Cleavage of an engulfment peptidoglycan hydrolase by a sporulation signature protease in Clostridioides difficile.

In the model organism Bacillus subtilis, a signaling protease produced in the forespore, SpoIVB, is essential for the activation of the sigma factor σK, which is produced in the mother cell as an inactive pro-protein, pro-σK. SpoIVB has a second function essential to sporulation, most likely during cortex synthesis. The cortex is composed of peptidoglycan (PG) and is essential for the spore's heat resistance and dormancy. Surprisingly, the genome of the intestinal pathogen Clostridioides difficile, in which σK is produced without a pro-sequence, encodes two SpoIVB paralogs, SpoIVB1 and SpoIVB2. Here, we show that spoIVB1 is dispensable for sporulation, while a spoIVB2 in-frame deletion mutant fails to produce heat-resistant spores. The spoIVB2 mutant enters sporulation, undergoes asymmetric division, and completes engulfment of the forespore by the mother cell but fails to synthesize the spore cortex. We show that SpoIIP, a PG hydrolase and part of the engulfasome, the machinery essential for engulfment, is cleaved by SpoIVB2 into an inactive form. Within the engulfasome, the SpoIIP amidase activity generates the substrates for the SpoIID lytic transglycosylase. Thus, following engulfment completion, the cleavage and inactivation of SpoIIP by SpoIVB2 curtails the engulfasome hydrolytic activity, at a time when synthesis of the spore cortex peptidoglycan begins. SpoIVB2 is also required for normal late gene expression in the forespore by a currently unknown mechanism. Together, these observations suggest a role for SpoIVB2 in coordinating late morphological and gene expression events between the forespore and the mother cell.

A sporulation signature protease is required for assembly of the spore surface layers, germination and host colonization in Clostridioides difficile.

A genomic signature for endosporulation includes a gene coding for a protease, YabG, which in the model organism Bacillus subtilis is involved in assembly of the spore coat. We show that in the human pathogen Clostridioidesm difficile, YabG is critical for the assembly of the coat and exosporium layers of spores. YabG is produced during sporulation under the control of the mother cell-specific regulators σE and σK and associates with the spore surface layers. YabG shows an N-terminal SH3-like domain and a C-terminal domain that resembles single domain response regulators, such as CheY, yet is atypical in that the conserved phosphoryl-acceptor residue is absent. Instead, the CheY-like domain carries residues required for activity, including Cys207 and His161, the homologues of which form a catalytic diad in the B. subtilis protein, and also Asp162. The substitution of any of these residues by Ala, eliminates an auto-proteolytic activity as well as interdomain processing of CspBA, a reaction that releases the CspB protease, required for proper spore germination. An in-frame deletion of yabG or an allele coding for an inactive protein, yabGC207A, both cause misassemby of the coat and exosporium and the formation of spores that are more permeable to lysozyme and impaired in germination and host colonization. Furthermore, we show that YabG is required for the expression of at least two σK-dependent genes, cotA, coding for a coat protein, and cdeM, coding for a key determinant of exosporium assembly. Thus, YabG also impinges upon the genetic program of the mother cell possibly by eliminating a transcriptional repressor. Although this activity has not been described for the B. subtilis protein and most of the YabG substrates vary among sporeformers, the general role of the protease in the assembly of the spore surface is likely to be conserved across evolutionary distance.

Clostridioides difficile Sporulation.

Some members of the Firmicutes phylum, including many members of the human gut microbiota, are able to differentiate a dormant and highly resistant cell type, the endospore (hereinafter spore for simplicity). Spore-formers can colonize virtually any habitat and, because of their resistance to a wide variety of physical and chemical insults, spores can remain viable in the environment for long periods of time. In the anaerobic enteric pathogen Clostridioides difficile the aetiologic agent is the oxygen-resistant spore, while the toxins produced by actively growing cells are the main cause of the disease symptoms. Here, we review the regulatory circuits that govern entry into sporulation. We also cover the role of spores in the infectious cycle of C. difficile in relation to spore structure and function and the main control points along spore morphogenesis.

Structural insights into ring-building motif domains involved in bacterial sporulation.

Components of specialized secretion systems, which span the inner and outer membranes in Gram-negative bacteria, include ring-forming proteins whose oligomerization was proposed to be promoted by domains called RBM for "Ring-Building Motifs". During spore formation in Gram-positive bacteria, a transport system called the SpoIIIA-SpoIIQ complex also assembles in the double membrane that surrounds the forespore following its endocytosis by the mother cell. The presence of RBM domains in some of the SpoIIIA proteins led to the hypothesis that they would assemble into rings connecting the two membranes and form a conduit between the mother cell and forespore. Among them, SpoIIIAG forms homo-oligomeric rings in vitro but the oligomerization of other RBM-containing SpoIIIA proteins, including SpoIIIAH, remains to be demonstrated. In this work, we identified RBM domains in the YhcN/YlaJ family of proteins that are not related to the SpoIIIA-SpoIIQ complex. We solved the crystal structure of YhcN from Bacillus subtilis, which confirmed the presence of a RBM fold, flanked by additional secondary structures. As the protein did not show any oligomerization ability in vitro, we investigated the structural determinants of ring formation in SpoIIIAG, SpoIIIAH and YhcN. We showed that in vitro, the conserved core of RBM domains alone is not sufficient for oligomerization while the ß-barrel forming region in SpoIIIAG forms rings on its own. This work suggests that some RBMs might indeed participate in the assembly of homomeric rings but others might have evolved toward other functions.

Temporal and spatial regulation of protein cross-linking by the pre-assembled substrates of a Bacillus subtilis spore coat transglutaminase.

In many cases protein assemblies are stabilized by covalent bonds, one example of which is the formation of intra- or intermolecular ε-(γ-glutamyl)lysil cross-links catalyzed by transglutaminases (TGases). Because of the potential for unwanted cross-linking reactions, the activities of many TGases have been shown to be tightly controlled. Bacterial endospores are highly resilient cells in part because they are surrounded by a complex protein coat. Proteins in the coat that surrounds Bacillus subtilis endospores are crosslinked by a TGase (Tgl). Unlike other TGases, however, Tgl is produced in an active form, and efficiently catalyzes amine incorporation and protein cross-linking in vitro with no known additional requirements. The absence of regulatory factors raises questions as to how the activity of Tgl is controlled during spore coat assembly. Here, we show that substrates assembled onto the spore coat prior to Tgl production govern the localization of Tgl to the surface of the developing spore. We also show that Tgl residues important for substrate recognition are crucial for its localization. We identified the glutamyl (Q) and lysil (K) substrate docking sites and we show that residues on the Q side of Tgl are more important for the assembly of Tgl than those on the K side. Thus, the first step in the reaction cycle, the interaction with Q-substrates and formation of an acyl-enzyme intermediate, is also the determinant step in the localization of Tgl. Consistent with the idea that Tg exerts a "spotwelding" activity, cross-linking pre-formed assemblies, we show that C30 is an oblong hexamer in solution that is cross-linked in vitro into high molecular weight forms. Moreover, during the reaction, Tgl becomes part of the cross-linked products. We suggest that the dependency of Tgl on its substrates is used to accurately control the time, location and extent of the enzyme´s activity, directed at the covalent fortification of pre-assembled complexes at the surface of the developing spore.

Clostridioides difficile para-Cresol Production Is Induced by the Precursor para-Hydroxyphenylacetate.

Clostridioides difficile is an etiological agent for antibiotic-associated diarrheal disease. C. difficile produces a phenolic compound, para-cresol, which selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. C. difficile decarboxylates para-hydroxyphenylacetate (p-HPA) to produce p-cresol by the action of the HpdBCA decarboxylase encoded by the hpdBCA operon. Here, we investigate regulation of the hpdBCA operon and directly compare three independent reporter systems; SNAP-tag, glucuronidase gusA, and alkaline phosphatase phoZ reporters to detect basal and inducible expression. We show that expression of hpdBCA is upregulated in response to elevated p-HPA. In silico analysis identified three putative promoters upstream of hpdBCA operon-P1, P2, and Pσ54; only the P1 promoter was responsible for both basal and p-HPA-inducible expression of hpdBCA We demonstrated that turnover of tyrosine, a precursor for p-HPA, is insufficient to induce expression of the hpdBCA operon above basal levels because it is inefficiently converted to p-HPA in minimal media. We show that induction of the hpdBCA operon in response to p-HPA occurs in a dose-dependent manner. We also identified an inverted palindromic repeat (AAAAAG-N13-CTTTTT) upstream of the hpdBCA start codon (ATG) that is essential for inducing transcription of the hpdBCA operon in response to p-HPA, which drives the production of p-cresol. This provides insights into the regulatory control of p-cresol production, which affords a competitive advantage for C. difficile over other intestinal bacteria, promoting dysbiosis.IMPORTANCEClostridioides difficile infection results from antibiotic-associated dysbiosis. para-Cresol, a phenolic compound produced by C. difficile, selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. Here, we demonstrate that expression of the hpdBCA operon, encoding the HpdBCA decarboxylase which converts p-HPA to p-cresol, is upregulated in response to elevated exogenous p-HPA, with induction occurring between >0.1 and ≤0.25 mg/ml. We determined a single promoter and an inverted palindromic repeat responsible for basal and p-HPA-inducible hpdBCA expression. We identified turnover of tyrosine, a p-HPA precursor, does not induce hpdBCA expression above basal level, indicating that exogenous p-HPA was required for p-cresol production. Identifying regulatory controls of p-cresol production will provide novel therapeutic targets to prevent p-cresol production, reducing C. difficile's competitive advantage.

From Root to Tips: Sporulation Evolution and Specialization in Bacillus subtilis and the Intestinal Pathogen Clostridioides difficile.

Bacteria of the Firmicutes phylum are able to enter a developmental pathway that culminates with the formation of highly resistant, dormant endospores. Endospores allow environmental persistence, dissemination and for pathogens, are also infection vehicles. In both the model Bacillus subtilis, an aerobic organism, and in the intestinal pathogen Clostridioides difficile, an obligate anaerobe, sporulation mobilizes hundreds of genes. Their expression is coordinated between the forespore and the mother cell, the two cells that participate in the process, and is kept in close register with the course of morphogenesis. The evolutionary mechanisms by which sporulation emerged and evolved in these two species, and more broadly across Firmicutes, remain largely unknown. Here, we trace the origin and evolution of sporulation using the genes known to be involved in the process in B. subtilis and C. difficile, and estimating their gain-loss dynamics in a comprehensive bacterial macroevolutionary framework. We show that sporulation evolution was driven by two major gene gain events, the first at the base of the Firmicutes and the second at the base of the B. subtilis group and within the Peptostreptococcaceae family, which includes C. difficile. We also show that early and late sporulation regulons have been coevolving and that sporulation genes entail greater innovation in B. subtilis with many Bacilli lineage-restricted genes. In contrast, C. difficile more often recruits new sporulation genes by horizontal gene transfer, which reflects both its highly mobile genome, the complexity of the gut microbiota, and an adjustment of sporulation to the gut ecosystem.

A LysM Domain Intervenes in Sequential Protein-Protein and Protein-Peptidoglycan Interactions Important for Spore Coat Assembly in Bacillus subtilis.

At a late stage in spore development in Bacillus subtilis, the mother cell directs synthesis of a layer of peptidoglycan known as the cortex between the two forespore membranes, as well as the assembly of a protective protein coat at the surface of the forespore outer membrane. SafA, the key determinant of inner coat assembly, is first recruited to the surface of the developing spore and then encases the spore under the control of the morphogenetic protein SpoVID. SafA has a LysM peptidoglycan-binding domain, SafALysM, and localizes to the cortex-coat interface in mature spores. SafALysM is followed by a region, A, required for an interaction with SpoVID and encasement. We now show that residues D10 and N30 in SafALysM, while involved in the interaction with peptidoglycan, are also required for the interaction with SpoVID and encasement. We further show that single alanine substitutions on residues S11, L12, and I39 of SafALysM that strongly impair binding to purified cortex peptidoglycan affect a later stage in the localization of SafA that is also dependent on the activity of SpoVE, a transglycosylase required for cortex formation. The assembly of SafA thus involves sequential protein-protein and protein-peptidoglycan interactions, mediated by the LysM domain, which are required first for encasement then for the final localization of the protein in mature spores.IMPORTANCEBacillus subtilis spores are encased in a multiprotein coat that surrounds an underlying peptidoglycan layer, the cortex. How the connection between the two layers is enforced is not well established. Here, we elucidate the role of the peptidoglycan-binding LysM domain, present in two proteins, SafA and SpoVID, that govern the localization of additional proteins to the coat. We found that SafALysM is a protein-protein interaction module during the early stages of coat assembly and a cortex-binding module at late stages in morphogenesis, with the cortex-binding function promoting a tight connection between the cortex and the coat. In contrast, SpoVIDLysM functions only as a protein-protein interaction domain that targets SpoVID to the spore surface at the onset of coat assembly.

SpoVID functions as a non-competitive hub that connects the modules for assembly of the inner and outer spore coat layers in Bacillus subtilis.

During sporulation in Bacillus subtilis, a group of mother cell-specific proteins guides the assembly of the coat, a multiprotein structure that protects the spore and influences many of its environmental interactions. SafA and CotE behave as party hubs, governing assembly of the inner and outer coat layers. Targeting of coat proteins to the developing spore is followed by encasement. Encasement by SafA and CotE requires E, a region of 11 amino acids in the encasement protein SpoVID, with which CotE interacts directly. Here, we identified two single alanine substitutions in E that prevent binding of SafA, but not of CotE, to SpoVID, and block encasement. The substitutions result in the accumulation of SafA, CotE and their dependent proteins at the mother cell proximal spore pole, phenocopying a spoVID null mutant and suggesting that mislocalized SafA acts as an attractor for the rest of the coat. The requirement for E in SafA binding is bypassed by a peptide with the sequence of E provided in trans. We suggest that E allows binding of SafA to a second region in SpoVID, enabling CotE to interact with E and SpoVID to function as a non-competitive hub during spore encasement.

A Recombination Directionality Factor Controls the Cell Type-Specific Activation of σK and the Fidelity of Spore Development in Clostridium difficile.

The strict anaerobe Clostridium difficile is the most common cause of nosocomial diarrhea, and the oxygen-resistant spores that it forms have a central role in the infectious cycle. The late stages of sporulation require the mother cell regulatory protein σK. In Bacillus subtilis, the onset of σK activity requires both excision of a prophage-like element (skinBs) inserted in the sigK gene and proteolytical removal of an inhibitory pro-sequence. Importantly, the rearrangement is restricted to the mother cell because the skinBs recombinase is produced specifically in this cell. In C. difficile, σK lacks a pro-sequence but a skinCd element is present. The product of the skinCd gene CD1231 shares similarity with large serine recombinases. We show that CD1231 is necessary for sporulation and skinCd excision. However, contrary to B. subtilis, expression of CD1231 is observed in vegetative cells and in both sporangial compartments. Nevertheless, we show that skinCd excision is under the control of mother cell regulatory proteins σE and SpoIIID. We then demonstrate that σE and SpoIIID control the expression of the skinCd gene CD1234, and that this gene is required for sporulation and skinCd excision. CD1231 and CD1234 appear to interact and both proteins are required for skinCd excision while only CD1231 is necessary for skinCd integration. Thus, CD1234 is a recombination directionality factor that delays and restricts skinCd excision to the terminal mother cell. Finally, while the skinCd element is not essential for sporulation, deletion of skinCd results in premature activity of σK and in spores with altered surface layers. Thus, skinCd excision is a key element controlling the onset of σK activity and the fidelity of spore development.

Autoregulation of SafA Assembly through Recruitment of a Protein Cross-Linking Enzyme.

The coat of Bacillus subtilis spores is a multiprotein protective structure that also arbitrates many of the environmental interactions of the spore. The coat assembles around the cortex peptidoglycan layer and is differentiated into an inner and an outer layer and a crust. SafA governs assembly of the inner coat, whereas CotE drives outer coat assembly. SafA localizes to the cortex-coat interface. Both SafA and its short form C30 are substrates for Tgl, a coat-associated transglutaminase that cross-links proteins through ε-(γ-glutamyl)lysyl isopeptide bonds. We show that SafA and C30 are distributed between the coat and cortex layers. The deletion of tgl increases the extractability of SafA, mainly from the cortex. Tgl itself is mostly located in the inner coat and cortex. The localization of Tgl-cyan fluorescent protein (Tgl-CFP) is strongly, but not exclusively, dependent on safA However, the association of Tgl with the cortex requires safA Together, our results suggest an assembly pathway in which Tgl is first recruited to the forming spore in a manner that is only partially dependent on SafA and then is drafted to the cortex by SafA. Tgl, in turn, promotes the conversion of coat- and cortex-associated SafA into forms that resist extraction, possibly by catalyzing the cross-linking of SafA to other coat proteins, to the cortex, and/or to cortex-associated proteins. Therefore, the final assembly state of SafA relies on an autoregulatory pathway that requires the subcellular localization of a protein cross-linking enzyme. Tgl most likely exerts a "spotwelding" activity, cross-linking preformed complexes in the cortex and inner coat layers of spores.IMPORTANCE In this work, we show how two proteins work together to determine their subcellular location within the coat of bacterial endospores. Bacillus subtilis endospores are surrounded by a multilayer protein coat composed of over 80 proteins, which surrounds an underlying peptidoglycan layer (the spore cortex) protecting it from lytic enzymes. How specific coat proteins are targeted to specific layers of the coat is not well understood. We found that the protein SafA recruits a protein-cross-linking enzyme (a transglutaminase) to the cortex and inner layers of the coat, where both are cemented, by cross-linking, into macromolecular complexes.

Imipenem Resistance in Clostridium difficile Ribotype 017, Portugal.

We describe imipenem-resistant and imipenem-susceptible clinical isolates of Clostridium difficile ribotype 017 in Portugal. All ribotype 017 isolates carried an extra penicillin-binding protein gene, pbp5, and the imipenem-resistant isolates had additional substitutions near the transpeptidase active sites of pbp1 and pbp3. These clones could disseminate and contribute to imipenem resistance.

Dual-specificity anti-sigma factor reinforces control of cell-type specific gene expression in Bacillus subtilis.

Gene expression during spore development in Bacillus subtilis is controlled by cell type-specific RNA polymerase sigma factors. σFand σE control early stages of development in the forespore and the mother cell, respectively. When, at an intermediate stage in development, the mother cell engulfs the forespore, σF is replaced by σG and σE is replaced by σK. The anti-sigma factor CsfB is produced under the control of σF and binds to and inhibits the auto-regulatory σG, but not σF. A position in region 2.1, occupied by an asparagine in σG and by a glutamate in οF, is sufficient for CsfB discrimination of the two sigmas, and allows it to delay the early to late switch in forespore gene expression. We now show that following engulfment completion, csfB is switched on in the mother cell under the control of σK and that CsfB binds to and inhibits σE but not σK, possibly to facilitate the switch from early to late gene expression. We show that a position in region 2.3 occupied by a conserved asparagine in σE and by a conserved glutamate in σK suffices for discrimination by CsfB. We also show that CsfB prevents activation of σG in the mother cell and the premature σG-dependent activation of σK. Thus, CsfB establishes negative feedback loops that curtail the activity of σE and prevent the ectopic activation of σG in the mother cell. The capacity of CsfB to directly block σE activity may also explain how CsfB plays a role as one of the several mechanisms that prevent σE activation in the forespore. Thus the capacity of CsfB to differentiate between the highly similar σF/σG and σE/σK pairs allows it to rinforce the cell-type specificity of these sigma factors and the transition from early to late development in B. subtilis, and possibly in all sporeformers that encode a CsfB orthologue.

The SpoIIQ-SpoIIIAH complex of Clostridium difficile controls forespore engulfment and late stages of gene expression and spore morphogenesis.

Engulfment of the forespore by the mother cell is a universal feature of endosporulation. In Bacillus subtilis, the forespore protein SpoIIQ and the mother cell protein SpoIIIAH form a channel, essential for endosporulation, through which the developing spore is nurtured. The two proteins also form a backup system for engulfment. Unlike in B. subtilis, SpoIIQ of Clostridium difficile has intact LytM zinc-binding motifs. We show that spoIIQ or spoIIIAH deletion mutants of C. difficile result in anomalous engulfment, and that disruption of the SpoIIQ LytM domain via a single amino acid substitution (H120S) impairs engulfment differently. SpoIIQ and SpoIIQ(H120S) interact with SpoIIIAH throughout engulfment. SpoIIQ, but not SpoIIQ(H120S) , binds Zn(2+) , and metal absence alters the SpoIIQ-SpoIIIAH complex in vitro. Possibly, SpoIIQ(H120S) supports normal engulfment in some cells but not a second function of the complex, required following engulfment completion. We show that cells of the spoIIQ or spoIIIAH mutants that complete engulfment are impaired in post-engulfment, forespore and mother cell-specific gene expression, suggesting a channel-like function. Both engulfment and a channel-like function may be ancestral functions of SpoIIQ-SpoIIIAH while the requirement for engulfment was alleviated through the emergence of redundant mechanisms in B. subtilis and related organisms.

Sporulation Temperature Reveals a Requirement for CotE in the Assembly of both the Coat and Exosporium Layers of Bacillus cereus Spores.

The Bacillus cereus spore surface layers consist of a coat surrounded by an exosporium. We investigated the interplay between the sporulation temperature and the CotE morphogenetic protein in the assembly of the surface layers of B. cereus ATCC 14579 spores and on the resulting spore properties. The cotE deletion affects the coat and exosporium composition of the spores formed both at the suboptimal temperature of 20°C and at the optimal growth temperature of 37°C. Transmission electron microscopy revealed that ΔcotE spores had a fragmented and detached exosporium when formed at 37°C. However, when produced at 20°C, ΔcotE spores showed defects in both coat and exosporium attachment and were susceptible to lysozyme and mutanolysin. Thus, CotE has a role in the assembly of both the coat and exosporium, which is more important during sporulation at 20°C. CotE was more represented in extracts from spores formed at 20°C than at 37°C, suggesting that increased synthesis of the protein is required to maintain proper assembly of spore surface layers at the former temperature. ΔcotE spores formed at either sporulation temperature were impaired in inosine-triggered germination and resistance to UV-C and H2O2 and were less hydrophobic than wild-type (WT) spores but had a higher resistance to wet heat. While underscoring the role of CotE in the assembly of B. cereus spore surface layers, our study also suggests a contribution of the protein to functional properties of additional spore structures. Moreover, it also suggests a complex relationship between the function of a spore morphogenetic protein and environmental factors such as the temperature during spore formation.

Genome-wide analysis of cell type-specific gene transcription during spore formation in Clostridium difficile.

Clostridium difficile, a Gram positive, anaerobic, spore-forming bacterium is an emergent pathogen and the most common cause of nosocomial diarrhea. Although transmission of C. difficile is mediated by contamination of the gut by spores, the regulatory cascade controlling spore formation remains poorly characterized. During Bacillus subtilis sporulation, a cascade of four sigma factors, σ(F) and σ(G) in the forespore and σ(E) and σ(K) in the mother cell governs compartment-specific gene expression. In this work, we combined genome wide transcriptional analyses and promoter mapping to define the C. difficile σ(F), σ(E), σ(G) and σ(K) regulons. We identified about 225 genes under the control of these sigma factors: 25 in the σ(F) regulon, 97 σ(E)-dependent genes, 50 σ(G)-governed genes and 56 genes under σ(K) control. A significant fraction of genes in each regulon is of unknown function but new candidates for spore coat proteins could be proposed as being synthesized under σ(E) or σ(K) control and detected in a previously published spore proteome. SpoIIID of C. difficile also plays a pivotal role in the mother cell line of expression repressing the transcription of many members of the σ(E) regulon and activating sigK expression. Global analysis of developmental gene expression under the control of these sigma factors revealed deviations from the B. subtilis model regarding the communication between mother cell and forespore in C. difficile. We showed that the expression of the σ(E) regulon in the mother cell was not strictly under the control of σ(F) despite the fact that the forespore product SpoIIR was required for the processing of pro-σ(E). In addition, the σ(K) regulon was not controlled by σ(G) in C. difficile in agreement with the lack of pro-σ(K) processing. This work is one key step to obtain new insights about the diversity and evolution of the sporulation process among Firmicutes.

The spore differentiation pathway in the enteric pathogen Clostridium difficile.

Endosporulation is an ancient bacterial developmental program that culminates with the differentiation of a highly resistant endospore. In the model organism Bacillus subtilis, gene expression in the forespore and in the mother cell, the two cells that participate in endospore development, is governed by cell type-specific RNA polymerase sigma subunits. σ(F) in the forespore, and σ(E) in the mother cell control early stages of development and are replaced, at later stages, by σ(G) and σ(K), respectively. Starting with σ(F), the activation of the sigma factors is sequential, requires the preceding factor, and involves cell-cell signaling pathways that operate at key morphological stages. Here, we have studied the function and regulation of the sporulation sigma factors in the intestinal pathogen Clostridium difficile, an obligate anaerobe in which the endospores are central to the infectious cycle. The morphological characterization of mutants for the sporulation sigma factors, in parallel with use of a fluorescence reporter for single cell analysis of gene expression, unraveled important deviations from the B. subtilis paradigm. While the main periods of activity of the sigma factors are conserved, we show that the activity of σ(E) is partially independent of σ(F), that σ(G) activity is not dependent on σ(E), and that the activity of σ(K) does not require σ(G). We also show that σ(K) is not strictly required for heat resistant spore formation. In all, our results indicate reduced temporal segregation between the activities of the early and late sigma factors, and reduced requirement for the σ(F)-to-σ(E), σ(E)-to-σ(G), and σ(G)-to-σ(K) cell-cell signaling pathways. Nevertheless, our results support the view that the top level of the endosporulation network is conserved in evolution, with the sigma factors acting as the key regulators of the pathway, established some 2.5 billion years ago upon its emergence at the base of the Firmicutes Phylum.

Structural and Functional Characterization of an Ancient Bacterial Transglutaminase Sheds Light on the Minimal Requirements for Protein Cross-Linking.

Transglutaminases are best known for their ability to catalyze protein cross-linking reactions that impart chemical and physical resilience to cellular structures. Here, we report the crystal structure and characterization of Tgl, a transglutaminase from the bacterium Bacillus subtilis. Tgl is produced during sporulation and cross-links the surface of the highly resilient spore. Tgl-like proteins are found only in spore-forming bacteria of the Bacillus and Clostridia classes, indicating an ancient origin. Tgl is a single-domain protein, produced in active form, and the smallest transglutaminase characterized to date. We show that Tgl is structurally similar to bacterial cell wall endopeptidases and has an NlpC/P60 catalytic core, thought to represent the ancestral unit of the cysteine protease fold. We show that Tgl functions through a unique partially redundant catalytic dyad formed by Cys116 and Glu187 or Glu115. Strikingly, the catalytic Cys is insulated within a hydrophobic tunnel that traverses the molecule from side to side. The lack of similarity of Tgl to other transglutaminases together with its small size suggests that an NlpC/P60 catalytic core and insulation of the active site during catalysis may be essential requirements for protein cross-linking.

Sporulation during growth in a gut isolate of Bacillus subtilis.

Sporulation by Bacillus subtilis is a cell density-dependent response to nutrient deprivation. Central to the decision of entering sporulation is a phosphorelay, through which sensor kinases promote phosphorylation of Spo0A. The phosphorelay integrates both positive and negative signals, ensuring that sporulation, a time- and energy-consuming process that may bring an ecological cost, is only triggered should other adaptations fail. Here we report that a gastrointestinal isolate of B. subtilis sporulates with high efficiency during growth, bypassing the cell density, nutritional, and other signals that normally make sporulation a post-exponential-phase response. Sporulation during growth occurs because Spo0A is more active per cell and in a higher fraction of the population than in a laboratory strain. This in turn, is primarily caused by the absence from the gut strain of the genes rapE and rapK, coding for two aspartyl phosphatases that negatively modulate the flow of phosphoryl groups to Spo0A. We show, in line with recent results, that activation of Spo0A through the phosphorelay is the limiting step for sporulation initiation in the gut strain. Our results further suggest that the phosphorelay is tuned to favor sporulation during growth in gastrointestinal B. subtilis isolates, presumably as a form of survival and/or propagation in the gut environment.

A negative feedback loop that limits the ectopic activation of a cell type-specific sporulation sigma factor of Bacillus subtilis.

Two highly similar RNA polymerase sigma subunits, σ(F) and σ(G), govern the early and late phases of forespore-specific gene expression during spore differentiation in Bacillus subtilis. σ(F) drives synthesis of σ(G) but the latter only becomes active once engulfment of the forespore by the mother cell is completed, its levels rising quickly due to a positive feedback loop. The mechanisms that prevent premature or ectopic activation of σ(G) while discriminating between σ(F) and σ(G) in the forespore are not fully comprehended. Here, we report that the substitution of an asparagine by a glutamic acid at position 45 of σ(G) (N45E) strongly reduced binding by a previously characterized anti-sigma factor, CsfB (also known as Gin), in vitro, and increased the activity of σ(G) in vivo. The N45E mutation caused the appearance of a sub-population of pre-divisional cells with strong activity of σ(G). CsfB is normally produced in the forespore, under σ(F) control, but sigGN45E mutant cells also expressed csfB and did so in a σ(G)-dependent manner, autonomously from σ(F). Thus, a negative feedback loop involving CsfB counteracts the positive feedback loop resulting from ectopic σ(G) activity. N45 is invariant in the homologous position of σ(G) orthologues, whereas its functional equivalent in σ(F) proteins, E39, is highly conserved. While CsfB does not bind to wild-type σ(F), a E39N substitution in σ(F) resulted in efficient binding of CsfB to σ(F). Moreover, under certain conditions, the E39N alteration strongly restrains the activity of σ(F) in vivo, in a csfB-dependent manner, and the efficiency of sporulation. Therefore, a single amino residue, N45/E39, is sufficient for the ability of CsfB to discriminate between the two forespore-specific sigma factors in B. subtilis.