demonstrated using a CotE–SNAP protein fusion (Pereira et al., 2013). In agreement, analysis of sigG mutant cells by transmission electron microscopy (TEM) shows deposition of at least some coat around the forespore (Fimlaid et al., 2013; Pereira et al., 2013). Thus, the absence of regulation of SigK targets by SigG may be related to the lack of control of SigK by processing of a preprotein in C. difficile (Fig. 5). FEMS Microbiol Lett 358 (2014) 1–10
The sporulation regulatory network in C. difficile
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(a)
(b)
Fig. 5. Communication between forespore and mother cell in C. difficile. (a) Model of the regulatory network controlling sporulation. The parallel vertical lines represent the two membranes separating the forespore (right) and the mother cell (left). IFM and OFM mean outerforespore membrane and inner-forespore membrane, respectively. Proteins associated with the membrane or located into the intermembrane space are illustrated as embedded in the parallel vertical lines. The four sigma factors are encircled (by oval boxes), while diamond-shaped green box corresponds to pro-SigE. Orange square boxes correspond to transcriptional regulators. The dotted line represents the engulfment completion. Black solid arrows and white arrows indicate activation at the transcriptional level or at the level of protein activity, respectively. Question marks represent mechanisms that are not yet fully understood. (b) Schematic representation of the intercellular communication pathways leading to sigma factor activation during sporulation. In C. difficile, the activation of SigE is partially independent on SigF (represented by a broken arrow), while the activation of SigG is independent of SigE and the activation of SigK also does not seem to depend on SigG (represented by crossed arrows). The figure includes a schematic representation of the deduced spatial pattern of transcription of a PspoIIIAA– SNAP fusion in a sigF mutant, of a PsspA–SNAP fusion in a sigE mutant or of a PcotE–SNAP fusion in a sigG mutant.
Control of SigK target genes by SigF
Interestingly, SigF controls some SigK targets in C. difficile (Fimlaid et al., 2013; Saujet et al., 2013). Indeed, several genes belonging to the SigK regulon and sigK itself are downregulated in the sigF mutant (Fimlaid et al., 2013; Saujet et al., 2013). Furthermore, SigK, CotE, and CdeC are not detected in the sigF mutant, while they are detected in a sigG mutant even though both cotE and cdeC transcripts could be detected at significant levels in a sigF mutant (Fimlaid et al., 2013). The effect of SigF on the expression of SigK targets could be mediated by the indirect control of SigK synthesis or activity by SigF. This might be also due to a decrease in SigE activity in the absence of SigF (Fig. 5). The mechanism of the control of SigK by SigF remains to be deciphered.
Potential role of the skin element excision in the regulation of sigK expression in C. difficile Some divergences are observed in the late stages of the sporulation process between B. subtilis and C. difficile but also among Clostridia. It is worth noting that the genus FEMS Microbiol Lett 358 (2014) 1–10
Clostridium is highly diverse. Clostridium difficile (recently re-named Peptoclostridium difficile) is a member of the Peptosptreptococcae family, while other Clostridia such as Clostridium perfringens, Clostridium botulinum, and Clostridium acetobutylicum belong to the Clostridiaceae family (Yutin & Galperin, 2013). The sigK mutants of B. subtilis and C. difficile are blocked in late sporulation phase (Piggot & Coote, 1976; Hilbert & Piggot, 2004; Pereira et al., 2013), while the sigK mutants of C. perfringens and C. acetobutylicum are arrested before asymmetric division (Harry et al., 2009; Al-Hinai et al., 2014). In correlation with these phenotypes, there are several major differences between B. subtilis and Clostridia regarding (1) the timing of sigK expression, (2) the presence of a pro-SigK protein, and (3) the insertion of a prophagelike mobile element (skin) into sigK. In C. difficile and B. subtilis, sigK is expressed late during sporulation (Pereira et al., 2013), while in C. perfringens, C. botulinum, and C. acetobutylicum, sigK is also expressed earlier and before asymmetric division in C. perfringens and C. acetobutylicum (Harry et al., 2009; Kirk et al., 2012; Al-Hinai et al., 2014). In B. subtilis, SigK activity depends on the cleavage of a pro-sequence, a step controlled by SigG (Errington, 2003). Interestingly, ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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the pro-SigK form is present in Clostridiaceae but absent in several Peptosptreptococcae including C. difficile, Clostridium sordelii, and Clostridium bifermentans (de Hoon et al., 2010) but does not seem sufficient to prevent early sigK expression and activity in C. perfringens, C. botulinum, and C. acetobutylicum [in the latter organism, SigK functions both early and late in sporulation; (Al-Hinai et al., 2014)]. In C. difficile and B. subtilis, the sigK gene is interrupted by a skin element absent in other Clostridia (de Hoon et al., 2010). So, the skin element may partly explain the late expression of sigK and the morphology of the sigK mutant in B. subtilis and C. difficile. The skin elements of B. subtilis and C. difficile have different sizes and are inserted at different sites and in opposite orientation, indicating that skin integration into sigK has occurred independently during evolution (Haraldsen & Sonenshein, 2003). The skinCd element is excised during sporulation (Haraldsen & Sonenshein, 2003). The CD1231 gene, located within the skin (Fig. 4), codes for a protein similar to the SpoIVCA site-specific recombinase of the skinBs (Kunkel et al., 1990; Haraldsen & Sonenshein, 2003). In the absence of a pro-sequence for SigK, the skinCd excision is likely an important element in the regulation of SigK activity in C. difficile. Then, it is important to better understand the regulation of skin excision in this organism. In B. subtilis, SigE and SpoIIID are required for the expression of the spoIVCA gene (Eichenberger et al., 2004). Surprisingly, neither SigE nor SpoIIID controls CD1231 expression (Saujet et al., 2013). Another factor may modulate CD1231 synthesis or activity, or another recombinase responsible for skinCd excision may be at play in C. difficile.
Conclusion In B. subtilis, activation of the sporulation sigma factors coincides with the completion of key morphological steps during spore formation (asymmetric division, engulfment completion) and intercompartmental communication pathways strictly coordinate the mother cell and forespore lines of expression and their coupling to the course of morphogenesis (Fig. 2). Important deviations from the B. subtilis paradigm exist in C. difficile especially concerning (1) the communication between the forespore and the mother cell and (2) a weaker connection between gene expression and morphogenesis (Fig. 5). First, the SigE regulon is not strictly under SigF control although SpoIIR is essential for pro-SigE cleavage. Residual SigF-independent spoIIR expression is probably responsible for pro-SigE processing in the sigF mutant. Second, sigG is expressed earlier (soon after asymmetric division) and a strict requirement for SigE is not observed for the production and activity of SigG although the increase in SigG activity following ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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engulfment completion requires SigE and possibly the formation of the SpoIIIAH–SpoIIQ channel. Third, SigK activity is independent on SigG. However, a control of SigK activity by the forespore seems to be maintained through a SigF-dependent mechanism and the role of the skin element in the timing of production of SigK remains to be clarified. Other regulatory proteins and/or small noncoding RNAs could play a role in this regulatory cascade. Interestingly, a strain depleted for the RNA chaperon, Hfq, has a better ability to sporulate. This effect is probably mediated through its activity on one or several noncoding RNAs (Boudry et al., 2014). Spore-forming Firmicutes have different lifestyles, and the most significant variations in the sporulation process are observed at the interface with their environment: signal transduction pathways triggering sporulation, coat proteins, and germination signals. Based on the fact that anaerobic bacteria existed before aerobes, it is tempting to speculate that the first spore-forming bacteria, which appeared at the basis of the Firmicutes phylum, were anaerobes (Galperin et al., 2012; Abecasis et al., 2013). The developmental control in C. difficile and probably in other Clostridia might reflect a more ancestral version of the sporulation gene regulatory network, while a more sophisticated system of control with a tight coupling between morphogenesis and gene expression would have been gradually introduced during evolution for Bacilli. Differences in the period of sporulation sigma factor activation may also be linked to the coordination with other processes such as solventogenesis, biofilm formation, or toxin production (Paredes et al., 2005; Harry et al., 2009; Semenyuk et al., 2014). The less tight control of sporulation in C. difficile might also allow heterogeneity in the population, a possible adaptive strategy during gut colonization.
References Abecasis AB, Serrano M, Alves R, Quintais L, Pereira-Leal JB & Henriques AO (2013) A genomic signature and the identification of new sporulation genes. J Bacteriol 195: 2101–2115. Al-Hinai MA, Jones SW & Papoutsakis ET (2014) sigmaK of Clostridium acetobutylicum is the first known sporulation-specific sigma factor with two developmentally separated roles, one early and one late in sporulation. J Bacteriol 196: 287–299. Bagyan I, Hobot J & Cutting S (1996) A compartmentalized regulator of developmental gene expression in Bacillus subtilis. J Bacteriol 178: 4500–4507. Baldus JM, Green BD, Youngman P & Moran CP Jr (1994) Phosphorylation of Bacillus subtilis transcription factor Spo0A stimulates transcription from the spoIIG promoter by enhancing binding to weak 0A boxes. J Bacteriol 176: 296–306.
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Boudry P, Gracia C, Monot M, Caillet J, Saujet L, Hajnsdorf E, Dupuy B, Martin-Verstraete I & Soutourina O (2014) Pleiotropic role of the RNA chaperone protein Hfq in the human pathogen Clostridium difficile. J Bacteriol in press. Britton RA, Eichenberger P, Gonzalez-Pastor JE, Fawcett P, Monson R, Losick R & Grossman AD (2002) Genome-wide analysis of the stationary-phase sigma factor (sigma-H) regulon of Bacillus subtilis. J Bacteriol 184: 4881–4890. Buckner CM, Schyns G & Moran CP Jr (1998) A region in the Bacillus subtilis transcription factor Spo0A that is important for spoIIG promoter activation. J Bacteriol 180: 3578–3583. Carroll KC & Bartlett JG (2011) Biology of Clostridium difficile: implications for epidemiology and diagnosis. Annu Rev Microbiol 65: 501–521. de Hoon MJ, Eichenberger P & Vitkup D (2010) Hierarchical evolution of the bacterial sporulation network. Curr Biol 20: R735–R745. Deakin LJ, Clare S, Fagan RP, Dawson LF, Pickard DJ, West MR, Wren BW, Fairweather NF, Dougan G & Lawley TD (2012) The Clostridium difficile spo0A gene is a persistence and transmission factor. Infect Immun 80: 2704–2711. Edwards AN & McBride SM (2014) Initiation of sporulation in Clostridium difficile: a twist on the classic model. FEMS Microbiol Lett, doi: 10.1111/1574-6968.12499. Eichenberger P, Fujita M, Jensen ST et al. (2004) The program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis. PLoS Biol 2: e328. Errington J (2003) Regulation of endospore formation in Bacillus subtilis. Nat Rev Microbiol 1: 117–126. Fimlaid KA, Bond JP, Schutz KC, Putnam EE, Leung JM, Lawley TD & Shen A (2013) Global analysis of the sporulation pathway of Clostridium difficile. PLoS Genet 9: e1003660. Galperin MY, Mekhedov SL, Puigbo P, Smirnov S, Wolf YI & Rigden DJ (2012) Genomic determinants of sporulation in Bacilli and Clostridia: towards the minimal set of sporulation-specific genes. Environ Microbiol 14: 2870–2890. Haraldsen JD & Sonenshein AL (2003) Efficient sporulation in Clostridium difficile requires disruption of the sigmaK gene. Mol Microbiol 48: 811–821. Harry KH, Zhou R, Kroos L & Melville SB (2009) Sporulation and enterotoxin (CPE) synthesis are controlled by the sporulation-specific sigma factors SigE and SigK in Clostridium perfringens. J Bacteriol 191: 2728–2742. Higgins D & Dworkin J (2012) Recent progress in Bacillus subtilis sporulation. FEMS Microbiol Rev 36: 131–148. Hilbert DW & Piggot PJ (2004) Compartmentalization of gene expression during Bacillus subtilis spore formation. Microbiol Mol Biol Rev 68: 234–262. Janoir C, Deneve C, Bouttier S et al. (2013) Adaptive strategies and pathogenesis of Clostridium difficile from in vivo transcriptomics. Infect Immun 81: 3757–3769. Karow ML, Glaser P & Piggot PJ (1995) Identification of a gene, spoIIR, that links the activation of sigma E to the
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transcriptional activity of sigma F during sporulation in Bacillus subtilis. P Natl Acad Sci USA 92: 2012–2016. Kirk DG, Dahlsten E, Zhang Z, Korkeala H & Lindstrom M (2012) Involvement of Clostridium botulinum ATCC 3502 sigma factor K in early-stage sporulation. Appl Environ Microbiol 78: 4590–4596. Kroos L, Kunkel B & Losick R (1989) Switch protein alters specificity of RNA polymerase containing a compartment-specific sigma factor. Science 243: 526–529. Kunkel B, Kroos L, Poth H, Youngman P & Losick R (1989) Temporal and spatial control of the mother-cell regulatory gene spoIIID of Bacillus subtilis. Genes Dev 3: 1735–1744. Kunkel B, Losick R & Stragier P (1990) The Bacillus subtilis gene for the development transcription factor sigma K is generated by excision of a dispensable DNA element containing a sporulation recombinase gene. Genes Dev 4: 525–535. Lawley TD, Clare S, Walker AW et al. (2009) Antibiotic treatment of Clostridium difficile carrier mice triggers a supershedder state, spore-mediated transmission, and severe disease in immunocompromised hosts. Infect Immun 77: 3661–3669. Londono-Vallejo JA & Stragier P (1995) Cell-cell signaling pathway activating a developmental transcription factor in Bacillus subtilis. Genes Dev 9: 503–508. Molle V, Fujita M, Jensen ST, Eichenberger P, Gonzalez-Pastor JE, Liu JS & Losick R (2003) The Spo0A regulon of Bacillus subtilis. Mol Microbiol 50: 1683–1701. Paredes CJ, Alsaker KV & Papoutsakis ET (2005) A comparative genomic view of clostridial sporulation and physiology. Nat Rev Microbiol 3: 969–978. Partridge SR & Errington J (1993) The importance of morphological events and intercellular interactions in the regulation of prespore-specific gene expression during sporulation in Bacillus subtilis. Mol Microbiol 8: 945–955. Pereira FC, Saujet L, Tome AR, Serrano M, Monot M, Couture-Tosi E, Martin-Verstraete I, Dupuy B & Henriques AO (2013) The spore differentiation pathway in the enteric pathogen Clostridium difficile. PLoS Genet 9: e1003782. Pettit LJ, Browne HP, Yu L et al. (2014) Functional genomics reveals that Clostridium difficile Spo0A coordinates sporulation, virulence and metabolism. BMC Genomics 15: 160. Piggot PJ & Coote JG (1976) Genetic aspects of bacterial endospore formation. Bacteriol Rev 40: 908–962. Rosenbusch KE, Bakker D, Kuijper EJ & Smits WK (2012) C. difficile 630Deltaerm Spo0A regulates sporulation, but does not contribute to toxin production, by direct high-affinity binding to target DNA. PLoS One 7: e48608. Saujet L, Monot M, Dupuy B, Soutourina O & Martin-Verstraete I (2011) The key sigma factor of transition phase, SigH, controls sporulation, metabolism, and virulence factor expression in Clostridium difficile. J Bacteriol 193: 3186–3196.
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Saujet L, Pereira FC, Serrano M, Soutourina O, Monot M, Shelyakin PV, Gelfand MS, Dupuy B, Henriques AO & Martin-Verstraete I (2013) Genome-wide analysis of cell type-specific gene transcription during spore formation in Clostridium difficile. PLoS Genet 9: e1003756. Semenyuk EG, Laning ML, Foley J, Johnston PF, Knight KL, Gerding DN & Driks A (2014) Spore formation and toxin production in Clostridium difficile biofilms. PLoS ONE 9: e87757. Sorg JA & Sonenshein AL (2008) Bile salts and glycine as cogerminants for Clostridium difficile spores. J Bacteriol 190: 2505–2512. Steil L, Serrano M, Henriques AO & Volker U (2005) Genome-wide analysis of temporally regulated and compartment-specific gene expression in sporulating cells of Bacillus subtilis. Microbiology 151: 399–420. Stragier P (2002) A Gene Odyssey: Exploring the Genomes of Endospore-Forming Bacteria. ASM Press, Washington, DC.
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Stragier P & Losick R (1996) Molecular genetics of sporulation in Bacillus subtilis. Annu Rev Genet 30: 297–341. Traag B, Ramirez-Peralta A, Wang Erickson AF, Setlow P & Losick R (2013) A novel RNA Polymerase-binding protein involved in spore germination in Bacillus subtilis. Mol Microbiol 89: 113–122. Underwood S, Guan S, Vijayasubhash V, Baines SD, Graham L, Lewis RJ, Wilcox MH & Stephenson K (2009) Characterization of the sporulation initiation pathway of Clostridium difficile and its role in toxin production. J Bacteriol 191: 7296–7305. Wang ST, Setlow B, Conlon EM, Lyon JL, Imamura D, Sato T, Setlow P, Losick R & Eichenberger P (2006) The forespore line of gene expression in Bacillus subtilis. J Mol Biol 358: 16–37. Yutin N & Galperin MY (2013) A genomic update on clostridial phylogeny: gram-negative spore formers and other misplaced clostridia. Environ Microbiol 15: 2631–2641.
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The regulatory network controlling spore formation in Clostridium difficile tima C. Pereira3, Adriano O. Henriques3 & Isabelle Martin-Verstraete1,2 Laure Saujet1,2, Fa Laboratoire Pathogenese des Bacteries Anaerobies, Institut Pasteur, Paris, France; 2University Paris Diderot, Sorbonne Paris Cit e, Cellule Pasteur, gica, Universidade Nova de Lisboa, Oeiras, Portugal Paris, France; and 3Microbial Development Laboratory, Instituto de Tecnologia Quımica e Biolo
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Correspondence: Isabelle Martin-Verstraete, Laboratoire Pathogen ese des Bacteries Ana erobies, Institut Pasteur, 28 rue du Dr Roux, 75724, Paris Cedex 15, France. Tel.: +33 1 40613561; fax: +33 1 40613123; e-mail: [email protected] Received 19 May 2014; revised 9 July 2014; accepted 13 July 2014. Final version published online 12 August 2014. DOI: 10.1111/1574-6968.12540 Editor: Andre Klier
MICROBIOLOGY LETTERS
Keywords sigma factors; developmental sporulation program; communication; forespore; mother cell; pathogenic Clostridia.
Abstract Clostridium difficile, a Gram-positive, anaerobic, spore-forming bacterium, is a major cause of nosocomial infections such as antibiotic-associated diarrhea. Spores are the vector of its transmission and persistence in the environment. Despite the importance of spores in the infectious cycle of C. difficile, little was known until recently about the control of spore development in this enteropathogen. In this review, we describe recent advances in our understanding of the regulatory network controlling C. difficile sporulation. The comparison with the model organism Bacillus subtilis highlights major differences in the signaling pathways between the forespore and the mother cell and a weaker connection between morphogenesis and gene expression. Indeed, the activation of the SigE regulon in the mother cell is partially independent of SigF although the forespore protein SpoIIR, itself partially independent of SigF, is essential for pro-SigE processing. Furthermore, SigG activity is not strictly dependent on SigE. Finally, SigG is dispensable for SigK activation in agreement with the absence of a pro-SigK sequence. The excision of the C. difficile skin element is also involved in the regulation of SigK activity. The C. difficile sporulation process might be a simpler, more ancestral version of the program characterized for B. subtilis.
Introduction Endosporulation is an ancient bacterial cell differentiation process allowing the conversion of a vegetative cell in a mature spore through a series of several morphological steps (Stragier & Losick, 1996). Bacterial endospores (or spores) are formed by many Bacilli, Clostridia, and related organisms. The spores have the ability to withstand extreme physical and chemical conditions, and their resistance properties allow them to survive in a variety of environments, often for very long periods of time. Clostridium difficile, a Gram-positive, anaerobic, sporeforming bacterium, is the major cause of antibioticassociated diarrhea. The two main risk factors for C. difficile infection (CDI) are antibiotic treatment and hospitalization of elderly and immunocompromised patients. Disruption of the intestinal flora caused by antibiotherapy increases the risk of CDI. Indeed, after ingestion of spores, C. difficile germinates in the intestine in the presence of bile salts (Sorg & Sonenshein, 2008). Vegetative FEMS Microbiol Lett 358 (2014) 1–10
forms multiply and produce two toxins, which are the major virulence factors (Carroll & Bartlett, 2011) and cause enterocyte lysis and inflammation leading to diarrhea, colitis, pseudomembranous colitis, or more severe symptoms including bowel perforation, sepsis, and death. Clostridium difficile also forms spores in the gut that will be released into the environment (Fig. 1) (Lawley et al., 2009; Janoir et al., 2013). The spore, which is the vector of transmission and persistence of C. difficile in the environment or in the host, is therefore central to the infectious cycle (Deakin et al., 2012). Sporulation shares morphological and molecular similarities among the Bacilli and Clostridia (Paredes et al., 2005). All the morphological steps of the sporulation process observed in B. subtilis (Fig. 2) are conserved in C. difficile (Fimlaid et al., 2013; Pereira et al., 2013). The developmental program of sporulation is mainly governed by the sequential appearance of four sporulation sigma factors, SigF and SigG in the forespore and SigE and SigK in the mother cell. The main periods of their activity in ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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Fig. 1. The role of spores in the infectious cycle of C. difficile. Red arrows indicate events of the infectious cycle that are linked to spores or their germination. CA means cholate and CDCA chenodeoxycholate.
the initiation of transcription follow the B. subtilis model. SigF and SigE control early stages of sporulation, mainly prior to engulfment completion, and are replaced by SigG and SigK following engulfment completion. The SigF, SigE, SigG, and SigK regulons of C. difficile have recently been characterized by genome-wide analysis (Fimlaid et al., 2013; Saujet et al., 2013). SigF is involved in the transcription of genes encoding proteins involved in communication between the mother cell and the forespore (spoIIR, spoIIQ, spoIVB). SigE plays a crucial role in forespore engulfment, in the synthesis of spore protective layers (cortex and coat proteins), in the production of the late sigma factor SigK and of the metabolites and energy needed to nurture the forespore. sigF and sigE mutants are blocked at the asymmetric septation stage. The SigG form of RNA polymerase transcribes genes encoding proteins mainly involved in stress resistance (SASP proteins, dipicolinic acid uptake, cortex modification). A sigG mutant produces spore coat proteins. This mutant does not complete engulfment in 90% of the cells in one study (Fimlaid et al., 2013) or is blocked just after engulfment completion in another (Pereira et al., 2013), but different strains and growth conditions (medium, liquid/plate) were used. Finally, SigK plays a crucial role in the synthesis of the spore coat and exosporium, thought to contain many SigK-dependent proteins that are specific to C. difficile. SigK is also essential for spore release into the environment. Unlike in B. subtilis, a sigK mutant assembles the cortex layer. Thus, the main functions of the cell type-specific SigF, SigE, SigG, and SigK conform to some extent to those described for B. subtilis. ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
However, the sporulation initiation pathway of C. difficile and B. subtilis shows major differences. Spo0A and SigH are present and required for sporulation in C. difficile (Underwood et al., 2009; Saujet et al., 2011), but AbrB and the phosphorelay proteins, Spo0F and Spo0B, are absent. The sporulation initiation pathway remains in C. difficile and other Clostridia as a two-component system with Spo0A and associated kinases (Paredes et al., 2005; Underwood et al., 2009; Pettit et al., 2014). SigH is involved in the transcription of spo0A as observed in B. subtilis and of CD2492, encoding one of its associated kinases (Saujet et al., 2011; Edwards & McBride, 2014). Here, we will describe recent advances in the study of the C. difficile sporulation regulatory cascade highlighting major differences from the B. subtilis model.
The forespore regulatory network In B. subtilis and C. difficile, the spoIIAA–spoIIAB–sigF operon is positively controlled by SigH and Spo0A (Britton et al., 2002; Molle et al., 2003; Saujet et al., 2011). In C. difficile, sigF is expressed in the predivisional cell and following asymmetric division in the forespore and in the mother cell (Pereira et al., 2013). However, the expression of gpr (a SigF target) is detected only in the forespore soon after asymmetric division (Fig. 3). The confinement of SigF activity to the forespore indicates the existence of a post-transcriptional control of SigF activity. In B. subtilis, SigF is held inactive by the antisigma factor SpoIIAB until the phosphatase SpoIIE dephosphorylates the antianti-sigma factor SpoIIAA, leading to the release of active FEMS Microbiol Lett 358 (2014) 1–10
The sporulation regulatory network in C. difficile
Fig. 2. Morphological stages and compartmentalized gene expression of B. subtilis sporulation. The process begins with an asymmetric cell division (a). The mother cell membrane then migrates around the forespore (b), engulfing it. At the end of this process, the forespore becomes a free protoplast in the mother cell cytoplasm (c). The cortex peptidoglycan (blue) and coat (red) layers are then synthesized and deposited around the developing spore (d). Upon mother cell lysis, a mature spore is released to the surrounding environment, where it remains in a dormant state (e). The spore can then germinate (f). The compartment and main periods of activity of the sporulation SigF (F), SigE (E), SigG (G), and SigK (K) sigma factors are indicated. Intercompartmental signaling pathways that operate at critical stages of morphogenesis are shown by solid or broken arrows. The broken arrow indicates that E is likely to be required to maintain late forespore gene expression, rather than for the specific activation of G. E and K are activated by proteolysis of an inactive pro-protein. These pathways link the forespore and mother cell lines of gene expression and also result in the tight coupling between gene expression and morphogenesis. PD, predivisional cell; MC, mother cell; FS, forespore.
SigF following asymmetric division (Errington, 2003). The control of SigF activity is also mediated by a transient genetic asymmetry with the origin-proximal region of the chromosome that enters the forespore first and is only followed by the remainder of the chromosome containing the FEMS Microbiol Lett 358 (2014) 1–10
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spoIIAA operon after a delay (Errington, 2003). The SpoIIAA, SpoIIAB, and SpoIIE proteins are present in C. difficile. It seems likely that these proteins modulate SigF activity in C. difficile although an experimental demonstration is still lacking. The spoIIAA operon is not close to the origin of replication, but the possible existence of a transient genetic asymmetry remains to be established in C. difficile. In C. difficile, the expression of sigG and of its target sspA is confined to the forespore (Fig. 3) (Pereira et al., 2013). In B. subtilis, the sigG gene is part of an operon (spoIIGA–sigE–sigG) transcribed from two promoters: a SigA promoter located upstream of spoIIGA and a second promoter located upstream of sigG, recognized first by SigF and later by SigG (Hilbert & Piggot, 2004). In C. difficile, a SigA-dependent promoter upstream of spoIIGA and a second promoter upstream of sigG, corresponding to a forespore sigma factor consensus sequence, have been mapped (Saujet et al., 2011, 2013). Several results strongly suggest that sigG is transcribed from at least two promoters: one in front of spoIIGA recognized by SigA, and one upstream of sigG recognized by SigF and maybe also later by SigG (Fimlaid et al., 2013; Pereira et al., 2013; Saujet et al., 2013). Further studies are necessary to elucidate the regulation of sigG transcription in C. difficile. SigF regulates many SigG-controlled genes in genomewide analyses (Fimlaid et al., 2013; Saujet et al., 2013). In the sigF and sigG mutants, the sspA gene (a SigG target) is not detected using a reporter SNAP fusion (Pereira et al., 2013) and the SspA protein is not produced (Fimlaid et al., 2013). Then, SigF directly or indirectly controls the expression of the SigG regulon. Both SigF and SigG might dually control some SigG targets as observed in B. subtilis (Steil et al., 2005; Wang et al., 2006). This control might also be due to the reduced accumulation of SigG in the sigF mutant (Fimlaid et al., 2013). However, the reduced SigG synthesis in this mutant is probably not sufficient to explain the strict requirement of SigF for sigG target expression, suggesting the existence of another level of control (Fimlaid et al., 2013; Saujet et al., 2013). The positive regulation of SigG target genes by SigF could be exerted at the level of SigG activity (Fimlaid et al., 2013; Saujet et al., 2013) (Fig. 3). Further studies will be necessary to elucidate the molecular mechanism involved in the control of SigG targets by SigF in C. difficile. In B. subtilis, SigG-containing RNA polymerase transcribes spoVT and SpoVT regulates the expression of about half of the genes in the SigG regulon, either as an activator or as a repressor (Wang et al., 2006). In C. difficile, SpoVT is absent in a sigF mutant and is detected in a sigG mutant at a reduced level, suggesting that both SigF and SigG are involved in spoVT expression (Fimlaid et al., 2013). The C. difficile spoVT mutant completes engulfment and forms phase dark immature spores, ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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Fig. 3. The forespore regulatory network in C. difficile. The upper part of the figure represents the cell before asymmetric division. The parallel vertical lines represent the two membranes separating the forespore (right) and the mother cell (left). IFM and OFM mean outer-forespore membrane and inner-forespore membrane, respectively. The sigma factors correspond to oval boxes. Diamond-shaped yellow box corresponds to the inactive form of SigF. The figure also includes a schematic representation of the deduced spatial pattern of transcription of a Pgpr–SNAP or PsspA–SNAP fusions. The Pgpr–SNAP and PsspA–SNAP fusions are specifically controlled by SigF and SigG, respectively. Solid arrows indicate activation at the transcriptional level, and crossed arrows correspond to inactivation processes. White arrows indicate activation at the level of protein activity. Question marks represent mechanisms that are not yet fully understood or demonstrated.
suggesting that the cortex is reduced or absent; accordingly, the mutant fails to produce heat-resistant spores (Saujet et al., 2013). The phenotype of the spoVT mutant of C. difficile differs from that of the spoVT B. subtilis mutant, which forms phase bright spores while showing a reduced ability to sporulate (Bagyan et al., 1996). Finally, SpoVT activates the expression of two SigG-controlled genes, sspA and sspB, and represses the expression of spoIIR and gpr, two members of the SigF regulon (Saujet et al., 2013). In B. subtilis, RsfA represses spoIIR transcription, but this regulator is absent in C. difficile. While we cannot exclude an indirect effect caused by the block in sporulation in the C. difficile spoVT mutant, we hypothesize that the negative effect seen on spoIIR and gpr indicates that SpoVT might play the role of RsfA in the negative control of SigF targets (Saujet et al., 2013). The positive effect of SpoVT on sspA and sspB expression is not sufficient to explain the phase gray spore phenotype of the spoVT mutant. However, no differential expression of other tested genes (pdaA, spoVAC, sigG) is observed under similar conditions. A transcriptome analysis of the spoVT mutant compared with the wildtype strain as well as the visualization of the cortex structure of this mutant by electron transmission microscopy will help to pinpoint its role. ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
In B. subtilis, besides RsfA and SpoVT, YlyA plays a role in the forespore line of gene expression; YlyA binds to RNA polymerase and activates a set of genes required for proper spore germination (Traag et al., 2013). CD2599, an ortholog of ylyA, is a member of the SigG regulon as in B. subtilis (Fimlaid et al., 2013; Traag et al., 2013). Thus, and despite the low degree of similarity with its B. subtilis counterpart, CD2599 may act as an additional forespore-specific regulator in C. difficile. Its role remains to be elucidated.
The mother cell regulatory network In B. subtilis, sigE transcription is activated by Spo0A and initiated at the SigA-dependent promoter located upstream of spoIIGA (Baldus et al., 1994; Buckner et al., 1998; Hilbert & Piggot, 2004). In turn, SigE is involved in the transcription of numerous genes including spoIIID and gerR encoding two mother cell regulators. SpoIIID and SigE jointly activate the expression of sigK (Kroos et al., 1989; Kunkel et al., 1989). Then, the SigK form of RNA polymerase transcribes gerE, encoding the GerE regulator controlling the expression of SigK-dependent genes (Eichenberger et al., 2004). In C. difficile, sigE expression is dependent on Spo0A and is detected in predivisional FEMS Microbiol Lett 358 (2014) 1–10
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The sporulation regulatory network in C. difficile
cells (Rosenbusch et al., 2012; Fimlaid et al., 2013; Pereira et al., 2013; Saujet et al., 2013; Pettit et al., 2014). However, the expression of spoIIIAA and sigK (SigE targets) is detected only in the mother cell just after asymmetric division (Fig. 4), indicating that SigE activity is confined to the mother cell (Pereira et al., 2013). sigK is transcribed from two distinct promoters recognized by SigE and SigK (Pereira et al., 2013; Saujet et al., 2013), suggesting that sigK is first transcribed by the SigE-RNA polymerase and then by SigK at later stages of sporulation (Fig. 4). Disruption of sigE prevents expression of a PsigK–SNAP transcriptional fusion (Pereira et al., 2013) and SigK production (Fimlaid et al., 2013). However, disruption of sigK did not seem to significantly reduce expression of a PsigK–SNAP in the condition tested. In C. difficile, SpoIIID is present but GerR and GerE are absent. SigE is involved in the transcription of spoIIID (Saujet et al., 2013) (Fig. 4). The C. difficile spoIIID mutant is blocked after engulfment completion and produces about 103 fewer heat-resistant spores than the wild-type strain (Saujet et al., 2013). SpoIIID negatively controls the expression of members of the SigE regulon (including spoIVA, sipL, cotB and the spoIIIA and spm operons) and positively controls the expression of sigK (Fig. 4) and of 11 members of the SigK regulon (Saujet et al., 2013). The positive regulation of SigK-controlled genes by SpoIIID involves either a direct binding of SpoIIID to their promoter regions or an indirect effect via the positive control of sigK transcription by SpoIIID. So, SpoIIID plays a major role in the control of gene expression in the mother cell in C. difficile by repressing the
expression of SigE targets and activating sigK and some of the SigK-controlled genes. Finally, four additional SigE-controlled genes code for transcription factors (Fimlaid et al., 2013). Further analysis will be necessary to determine their possible involvement in the mother cell line of gene expression.
Intercompartmental communication between the forespore and the mother cell One of the most interesting features of the B. subtilis sporulation network is the existence of signaling pathways allowing communication between the forespore and the mother cell (Fig. 2). This communication takes place at critical steps of spore morphogenesis and allows the coordinated deployment of the two genetic programs and their coupling to the course of morphogenesis. In B. subtilis, SigF activity in the forespore is necessary for pro-SigE cleavage in the mother cell. Then, the activity of SigG in the forespore requires the function of SigE in the mother cell. Finally, SigG is necessary for the cleavage and activation of SigK in the mother cell (Higgins & Dworkin, 2012). Partial SigF-independent activation of SigE in C. difficile: role of SpoIIR
In B. subtilis, SigF, which is required for the activation of pro-SigE, indirectly controls the SigE regulon. Indeed, SigF allows the production of the signaling protein SpoIIR, which is secreted across the forespore inner-membrane
Fig. 4. The mother cell regulatory network in C. difficile. The upper part of the figure represents the cell before asymmetric division. The parallel vertical lines represent the two membranes separating the forespore (right) and the mother cell (left). IFM and OFM mean outer-forespore membrane and innerforespore membrane, respectively. The sigma factors correspond to oval boxes. Diamondshaped green box corresponds to pro-SigE, the inactive form of SigE. The figure also includes a schematic representation of the deduced spatial pattern of transcription of a PspoIIIAA– SNAP or PcotE–SNAP fusions. The PspoIIIAA– SNAP and PcotE–SNAP fusions are specifically controlled by SigE and SigK, respectively. Black solid arrows and white arrows indicate activation at the transcriptional level or at the level of protein activity, respectively. Crossed arrow corresponds to transcriptional repression. Question marks represent mechanisms that are not yet fully understood.
FEMS Microbiol Lett 358 (2014) 1–10
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into the intermembrane space, where it triggers the SpoIIGA-dependent pro-SigE processing in the mother cell (Karow et al., 1995; Londono-Vallejo & Stragier, 1995). Surprisingly, the expression of the SigE regulon in the mother cell is not strictly under SigF control in C. difficile despite the fact that SpoIIR and SpoIIGA are present (Fimlaid et al., 2013; Pereira et al., 2013; Saujet et al., 2013). Indeed, SigE target genes are either not regulated (Saujet et al., 2013) or only slightly downregulated in a sigF mutant compared with the wild-type strain (Fimlaid et al., 2013). In addition, the PspoIIIAA–SNAP fusion is still expressed in the mother cell compartment of a sigF mutant, indicating that SigE is active in the absence of SigF, although in only a fraction of the cells (Pereira et al., 2013). In agreement with these results, both pro-SigE and processed SigE are detected in the sigF mutant, even if SigE is present in a reduced amount compared with the wild-type strain (Fimlaid et al., 2013; Saujet et al., 2013). As shown in B. subtilis, SpoIIR is required for pro-SigE processing likely through a control of SpoIIGA activity (Saujet et al., 2013). However, spoIIR expression is not strictly dependent on SigF (Fimlaid et al., 2013; Saujet et al., 2013). Indeed, in the sigF mutant, a residual expression of spoIIR that is Spo0Adependent is sufficient to maintain a SigE-dependent activity in the mother cell (Fig. 5) (Saujet et al., 2013). Nevertheless, no Spo0A box is identified in the spoIIR promoter region suggesting an indirect effect of Spo0A. A SigF promoter is found upstream of spoIIR, and its transcription is confined to the forespore and detected only after asymmetric division (Saujet et al., 2013). Presumably, another sigma factor can activate foresporespecific transcription of spoIIR in a fraction of the sporulating cells. Furthermore, expression of a PspoIIIAA– SNAP fusion is detected in only about 20% of the sigF cells (Pereira et al., 2013) in agreement with the idea that spoIIR is expressed independently of SigF in a fraction of cells. To conclude, these results indicate that SigF is not strictly required for SigE functionality in C. difficile, highlighting a major difference relative to the B. subtilis sporulation. Regulation of SigG activity by SigE
In B. subtilis, the main period of sigG transcription and activity takes place after engulfment completion (Partridge & Errington, 1993) and the expression of SigG targets in the forespore depends upon SigE activity in the mother cell. The requirement for SigE is at least in part dependent on the expression of the spoIIIA operon and the formation of the SpoIIIAH–SpoIIQ channel that links the cytoplasm of the two cells and is required for late, postengulfment completion, gene expression in the foreª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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spore (Hilbert & Piggot, 2004; Higgins & Dworkin, 2012). Surprisingly, sigG expression is detected just after asymmetric division and SigG activity begins prior to engulfment completion in C. difficile (Pereira et al., 2013). Furthermore, the SigG protein is detected and several SigG targets are expressed at normal levels in a sigE mutant (Fimlaid et al., 2013; Pereira et al., 2013; Saujet et al., 2013). Indeed, a PsspA–SNAP fusion is expressed in the forespore of this mutant (Pereira et al., 2013). Then, a strict requirement of SigE is not observed for the expression of the large majority of the SigG-controlled genes (Fig. 5). However, some SigG target genes are downregulated in the C. difficile sigE mutant (Saujet et al., 2013). This suggests that SigE participates in the control of the SigG regulon in C. difficile, probably through the control of the expression of the spoIIIAA operon as observed in B. subtilis (Higgins & Dworkin, 2012). Moreover, fluorescence from a PsspA–SNAP fusion enhances after engulfment completion, an increase that is prevented by disruption of sigE (Pereira et al., 2013). To conclude, SigG is active in the absence of SigE in C. difficile, but the increase in SigG activity after engulfment completion probably requires SigE and may rely on formation of the SpoIIIAH–SpoIIQ channel (Fig. 5). The SigK regulon is not controlled by SigG
In B. subtilis, SigG regulates the expression of the SigK regulon in the mother cell through the control of proSigK processing involving the SpoIVFB protease and SpoIVFA and BofA, two membrane proteins that form a complex with and control SpoIVFB activity (Higgins & Dworkin, 2012). SigK lacks a pro-sequence, and SpoIVFB, SpoIVFA, and BofA are absent in C. difficile (Stragier, 2002; Haraldsen & Sonenshein, 2003; Abecasis et al., 2013). Interestingly, the SigK regulon is not controlled by SigG using global approaches or a more targeted strategy coupling morphology and expression using a PcotE–SNAP fusion (Fimlaid et al., 2013; Pereira et al., 2013; Saujet et al., 2013). Moreover, accumulation of SigK as well as of three other SigK-controlled proteins (CotE, CdeC, and SleC) is detected in a sigG mutant (Fimlaid et al., 2013). In addition, the coat protein, CotE, is produced and assembled around the developing spore in the sigG mutant as demonstrated using a CotE–SNAP protein fusion (Pereira et al., 2013). In agreement, analysis of sigG mutant cells by transmission electron microscopy (TEM) shows deposition of at least some coat around the forespore (Fimlaid et al., 2013; Pereira et al., 2013). Thus, the absence of regulation of SigK targets by SigG may be related to the lack of control of SigK by processing of a preprotein in C. difficile (Fig. 5). FEMS Microbiol Lett 358 (2014) 1–10
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(a)
(b)
Fig. 5. Communication between forespore and mother cell in C. difficile. (a) Model of the regulatory network controlling sporulation. The parallel vertical lines represent the two membranes separating the forespore (right) and the mother cell (left). IFM and OFM mean outerforespore membrane and inner-forespore membrane, respectively. Proteins associated with the membrane or located into the intermembrane space are illustrated as embedded in the parallel vertical lines. The four sigma factors are encircled (by oval boxes), while diamond-shaped green box corresponds to pro-SigE. Orange square boxes correspond to transcriptional regulators. The dotted line represents the engulfment completion. Black solid arrows and white arrows indicate activation at the transcriptional level or at the level of protein activity, respectively. Question marks represent mechanisms that are not yet fully understood. (b) Schematic representation of the intercellular communication pathways leading to sigma factor activation during sporulation. In C. difficile, the activation of SigE is partially independent on SigF (represented by a broken arrow), while the activation of SigG is independent of SigE and the activation of SigK also does not seem to depend on SigG (represented by crossed arrows). The figure includes a schematic representation of the deduced spatial pattern of transcription of a PspoIIIAA– SNAP fusion in a sigF mutant, of a PsspA–SNAP fusion in a sigE mutant or of a PcotE–SNAP fusion in a sigG mutant.
Control of SigK target genes by SigF
Interestingly, SigF controls some SigK targets in C. difficile (Fimlaid et al., 2013; Saujet et al., 2013). Indeed, several genes belonging to the SigK regulon and sigK itself are downregulated in the sigF mutant (Fimlaid et al., 2013; Saujet et al., 2013). Furthermore, SigK, CotE, and CdeC are not detected in the sigF mutant, while they are detected in a sigG mutant even though both cotE and cdeC transcripts could be detected at significant levels in a sigF mutant (Fimlaid et al., 2013). The effect of SigF on the expression of SigK targets could be mediated by the indirect control of SigK synthesis or activity by SigF. This might be also due to a decrease in SigE activity in the absence of SigF (Fig. 5). The mechanism of the control of SigK by SigF remains to be deciphered.
Potential role of the skin element excision in the regulation of sigK expression in C. difficile Some divergences are observed in the late stages of the sporulation process between B. subtilis and C. difficile but also among Clostridia. It is worth noting that the genus FEMS Microbiol Lett 358 (2014) 1–10
Clostridium is highly diverse. Clostridium difficile (recently re-named Peptoclostridium difficile) is a member of the Peptosptreptococcae family, while other Clostridia such as Clostridium perfringens, Clostridium botulinum, and Clostridium acetobutylicum belong to the Clostridiaceae family (Yutin & Galperin, 2013). The sigK mutants of B. subtilis and C. difficile are blocked in late sporulation phase (Piggot & Coote, 1976; Hilbert & Piggot, 2004; Pereira et al., 2013), while the sigK mutants of C. perfringens and C. acetobutylicum are arrested before asymmetric division (Harry et al., 2009; Al-Hinai et al., 2014). In correlation with these phenotypes, there are several major differences between B. subtilis and Clostridia regarding (1) the timing of sigK expression, (2) the presence of a pro-SigK protein, and (3) the insertion of a prophagelike mobile element (skin) into sigK. In C. difficile and B. subtilis, sigK is expressed late during sporulation (Pereira et al., 2013), while in C. perfringens, C. botulinum, and C. acetobutylicum, sigK is also expressed earlier and before asymmetric division in C. perfringens and C. acetobutylicum (Harry et al., 2009; Kirk et al., 2012; Al-Hinai et al., 2014). In B. subtilis, SigK activity depends on the cleavage of a pro-sequence, a step controlled by SigG (Errington, 2003). Interestingly, ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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the pro-SigK form is present in Clostridiaceae but absent in several Peptosptreptococcae including C. difficile, Clostridium sordelii, and Clostridium bifermentans (de Hoon et al., 2010) but does not seem sufficient to prevent early sigK expression and activity in C. perfringens, C. botulinum, and C. acetobutylicum [in the latter organism, SigK functions both early and late in sporulation; (Al-Hinai et al., 2014)]. In C. difficile and B. subtilis, the sigK gene is interrupted by a skin element absent in other Clostridia (de Hoon et al., 2010). So, the skin element may partly explain the late expression of sigK and the morphology of the sigK mutant in B. subtilis and C. difficile. The skin elements of B. subtilis and C. difficile have different sizes and are inserted at different sites and in opposite orientation, indicating that skin integration into sigK has occurred independently during evolution (Haraldsen & Sonenshein, 2003). The skinCd element is excised during sporulation (Haraldsen & Sonenshein, 2003). The CD1231 gene, located within the skin (Fig. 4), codes for a protein similar to the SpoIVCA site-specific recombinase of the skinBs (Kunkel et al., 1990; Haraldsen & Sonenshein, 2003). In the absence of a pro-sequence for SigK, the skinCd excision is likely an important element in the regulation of SigK activity in C. difficile. Then, it is important to better understand the regulation of skin excision in this organism. In B. subtilis, SigE and SpoIIID are required for the expression of the spoIVCA gene (Eichenberger et al., 2004). Surprisingly, neither SigE nor SpoIIID controls CD1231 expression (Saujet et al., 2013). Another factor may modulate CD1231 synthesis or activity, or another recombinase responsible for skinCd excision may be at play in C. difficile.
Conclusion In B. subtilis, activation of the sporulation sigma factors coincides with the completion of key morphological steps during spore formation (asymmetric division, engulfment completion) and intercompartmental communication pathways strictly coordinate the mother cell and forespore lines of expression and their coupling to the course of morphogenesis (Fig. 2). Important deviations from the B. subtilis paradigm exist in C. difficile especially concerning (1) the communication between the forespore and the mother cell and (2) a weaker connection between gene expression and morphogenesis (Fig. 5). First, the SigE regulon is not strictly under SigF control although SpoIIR is essential for pro-SigE cleavage. Residual SigF-independent spoIIR expression is probably responsible for pro-SigE processing in the sigF mutant. Second, sigG is expressed earlier (soon after asymmetric division) and a strict requirement for SigE is not observed for the production and activity of SigG although the increase in SigG activity following ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
L. Saujet et al.
engulfment completion requires SigE and possibly the formation of the SpoIIIAH–SpoIIQ channel. Third, SigK activity is independent on SigG. However, a control of SigK activity by the forespore seems to be maintained through a SigF-dependent mechanism and the role of the skin element in the timing of production of SigK remains to be clarified. Other regulatory proteins and/or small noncoding RNAs could play a role in this regulatory cascade. Interestingly, a strain depleted for the RNA chaperon, Hfq, has a better ability to sporulate. This effect is probably mediated through its activity on one or several noncoding RNAs (Boudry et al., 2014). Spore-forming Firmicutes have different lifestyles, and the most significant variations in the sporulation process are observed at the interface with their environment: signal transduction pathways triggering sporulation, coat proteins, and germination signals. Based on the fact that anaerobic bacteria existed before aerobes, it is tempting to speculate that the first spore-forming bacteria, which appeared at the basis of the Firmicutes phylum, were anaerobes (Galperin et al., 2012; Abecasis et al., 2013). The developmental control in C. difficile and probably in other Clostridia might reflect a more ancestral version of the sporulation gene regulatory network, while a more sophisticated system of control with a tight coupling between morphogenesis and gene expression would have been gradually introduced during evolution for Bacilli. Differences in the period of sporulation sigma factor activation may also be linked to the coordination with other processes such as solventogenesis, biofilm formation, or toxin production (Paredes et al., 2005; Harry et al., 2009; Semenyuk et al., 2014). The less tight control of sporulation in C. difficile might also allow heterogeneity in the population, a possible adaptive strategy during gut colonization.
References Abecasis AB, Serrano M, Alves R, Quintais L, Pereira-Leal JB & Henriques AO (2013) A genomic signature and the identification of new sporulation genes. J Bacteriol 195: 2101–2115. Al-Hinai MA, Jones SW & Papoutsakis ET (2014) sigmaK of Clostridium acetobutylicum is the first known sporulation-specific sigma factor with two developmentally separated roles, one early and one late in sporulation. J Bacteriol 196: 287–299. Bagyan I, Hobot J & Cutting S (1996) A compartmentalized regulator of developmental gene expression in Bacillus subtilis. J Bacteriol 178: 4500–4507. Baldus JM, Green BD, Youngman P & Moran CP Jr (1994) Phosphorylation of Bacillus subtilis transcription factor Spo0A stimulates transcription from the spoIIG promoter by enhancing binding to weak 0A boxes. J Bacteriol 176: 296–306.
FEMS Microbiol Lett 358 (2014) 1–10
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Boudry P, Gracia C, Monot M, Caillet J, Saujet L, Hajnsdorf E, Dupuy B, Martin-Verstraete I & Soutourina O (2014) Pleiotropic role of the RNA chaperone protein Hfq in the human pathogen Clostridium difficile. J Bacteriol in press. Britton RA, Eichenberger P, Gonzalez-Pastor JE, Fawcett P, Monson R, Losick R & Grossman AD (2002) Genome-wide analysis of the stationary-phase sigma factor (sigma-H) regulon of Bacillus subtilis. J Bacteriol 184: 4881–4890. Buckner CM, Schyns G & Moran CP Jr (1998) A region in the Bacillus subtilis transcription factor Spo0A that is important for spoIIG promoter activation. J Bacteriol 180: 3578–3583. Carroll KC & Bartlett JG (2011) Biology of Clostridium difficile: implications for epidemiology and diagnosis. Annu Rev Microbiol 65: 501–521. de Hoon MJ, Eichenberger P & Vitkup D (2010) Hierarchical evolution of the bacterial sporulation network. Curr Biol 20: R735–R745. Deakin LJ, Clare S, Fagan RP, Dawson LF, Pickard DJ, West MR, Wren BW, Fairweather NF, Dougan G & Lawley TD (2012) The Clostridium difficile spo0A gene is a persistence and transmission factor. Infect Immun 80: 2704–2711. Edwards AN & McBride SM (2014) Initiation of sporulation in Clostridium difficile: a twist on the classic model. FEMS Microbiol Lett, doi: 10.1111/1574-6968.12499. Eichenberger P, Fujita M, Jensen ST et al. (2004) The program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis. PLoS Biol 2: e328. Errington J (2003) Regulation of endospore formation in Bacillus subtilis. Nat Rev Microbiol 1: 117–126. Fimlaid KA, Bond JP, Schutz KC, Putnam EE, Leung JM, Lawley TD & Shen A (2013) Global analysis of the sporulation pathway of Clostridium difficile. PLoS Genet 9: e1003660. Galperin MY, Mekhedov SL, Puigbo P, Smirnov S, Wolf YI & Rigden DJ (2012) Genomic determinants of sporulation in Bacilli and Clostridia: towards the minimal set of sporulation-specific genes. Environ Microbiol 14: 2870–2890. Haraldsen JD & Sonenshein AL (2003) Efficient sporulation in Clostridium difficile requires disruption of the sigmaK gene. Mol Microbiol 48: 811–821. Harry KH, Zhou R, Kroos L & Melville SB (2009) Sporulation and enterotoxin (CPE) synthesis are controlled by the sporulation-specific sigma factors SigE and SigK in Clostridium perfringens. J Bacteriol 191: 2728–2742. Higgins D & Dworkin J (2012) Recent progress in Bacillus subtilis sporulation. FEMS Microbiol Rev 36: 131–148. Hilbert DW & Piggot PJ (2004) Compartmentalization of gene expression during Bacillus subtilis spore formation. Microbiol Mol Biol Rev 68: 234–262. Janoir C, Deneve C, Bouttier S et al. (2013) Adaptive strategies and pathogenesis of Clostridium difficile from in vivo transcriptomics. Infect Immun 81: 3757–3769. Karow ML, Glaser P & Piggot PJ (1995) Identification of a gene, spoIIR, that links the activation of sigma E to the
FEMS Microbiol Lett 358 (2014) 1–10
9
transcriptional activity of sigma F during sporulation in Bacillus subtilis. P Natl Acad Sci USA 92: 2012–2016. Kirk DG, Dahlsten E, Zhang Z, Korkeala H & Lindstrom M (2012) Involvement of Clostridium botulinum ATCC 3502 sigma factor K in early-stage sporulation. Appl Environ Microbiol 78: 4590–4596. Kroos L, Kunkel B & Losick R (1989) Switch protein alters specificity of RNA polymerase containing a compartment-specific sigma factor. Science 243: 526–529. Kunkel B, Kroos L, Poth H, Youngman P & Losick R (1989) Temporal and spatial control of the mother-cell regulatory gene spoIIID of Bacillus subtilis. Genes Dev 3: 1735–1744. Kunkel B, Losick R & Stragier P (1990) The Bacillus subtilis gene for the development transcription factor sigma K is generated by excision of a dispensable DNA element containing a sporulation recombinase gene. Genes Dev 4: 525–535. Lawley TD, Clare S, Walker AW et al. (2009) Antibiotic treatment of Clostridium difficile carrier mice triggers a supershedder state, spore-mediated transmission, and severe disease in immunocompromised hosts. Infect Immun 77: 3661–3669. Londono-Vallejo JA & Stragier P (1995) Cell-cell signaling pathway activating a developmental transcription factor in Bacillus subtilis. Genes Dev 9: 503–508. Molle V, Fujita M, Jensen ST, Eichenberger P, Gonzalez-Pastor JE, Liu JS & Losick R (2003) The Spo0A regulon of Bacillus subtilis. Mol Microbiol 50: 1683–1701. Paredes CJ, Alsaker KV & Papoutsakis ET (2005) A comparative genomic view of clostridial sporulation and physiology. Nat Rev Microbiol 3: 969–978. Partridge SR & Errington J (1993) The importance of morphological events and intercellular interactions in the regulation of prespore-specific gene expression during sporulation in Bacillus subtilis. Mol Microbiol 8: 945–955. Pereira FC, Saujet L, Tome AR, Serrano M, Monot M, Couture-Tosi E, Martin-Verstraete I, Dupuy B & Henriques AO (2013) The spore differentiation pathway in the enteric pathogen Clostridium difficile. PLoS Genet 9: e1003782. Pettit LJ, Browne HP, Yu L et al. (2014) Functional genomics reveals that Clostridium difficile Spo0A coordinates sporulation, virulence and metabolism. BMC Genomics 15: 160. Piggot PJ & Coote JG (1976) Genetic aspects of bacterial endospore formation. Bacteriol Rev 40: 908–962. Rosenbusch KE, Bakker D, Kuijper EJ & Smits WK (2012) C. difficile 630Deltaerm Spo0A regulates sporulation, but does not contribute to toxin production, by direct high-affinity binding to target DNA. PLoS One 7: e48608. Saujet L, Monot M, Dupuy B, Soutourina O & Martin-Verstraete I (2011) The key sigma factor of transition phase, SigH, controls sporulation, metabolism, and virulence factor expression in Clostridium difficile. J Bacteriol 193: 3186–3196.
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Saujet L, Pereira FC, Serrano M, Soutourina O, Monot M, Shelyakin PV, Gelfand MS, Dupuy B, Henriques AO & Martin-Verstraete I (2013) Genome-wide analysis of cell type-specific gene transcription during spore formation in Clostridium difficile. PLoS Genet 9: e1003756. Semenyuk EG, Laning ML, Foley J, Johnston PF, Knight KL, Gerding DN & Driks A (2014) Spore formation and toxin production in Clostridium difficile biofilms. PLoS ONE 9: e87757. Sorg JA & Sonenshein AL (2008) Bile salts and glycine as cogerminants for Clostridium difficile spores. J Bacteriol 190: 2505–2512. Steil L, Serrano M, Henriques AO & Volker U (2005) Genome-wide analysis of temporally regulated and compartment-specific gene expression in sporulating cells of Bacillus subtilis. Microbiology 151: 399–420. Stragier P (2002) A Gene Odyssey: Exploring the Genomes of Endospore-Forming Bacteria. ASM Press, Washington, DC.
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L. Saujet et al.
Stragier P & Losick R (1996) Molecular genetics of sporulation in Bacillus subtilis. Annu Rev Genet 30: 297–341. Traag B, Ramirez-Peralta A, Wang Erickson AF, Setlow P & Losick R (2013) A novel RNA Polymerase-binding protein involved in spore germination in Bacillus subtilis. Mol Microbiol 89: 113–122. Underwood S, Guan S, Vijayasubhash V, Baines SD, Graham L, Lewis RJ, Wilcox MH & Stephenson K (2009) Characterization of the sporulation initiation pathway of Clostridium difficile and its role in toxin production. J Bacteriol 191: 7296–7305. Wang ST, Setlow B, Conlon EM, Lyon JL, Imamura D, Sato T, Setlow P, Losick R & Eichenberger P (2006) The forespore line of gene expression in Bacillus subtilis. J Mol Biol 358: 16–37. Yutin N & Galperin MY (2013) A genomic update on clostridial phylogeny: gram-negative spore formers and other misplaced clostridia. Environ Microbiol 15: 2631–2641.
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