Effects of Na+ adaptation on Bacillus cereus endospores inactivation and transcriptome changes.

As Bacillus cereus endospores exist in various vegetables grown in soil, the possibility of contamination in food products with high salt concentrations cannot be ignored. Recent studies revealed that harsh conditions affect the resistance of bacteria; thus, we investigated the developmental aspect of heat resistance of B. cereus after sporulation with high NaCl concentration. RNA sequencing was conducted for transcriptomic changes when B. cereus endospores formed at high salinity, and membrane fluidity and hydrophobicity were measured to verify the transcriptomic analysis. Our data showed that increasing NaCl concentration in sporulation media led to a decrease in heat resistance. Also, endospore hydrophobicity, membrane fluidity, and endospore density decreased with sporulation at higher NaCl concentrations. When the transcript changes of B. cereus sporulated at NaCl concentrations of 0.5 and 7% were analyzed by transcriptome analysis, it was confirmed that the NaCl 7% endospores had significantly lower expression levels (FDR<0.05) of genes related to sporulation stages 3 and 4, which led to a decrease in expression of spore-related genes such as coat proteins and small acid-soluble proteins. Our findings indicated that high NaCl concentrations inhibited sporulation stages 3 and 4, thereby preventing proper cell maturation in the forespores and adequate formation of the coat protein and cortex. This inhibition led to decreased endospore density and hydrophobicity, ultimately resulting in reduced heat resistance.resistanceWe expect that this study will be utilized as a baseline for further studies and enhance sterilization strategies.

EsxA, a type VII secretion system-dependent effector, reveals a novel function in the sporulation of Bacillus cereus ATCC14579.

BACKGROUND: Bacillus cereus is a Gram-positive, spore-forming bacterium that produces a spectrum of effectors integral to bacterial niche adaptation and the development of various infections. Among those is EsxA, whose secretion depends on the EssC component of the type VII secretion system (T7SS). EsxA's roles within the bacterial cell are poorly understood, although postulations indicate that it may be involved in sporulation. However, the T7SS repertoire in B. cereus has not been reported, and its functions are unestablished. METHODS: We used the type strain, B. cereus ATCC14579, to generate ΔessC mutant through homologous recombination using the homing endonuclease I-SceI mediated markerless gene replacement. Comparatively, we analyzed the culture supernatant of type strain and the ΔessC mutant through Liquid chromatography-tandem mass spectrometry (LC-MS/MS). We further generated T7SSb-specific gene mutations to explore the housekeeping roles of the T7SSb-dependent effectors. The sporulation process of B. cereus ATCC14579 and its mutants was observed microscopically through the classic Schaeffer-Fulton staining method. The spore viability of each strain in this study was established by enumerating the colony-forming units on LB agar. RESULTS: Through LC-MS/MS, we identified a pair of nearly identical (94%) effector proteins named EsxA belonging to the sagEsxA-like subfamily of the WXG100 protein superfamily in the culture supernatant of the wild type and none in the ΔessC mutant. Homology analysis of the T7SSb gene cluster among B. cereus strains revealed diversity from the 3' end of essC, encoding additional substrates. Deletions in esxA1 and esxA2 neither altered cellular morphology nor growth rate, but the ΔesxA1ΔesxA2 deletion resulted in significantly fewer viable spores and an overall slower sporulation process. Within 24 h culture, more than 80% of wild-type cells formed endospores compared to less than 5% in the ΔesxA1ΔesxA2 mutant. The maximum spore ratios for the wild type and ΔesxA1ΔesxA2 were 0.96 and 0.72, respectively. Altogether, these results indicated that EsxA1 and EsxA2 work cooperatively and are required for sporulation in B. cereus ATCC14567. CONCLUSION: B. cereus ATCC14579 possesses two nearly identical T7SSb-dependent effectors belonging to the sagEsxA-like proteins. Simultaneous deletion of genes encoding these effectors significantly delayed and reduced sporulation, a novel finding for EsxA.

Bacillus altitudinis 1.4 genome analysis-functional annotation of probiotic properties and immunomodulatory activity.

Probiotics are live microorganisms that, when administered in adequate quantities, provide health benefits to the host. In this study, phenotypic and genotypic methods were used to evaluate the probiotic properties of Bacillus altitudinis 1.4. The isolate was sensitive to all antimicrobials tested and presented a positive result in the hemolysis test. B. altitudinis 1.4 spores were more resistant than vegetative cells, when evaluated in simulation of cell viability in the gastrointestinal tract, as well as adhesion to the intestinal mucosa. The isolate was capable of self-aggregation and coaggregation with pathogens such as Escherichia coli ATCC 25922 and Salmonella Enteritidis ATCC 13076. Genomic analysis revealed the presence of genes with probiotic characteristics. From this study it was possible to evaluate the gene expression of pro-inflammatory and anti-inflammatory cytokines for different treatments. Viable vegetative cells of B. altitudinis 1.4 increased the transcription of pro-inflammatory factors, in addition to also increasing the transcription of IL-10, indicating a tendency to stimulate a pro-inflammatory profile. Given the results presented, B. altitudinis 1.4 showed potential to be applied in the incorporation of this microorganism into animal feed, since the spores could tolerate the feed handling and pelletization processes.

Identification of pathways to high-level vancomycin resistance in Clostridioides difficile that incur high fitness costs in key pathogenicity traits.

Clostridioides difficile is an important human pathogen, for which there are very limited treatment options, primarily the glycopeptide antibiotic vancomycin. In recent years, vancomycin resistance has emerged as a serious problem in several gram-positive pathogens, but high-level resistance has yet to be reported for C. difficile, although it is not known if this is due to constraints upon resistance evolution in this species. Here, we show that resistance to vancomycin can evolve rapidly under ramping selection but is accompanied by fitness costs and pleiotropic trade-offs, including sporulation defects that would be expected to severely impact transmission. We identified 2 distinct pathways to resistance, both of which are predicted to result in changes to the muropeptide terminal D-Ala-D-Ala that is the primary target of vancomycin. One of these pathways involves a previously uncharacterised D,D-carboxypeptidase, expression of which is controlled by a dedicated two-component signal transduction system. Our findings suggest that while C. difficile is capable of evolving high-level vancomycin resistance, this outcome may be limited clinically due to pleiotropic effects on key pathogenicity traits. Moreover, our data identify potential mutational routes to resistance that should be considered in genomic surveillance.

Translation in Bacillus subtilis is spatially and temporally coordinated during sporulation.

The transcriptional control of sporulation in Bacillus subtilis is reasonably well understood, but its translational control is underexplored. Here, we use RNA-seq, ribosome profiling and fluorescence microscopy to study the translational dynamics of B. subtilis sporulation. We identify two events of translation silencing and describe spatiotemporal changes in subcellular localization of ribosomes during sporulation. We investigate the potential regulatory role of ribosomes during sporulation using a strain lacking zinc-independent paralogs of three zinc-dependent ribosomal proteins (L31, L33 and S14). The mutant strain exhibits delayed sporulation, reduced germination efficiency, dysregulated translation of metabolic and sporulation-related genes, and disruptions in translation silencing, particularly in late sporulation.

Clostridium septicum manifests a bile salt germinant response mediated by Clostridioides difficile csp gene orthologs.

Clostridium septicum infections are highly predictive of certain malignancies in human patients. To initiate infections, C. septicum spores must first germinate and regain vegetative growth. Yet, what triggers the germination of C. septicum spores is still unknown. Here, we observe that C. septicum germinates in response to specific bile salts. Putative bile salt recognition genes are identified in C. septicum based on their similarity in sequence and organization to bile salt-responsive csp genes in Clostridioides difficile. Inactivating two of these csp orthologs (cspC-82 and cspC-1718) results in mutant spores that no longer germinate in the presence of their respective cognate bile salts. Additionally, inactivating the putative cspBA or sleC genes in C. septicum abrogates the germination response to all bile salt germinants, suggesting that both act at a convergent point downstream of cspC-82 and cspC-1718. Molecular dynamics simulations show that both CspC-82 and CspC-1718 bear a strong structural congruence with C. difficile's CspC. The existence of functional bile salt germination sensors in C. septicum may be relevant to the association between infection and malignancy.

The small acid-soluble proteins of Clostridioides difficile regulate sporulation in a SpoIVB2-dependent manner.

Clostridioides difficile is a pathogen whose transmission relies on the formation of dormant endospores. Spores are highly resilient forms of bacteria that resist environmental and chemical insults. In recent work, we found that C. difficile SspA and SspB, two small acid-soluble proteins (SASPs), protect spores from UV damage and, interestingly, are necessary for the formation of mature spores. Here, we build upon this finding and show that C. difficile sspA and sspB are required for the formation of the spore cortex layer. Moreover, using an EMS mutagenesis selection strategy, we identified mutations that suppressed the defect in sporulation of C. difficile SASP mutants. Many of these strains contained mutations in CDR20291_0714 (spoIVB2) revealing a connection between the SpoIVB2 protease and the SASPs in the sporulation pathway. This work builds upon the hypothesis that the small acid-soluble proteins can regulate gene expression.

The megaplasmid pCER270 of Bacillus cereus emetic strain affects the timing of the sporulation process, spore resistance properties, and germination.

The Bacillus cereus group includes closely related spore-forming Gram-positive bacteria. In this group, plasmids play a crucial role in species differentiation and are essential for pathogenesis and adaptation to ecological niches. The B. cereus emetic strains are characterized by the presence of the pCER270 megaplasmid, which encodes the non-ribosomal peptide synthetase for the production of cereulide, the emetic toxin. This plasmid carries several genes that may be involved in the sporulation process. Furthermore, a transcriptomic analysis has revealed that pCER270 influences the expression of chromosome genes, particularly under sporulation conditions. In this study, we investigated the role of pCER270 on spore properties in different species of the B. cereus group. We showed that pCER270 plays a role in spore wet heat resistance and germination, with varying degrees of impact depending on the genetic background. In addition, pCER270 ensures that sporulation occurs at the appropriate time by delaying the expression of sporulation genes. This regulation of sporulation timing is controlled by the pCER270-borne Rap-Phr system, which likely regulates the phosphorylation state of Spo0A. Acquisition of the pCER270 plasmid by new strains could give them an advantage in adapting to new environments and lead to the emergence of new pathogenic strains. IMPORTANCE: The acquisition of new mobile genetic elements, such as plasmids, is essential for the pathogenesis and adaptation of bacteria belonging to the Bacillus cereus group. This can confer new phenotypic traits and beneficial functions that enable bacteria to adapt to changing environments and colonize new ecological niches. Emetic B. cereus strains cause food poisoning linked to the production of cereulide, the emetic toxin whose synthesis is due to the presence of plasmid pCER270. In the environment, cereulide provides a competitive advantage in producing bacteria against various competitors or predators. This study demonstrates that pCER270 also regulates the sporulation process, resulting in spores with improved heat resistance and germination capacity. The transfer of plasmid pCER270 among different strains of the B. cereus group may enhance their adaptation to new environments. This raises the question of the emergence of new pathogenic strains, which could pose a serious threat to human health.

Is there a role for intestinal sporobiota in the antimicrobial resistance crisis?

Antimicrobial resistance (AMR) is a complex issue requiring specific, multi-sectoral measures to slow its spread. When people are exposed to antimicrobial agents, it can cause resistant bacteria to increase. This means that the use, misuse, and excessive use of antimicrobial agents exert selective pressure on bacteria, which can lead to the development of "silent" reservoirs of antimicrobial resistance genes. These genes can later be mobilized into pathogenic bacteria and contribute to the spread of AMR. Many socioeconomic and environmental factors influence the transmission and dissemination of resistance genes, such as the quality of healthcare systems, water sanitation, hygiene infrastructure, and pollution. The sporobiota is an essential part of the gut microbiota that plays a role in maintaining gut homeostasis. However, because spores are highly transmissible and can spread easily, they can be a vector for AMR. The sporobiota resistome, particularly the mobile resistome, is important for tracking, managing, and limiting the spread of antimicrobial resistance genes among pathogenic and commensal bacterial species.

RelQ-mediated alarmone signalling regulates growth, stress-induced biofilm formation and spore accumulation in Clostridioides difficile.

The bacterial stringent response (SR) is a conserved transcriptional reprogramming pathway mediated by the nucleotide signalling alarmones, (pp)pGpp. The SR has been implicated in antibiotic survival in Clostridioides difficile, a biofilm- and spore-forming pathogen that causes resilient, highly recurrent C. difficile infections. The role of the SR in other processes and the effectors by which it regulates C. difficile physiology are unknown. C. difficile RelQ is a clostridial alarmone synthetase. Deletion of relQ dysregulates C. difficile growth in unstressed conditions, affects susceptibility to antibiotic and oxidative stressors and drastically reduces biofilm formation. While wild-type C. difficile displays increased biofilm formation in the presence of sublethal stress, the ΔrelQ strain cannot upregulate biofilm production in response to stress. Deletion of relQ slows spore accumulation in planktonic cultures but accelerates it in biofilms. This work establishes biofilm formation and spore accumulation as alarmone-mediated processes in C. difficile and reveals the importance of RelQ in stress-induced biofilm regulation.

Potential risk of biofilm-forming Bacillus cereus group in fresh-cut lettuce production chain.

Bacillus cereus and Bacillus thuringiensis, which belong to the B. cereus group, are widely distributed in nature and can cause food poisoning symptoms. In this study, we collected 131 isolates belonging to the B. cereus group, comprising 124B. cereus and seven B. thuringiensis isolates, from fresh-cut lettuce production chain and investigated their potential risk by analyzing genotypic (enterotoxin and emetic toxin gene profiles) and phenotypic (antibiotic susceptibility, sporulation, and biofilm formation) characteristics. Enterotoxin genes were present only in B. cereus, whereas the emetic toxin gene was not detected in any of the B. cereus isolates. All isolates were susceptible to vancomycin, which is a last resort for treating B. cereus group infection symptoms, but generally resistant to ß-lactam antimicrobials, and had the ability to form spores (at an average sporulation rate of 24.6 %) and biofilms at 30 °C. Isolates that formed strong biofilms at 30 °C had a superior possibility of forming a dense biofilm by proliferating at 10 °C compared to other isolates. Additionally, confocal laser scanning microscopy (CLSM) images revealed a notable presence of spores within the submerged biofilm formed at 10 °C, and the strengthened attachment of biofilm inner cells to the substrate was further revealed through biofilm structure parameters analysis. Collectively, our study revealed the prevalence and contamination levels of B. cereus and B. thuringiensis at fresh-cut lettuce production chain and investigated their genotypic and phenotypic characteristics, aiming to provide valuable insights for the development of potential risk management strategies to ensure food safety, especially along the cold chain.

Spore-forming bacteria in gelatin: Characterization, identification by 16S rRNA and MALDI-TOF mass spectrometry (MS), and presence of heat resistance and virulence genes.

Gelatin, a versatile protein derived from collagen, is widely used in the food, pharmaceutical and medical sectors. However, bacterial contamination by spore-forming bacteria during gelatin processing represents a significant concern for product safety and quality. In this study, an investigation was carried out to explore the heat and chemical resistance, as well as the identification and characterization of spore-forming bacteria isolated from gelatin processing. The methodologies involved chemical resistance tests with drastic pH in microplates and thermal resistance tests in capillary tubes of various isolates obtained at different processing stages. In addition, phenotypic and genotypic analyses were carried out to characterize the most resistant isolates of spore-forming bacteria. The findings of this study revealed the presence of several species, including Bacillus cereus, Bacillus licheniformis, Bacillus sonorensis, Bacillus subtilis, Geobacillus stearothermophilus, and Clostridium sporogenes, with some isolates exhibiting remarkable chemical and heat resistances. In addition, a significant proportion of the most resistant isolates showed gelatinase activity (n = 19/21; 90.5 %) and the presence of heat resistance (n = 5/21; 23.8 %), and virulence genes (n = 11/21; 52.4 %). The results of this study suggest that interventions should be done in quality control practices and that process parameter adjustments and effective contamination reduction strategies should be implemented through gelatin processing.

How c-di-GMP controls progression through the Streptomyces life cycle.

Members of the antibiotic-producing bacterial genus Streptomyces undergo a complex developmental life cycle that culminates in the production of spores. Central to control of this cell differentiation process is signaling through the second messenger 3', 5'-cyclic diguanylic acid (c-di-GMP). So far, three proteins that are directly controlled by c-di-GMP in Streptomyces have been functionally and structurally characterized: the key developmental regulators BldD and σWhiG, and the glycogen-degrading enzyme GlgX. c-di-GMP signals through BldD and σWhiG, respectively, to control the two most dramatic transitions of the Streptomyces life cycle, the formation of the reproductive aerial hyphae and their differentiation into spore chains. Later in development, c-di-GMP activates GlgX-mediated degradation of glycogen, releasing stored carbon for spore maturation.

Surface display of the COE antigen of porcine epidemic diarrhoea virus on Bacillus subtilis spores.

Porcine epidemic diarrhoea virus (PEDV) infects pigs of all ages by invading small intestine, causing acute diarrhoea, vomiting, and dehydration with high morbidity and mortality among newborn piglets. However, current PEDV vaccines are not effective to protect the pigs from field epidemic strains because of poor mucosal immune response and strain variation. Therefore, it is indispensable to develop a novel oral vaccine based on epidemic strains. Bacillus subtilis spores are attractive delivery vehicles for oral vaccination on account of the safety, high stability, and low cost. In this study, a chimeric gene CotC-Linker-COE (CLE), comprising of the B. subtilis spore coat gene cotC fused to the core neutralizing epitope CO-26 K equivalent (COE) of the epidemic strain PEDV-AJ1102 spike protein gene, was constructed. Then recombinant B. subtilis displaying the CLE on the spore surface was developed by homologous recombination. Mice were immunized by oral route with B. subtilis 168-CLE, B. subtilis 168, or phosphate-buffered saline (PBS) as control. Results showed that the IgG antibodies and cytokine (IL-4, IFN-γ) levels in the B. subtilis 168-CLE group were significantly higher than the control groups. This study demonstrates that B. subtilis 168-CLE can generate specific systemic immune and mucosal immune responses and is a potential vaccine candidate against PEDV infection.

Polyphosphate Plays a Significant Role in the Maturation of Spores in Myxococcus xanthus.

Myxococcus xanthus synthesizes polyphosphates (polyPs) with polyphosphate kinase 1 (Ppk1) and degrades short- and long-chain polyPs with the exopolyphosphatases, Ppx1 and Ppx2, respectively. M. xanthus polyP:AMP phosphotransferase (Pap) generates ADP from AMP and polyPs. Pap expression is induced by an elevation in intracellular polyP concentration. M. xanthus synthesized polyPs during the stationary phase; the ppk1 mutant died earlier than the wild-type strain after the stationary phase. In addition, M. xanthus cells cultured in phosphate-starved medium, H2O2-supplemented medium, or amino acid-deficient medium increased the intracellular polyP levels by six- to ninefold after 6 h of incubation. However, the growth of ppk1 and ppx2 mutants in phosphate-starved medium and H2O2-supplemented medium was not significantly different from that of wild-type strain, nor was there a significant difference in fruiting body formation and sporulation in starvation condition. During development, no difference was observed in the adenylate energy charge (AEC) values in the wild-type, ppk1 mutant, and pap mutant strains until the second day of development. However, after day 3, the ppk1 and pap mutants had a lower ADP ratio and a higher AMP ratio compared to wild-type strain, and as a result, the AEC values of these mutants were lower than those of the wild-type strain. Spores of ppk1 and pap mutants in the nutrient medium germinated later than those of the wild-type strain. These results suggested that polyPs produced during development may play an important role in cellular energy homeostasis of the spores by being used to convert AMP to ADP via Pap.

Spores of Clostridioides difficile are toxin delivery vehicles.

Clostridioides difficile causes a wide range of intestinal diseases through the action of two main cytotoxins, TcdA and TcdB. Ingested spores germinate in the intestine establishing a population of cells that produce toxins and spores. The pathogenicity locus, PaLoc, comprises several genes, including those coding for TcdA/B, for the holin-like TcdE protein, and for TcdR, an auto-regulatory RNA polymerase sigma factor essential for tcdA/B and tcdE expression. Here we show that tcdR, tcdA, tcdB and tcdE are expressed in a fraction of the sporulating cells, in either the whole sporangium or in the forespore. The whole sporangium pattern is due to protracted expression initiated in vegetative cells by σD, which primes the TcdR auto-regulatory loop. In contrast, the forespore-specific regulatory proteins σG and SpoVT control TcdR production and tcdA/tcdB and tcdE expression in this cell. We detected TcdA at the spore surface, and we show that wild type and ΔtcdA or ΔtcdB spores but not ΔtcdR or ΔtcdA/ΔtcdB spores are cytopathic against HT29 and Vero cells, indicating that spores may serve as toxin-delivery vehicles. Since the addition of TcdA and TcdB enhance binding of spores to epithelial cells, this effect may occur independently of toxin production by vegetative cells.

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.

Rapid evolutionary tuning of endospore quantity versus quality trade-off via a phase-variable contingency locus.

The UV resistance of bacterial endospores is an important quality supporting their survival in inhospitable environments and therefore constitutes an essential driver of the ecological success of spore-forming bacteria. Nevertheless, the variability and evolvability of this trait are poorly understood. In this study, directed evolution and genetics approaches revealed that the Bacillus cereus pdaA gene (encoding the endospore-specific peptidoglycan-N-acetylmuramic acid deacetylase) serves as a contingency locus in which the expansion and contraction of short tandem repeats can readily compromise (PdaAOFF) or restore (PdaAON) the pdaA open reading frame. Compared with B. cereus populations in the PdaAON state, populations in the PdaAOFF state produced a lower yield of viable endospores but endowed them with vastly increased UV resistance. Moreover, selection pressures based on either quantity (i.e., yield of viable endospores) or quality (i.e., UV resistance of viable endospores) aspects could readily shift populations between PdaAON and PdaAOFF states, respectively. Bioinformatic analysis also revealed that pdaA homologs within the Bacillus and Clostridium genera are often equipped with several short tandem repeat regions, suggesting a wider implementation of the pdaA-mediated phase variability in other sporeformers as well. These results for the first time reveal (1) pdaA as a phase-variable contingency locus in the adaptive evolution of endospore properties and (2) bet-hedging between what appears to be a quantity versus quality trade-off in endospore crops.

Cyclic diguanylate differentially regulates the expression of virulence factors and pathogenesis-related phenotypes in Clostridioides difficile.

Clostridioides difficile infection (CDI) caused by toxigenic C. difficile is the leading cause of antimicrobial and healthcare-associated diarrhea. The pathogenicity of C. difficile relies on the synergistic effect of multiple virulence factors, including spores, flagella, type IV pili (T4P), toxins, and biofilm. Spores enable survival and transmission of C. difficile, while adhesion factors such as flagella and T4P allow C. difficile to colonize and persist in the host intestine. Subsequently, C. difficile produces the toxins TcdA and TcdB, causing pseudomembranous colitis and other C. difficile-associated diseases; adhesion factors bind to the extracellular matrix to form biofilm, allowing C. difficile to evade drug and immune system attack and cause recurrent infection. Cyclic diguanylate (c-di-GMP) is a near-ubiquitous second messenger that extensively regulates morphology, the expression of virulence factors, and multiple physiological processes in C. difficile. In this review, we summarize current knowledge of how c-di-GMP differentially regulates the expression of virulence factors and pathogenesis-related phenotypes in C. difficile. We highlight that C. difficile spore formation and expression of toxin and flagella genes are inhibited at high intracellular levels of c-di-GMP, while T4P biosynthesis, cell aggregation, and biofilm formation are induced. Recent studies have enhanced our understanding of the c-di-GMP signaling networks in C. difficile and provided insights for the development of c-di-GMP-dependent strategies against CDI.

SpoIIQ-dependent localization of SpoIIE contributes to septal stability and compartmentalization during the engulfment stage of Bacillus subtilis sporulation.

During spore development in bacteria, a polar septum separates two transcriptionally distinct cellular compartments, the mother cell and the forespore. The conserved serine phosphatase SpoIIE is known for its critical role in the formation of this septum and activation of compartment-specific transcription in the forespore. Signaling between the mother cell and forespore then leads to activation of mother cell transcription and a phagocytic-like process called engulfment, which involves dramatic remodeling of the septum and requires a balance between peptidoglycan synthesis and hydrolysis to ensure septal stability and compartmentalization. Using Bacillus subtilis, we identify an additional role for SpoIIE in maintaining septal stability and compartmentalization at the onset of engulfment. This role for SpoIIE is mediated by SpoIIQ, which anchors SpoIIE in the engulfing membrane. A SpoIIQ mutant (SpoIIQ Y28A) that fails to anchor SpoIIE, results in septal instability and miscompartmentalization during septal peptidoglycan hydrolysis, when other septal stabilization factors are absent. Our data support a model whereby SpoIIE and its interactions with the peptidoglycan synthetic machinery contribute to the stabilization of the asymmetric septum early in engulfment, thereby ensuring compartmentalization during spore development.IMPORTANCEBacterial sporulation is a complex process involving a vast array of proteins. Some of these proteins are absolutely critical and regulate key points in the developmental process. Once such protein is SpoIIE, known for its role in the formation of the polar septum, a hallmark of the early stages of sporulation, and activation of the first sporulation-specific sigma factor, σF, in the developing spore. Interestingly, SpoIIE has been shown to interact with SpoIIQ, an important σF-regulated protein that functions during the engulfment stage. However, the significance of this interaction has remained unclear. Here, we unveil the importance of the SpoIIQ-SpoIIE interaction and identify a role for SpoIIE in the stabilization of the polar septum and maintenance of compartmentalization at the onset of engulfment. In this way, we demonstrate that key sporulation proteins, like SpoIIQ and SpoIIE, function in multiple processes during spore development.