pSPObooster: A Plasmid System to Improve Sporulation Efficiency of Saccharomyces cerevisiae Lab Strains.

Common Saccharomyces cerevisiae lab yeast strains derived from S288C have meiotic defects and therefore are poor sporulators. Here, we developed a plasmid system containing corrected alleles of the MKT1 and RME1 genes to rescue the meiotic defects and show that standard BY4741 and BY4742 strains containing the plasmid display faster and more efficient sporulation. The plasmid, pSPObooster, can be maintained as an episome and easily cured or stably integrated into the genome at a single locus. We demonstrate the use of pSPObooster in low- and high-throughput yeast genetic manipulations and show that it can expedite both procedures without impacting strain behavior.

Cell shape and division septa positioning in filamentous Streptomyces require a functional cell wall glycopolymer ligase CglA.

The cell wall of monoderm bacteria consists of peptidoglycan and glycopolymers in roughly equal proportions and is crucial for cellular integrity, cell shape, and bacterial vitality. Despite the immense value of Streptomyces in biotechnology and medicine as antibiotic producers, we know very little about their cell wall biogenesis, composition, and functions. Here, we have identified the LCP-LytR_C domain protein CglA (Vnz_13690) as a key glycopolymer ligase, which specifically localizes in zones of cell wall biosynthesis in S. venezuelae. Reduced amount of glycopolymers in the cglA mutant results in enlarged vegetative hyphae and failures in FtsZ-rings formation and positioning. Consequently, division septa are misplaced leading to the formation of aberrant cell compartments, misshaped spores, and reduced cell vitality. In addition, we report our discovery that c-di-AMP signaling and decoration of the cell wall with glycopolymers are physiologically linked in Streptomyces since the deletion of cglA restores growth of the S. venezuelae disA mutant at high salt. Altogether, we have identified and characterized CglA as a novel component of cell wall biogenesis in Streptomyces, which is required for cell shape maintenance and cellular vitality in filamentous, multicellular bacteria.IMPORTANCEStreptomyces are our key producers of antibitiotics and other bioactive molecules and are, therefore, of high value for medicine and biotechnology. They proliferate by apical extension and branching of hyphae and undergo complex cell differentiation from filaments to spores during their life cycle. For both, growth and sporulation, coordinated cell wall biogenesis is crucial. However, our knowledge about cell wall biosynthesis, functions, and architecture in Streptomyces and in other Actinomycetota is still very limited. Here, we identify CglA as the key enzyme needed for the attachment of glycopolymers to the cell wall of S. venezuelae. We demonstrate that defects in the cell wall glycopolymer content result in loss of cell shape in these filamentous bacteria and show that division-competent FtsZ-rings cannot assemble properly and fail to be positioned correctly. As a consequence, cell septa placement is disturbed leading to the formation of misshaped spores with reduced viability.

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.

Functional Characterization of Ao4g24: An Uncharacterized Gene Involved in Conidiation, Trap Formation, Stress Response, and Secondary Metabolism in Arthrobotrys oligospora.

Arthrobotrys oligospora is a typical nematode-trapping (NT) fungus, which can secrete food cues to lure, capture, and digest nematodes by triggering the production of adhesive networks (traps). Based on genomic and proteomic analyses, multiple pathogenic genes and proteins involved in trap formation have been characterized; however, there are numerous uncharacterized genes that play important roles in trap formation. The functional studies of these unknown genes are helpful in systematically elucidating the complex interactions between A. oligospora and nematode hosts. In this study, we screened the gene AOL_s00004g24 (Ao4g24). This gene is similar to the SWI/SNF chromatin remodeling complex, which was found to play a potential role in trap formation in our previous transcriptome analysis. Here, we characterized the function of Ao4g24 by gene disruption, phenotypic analysis, and metabolomics. The deletion of Ao4g24 led to a remarkable decrease in conidia yield, trap formation, and secondary metabolites. Meanwhile, the absence of Ao4g24 influenced the mitochondrial membrane potential, ATP content, autophagy, ROS level, and stress response. These results indicate that Ao4g24 has crucial functions in sporulation, trap formation, and pathogenicity in NT fungi. Our study provides a reference for understanding the role of unidentified genes in mycelium growth and trap formation in NT fungi.

Engineering the cell wall reactive groups of Plant Growth Promoting Rhizobacteria by culture strategy for heavy metal removal.

This research delved into the effects of nutrient limitation on the level of sporulation and the cadmium adsorption capacity of the bacterium Bacillus sp. isolated from the rhizosphere of endemic soils in the Region of Valparaiso, Chile. The bacteria were subjected to nitrogen limitation in fed-batch mode and were compared to bacteria grown in batch culture without nutrient limitation. The cultures were carried out in a 3 L bioreactor with an external nitrogen supply of ammonium at a flow of 0.123 L h-1. The specific maximum growth rate was 0.42 h-1 in batch and 0.45 h-1 in the exponential phase of the fed-batch. The analysis of sporulation did not show any significant difference between the biomass coming from the fed-batch and batch cultures. It was found that maximum cadmium adsorption capacity varied with culture strategy. The dry biomass grown without nutrient limitation exhibited a maximum adsorption capacity for cadmium of 65.0 mgCd g-1biomass. Conversely, the limited biomass achieved a lower cadmium adsorption capacity of approximately 36.0 mgCd g-1biomass. FTIR analysis showed that nitrogen limitation induced changes in the composition of the outer cell wall, specifically an increase of deacetlylated polysaccharides, reducing the relative amount of secondary amines and proteins from the peptidoglycan matrix. Amino groups from acetylated polysaccharides and proteins have been associated elsewhere with greater cadmium affinity, which could explain the poor results obtained with the nitrogen-restricted biomass. This study shows that new physiological states displaying different adsorption capabilities were effectively obtained by engineering the cell coverage of the bacteria using varying culture strategies. The fed-batch culture proved to be a valuable tool for studying PGPR strains for biosorption and other applications. Exploring diverse nutrient limitations and other pollutants in this bacterium and other members of the PGPR family offer great opportunities to tailor biosorption strategies based on specific conditions, ultimately contributing to sustainable environmental solutions.

Role of glycogen metabolism in Clostridioides difficile virulence.

Glycogen plays a vital role as an energy reserve in various bacterial and fungal species. Clostridioides difficile possesses a glycogen metabolism operon that contains genes for both glycogen synthesis and utilization. In our investigation, we focused on understanding the significance of glycogen metabolism in the physiology and pathogenesis of C. difficile. To explore this, we engineered a C. difficile JIR8094 strain lacking glycogen synthesis capability by introducing a group II intron into the glgC gene, the operon's first component. Quantification of intracellular glycogen levels validated the impact of this modification. Interestingly, the mutant strain exhibited a 1.5-fold increase in toxin production compared with the parental strain, without significant changes in the sporulation rate. Our analysis also revealed that wild-type C. difficile spores contained glycogen, whereas spores from the mutant strain lacking stored glycogen showed increased sensitivity to physical and chemical treatments and had a shorter storage life. By suppressing glgP expression, the gene coding for glycogen-phosphorylase, via CRISPRi, we demonstrated that glycogen accumulation but not the utilization is needed for spore resilience in C. difficile. Transmission electron microscopy analysis revealed a significantly lower core/cortex ratio in glgC mutant strain spores. In hamster challenge experiments, both the parental and glgC mutant strains colonized hosts similarly; however, the mutant strain failed to induce infection relapse after antibiotic treatment cessation. These findings highlight the importance of glycogen metabolism in C. difficile spore resilience and suggest its role in disease relapse.IMPORTANCEThis study on the role of glycogen metabolism in Clostridioides difficile highlights its critical involvement in the pathogen's energy management, its pathogenicity, and its resilience. Our results also revealed that glycogen presence in spores is pivotal for their structural integrity and resistance to adverse conditions, which is essential for their longevity and infectivity. Importantly, the inability of the mutant strain to cause infection relapse in hamsters post-antibiotic treatment pinpoints a potential target for therapeutic interventions, highlighting the importance of glycogen in disease dynamics. This research thus significantly advances our understanding of C. difficile physiology and pathogenesis, offering new avenues for combating its persistence and recurrence.

Potential role of the methanolic extract for Olea europaea (stem and leaves) on sporulation of Eimeria papillata oocysts: In vitro study.

Many plants are efficient anticoccidial agents owing to their content of active chemicals. Drug-resistant Eimeria species have emerged as a result of excessive drug use. The current work aimed to investigate the oocysticidal activity (Eimeria papillata) of Olea europaea stem extract (OESE) and leaf extract (OELE) in vitro. The results of gas chromatography-mass spectrometry analysis for OELE and OESE showed the presence of 12 and 9 phytochemical compounds, respectively. Also, chemical examination revealed that the plant extracts are rich in phenols, flavonoids and tannins. Additionally, the best radical scavenging activity of OESE and OELE was at a concentration of 100 µg/ml, reaching 92.04 ± 0.02 and 92.4 ± 0.2%, respectively. The in vitro study revealed that concentrations of 200 mg/ml from OESE and OELE caused significant inhibition (100%) of process sporulation for E. papillata oocysts, in contrast to the other commercial products, which displayed varying degrees of suppression sporulation. Our findings showed that OESE and OELE have anticoccidial activity, which motivates further the conduction of in vivo studies in the search for a less expensive and more efficient cure.

Anti-Eimeria tenella activity of Ethanamizuril in vitro and in vivo.

The prevalence of chicken coccidiosis in the poultry industry is a significant concern, further exacerbated by the emergence of drug-resistant coccidia resulting from the indiscriminate use of medications. Ethanamizuril, a novel triazine anti-coccidial compound, has been used to combat drug resistance. Currently, it is known that Ethanamizuril acts on the second-generation merozoites and early gametogenesis stages of Eimeria. Limited information exists regarding its impact on the early merozoites and exogenous stage of Eimeria. In the present study, the anti-coccidial properties of Ethanamizuril were evaluated both in vitro and in vivo. The in vitro experiments demonstrated that Ethanamizuril effectively inhibits the sporulation of E. tenella oocysts in a dose-dependent manner and significantly reduces the sporozoite excystation rate. Furthermore, in vivo tests revealed that treatment with 10 mg/L Ethanamizuril in drinking water significantly decreased the copy number of first-generation and secondary-generation merozoites in the chicken cecum, indicating that it can inhibit the development of whole schizonts development. Moreover, treatment with Ethanamizuril demonstrated excellent protective efficacy with an anti-coccidial index (ACI) of 180, which was manifested through higher body weight gains, lighter cecal lesion, lower fecal oocyst shedding score and reduced liver index. Collectively, this study suggests that Ethanamizuril effectively treats E. tenella infection by inhibiting both endogenous and exogenous stages development.

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.

Alanine and glutamate catabolism collaborate to ensure the success of Bacillus subtilis sporulation.

Sporulation as a typical bacterial differentiation process has been studied for decades. However, two crucial aspects of sporulation, (i) the energy sources supporting the process, and (ii) the maintenance of spore dormancy throughout sporulation, are scarcely explored. Here, we reported the crucial role of RocG-mediated glutamate catabolism in regulating mother cell lysis, a critical step for sporulation completion of Bacillus subtilis, likely by providing energy metabolite ATP. Notably, rocG overexpression resulted in an excessive ATP accumulation in sporulating cells, leading to adverse effects on future spore properties, e.g. increased germination efficiency, reduced DPA content, and lowered heat resistance. Additionally, we revealed that Ald-mediated alanine metabolism was highly related to the inhibition of premature germination and the maintenance of spore dormancy during sporulation, which might be achieved by decreasing the typical germinant L-alanine concentration in sporulating environment. Our data inferred that sporulation of B. subtilis was a highly orchestrated biological process requiring a delicate balance in diverse metabolic pathways, hence ensuring both the completion of sporulation and production of high-quality spores.

The biology and pathogenicity of Clostridium perfringens type F: a common human enteropathogen with a new(ish) name.

SUMMARYIn the 2018-revised Clostridium perfringens typing classification system, isolates carrying the enterotoxin (cpe) and alpha toxin genes but no other typing toxin genes are now designated as type F. Type F isolates cause food poisoning and nonfoodborne human gastrointestinal (GI) diseases, which most commonly involve type F isolates carrying, respectivefooly, a chromosomal or plasmid-borne cpe gene. Compared to spores of other C. perfringens isolates, spores of type F chromosomal cpe isolates often exhibit greater resistance to food environment stresses, likely facilitating their survival in improperly prepared or stored foods. Multiple factors contribute to this spore resistance phenotype, including the production of a variant small acid-soluble protein-4. The pathogenicity of type F isolates involves sporulation-dependent C. perfringens enterotoxin (CPE) production. C. perfringens sporulation is initiated by orphan histidine kinases and sporulation-associated sigma factors that drive cpe transcription. CPE-induced cytotoxicity starts when CPE binds to claudin receptors to form a small complex (which also includes nonreceptor claudins). Approximately six small complexes oligomerize on the host cell plasma membrane surface to form a prepore. CPE molecules in that prepore apparently extend ß-hairpin loops to form a ß-barrel pore, allowing a Ca2+ influx that activates calpain. With low-dose CPE treatment, caspase-3-dependent apoptosis develops, while high-CPE dose treatment induces necroptosis. Those effects cause histologic damage along with fluid and electrolyte losses from the colon and small intestine. Sialidases likely contribute to type F disease by enhancing CPE action and, for NanI-producing nonfoodborne human GI disease isolates, increasing intestinal growth and colonization.

Response of a simulated aquatic fungal community to nanoplastics exposure and functional consequence on leaf decomposition.

Nanoplastics pose a potential threat to a wide variety of aquatic organisms. Despite the awareness of this existing hazard, the impact of nanoplastics on natural fungal communities remains a research gap. In this study, five dominant fungi species, isolated from a stream ecosystem, were used to explore the effects of different nano-polystyrene (nano-PS) particles concentrations on a simulated fungal community. Specifically, the evaluation was conducted regarding the fungal growth, reproductivity, structural composition, and ecological function in leaf litter decomposition. A 15-day exposure experiment showed that 100 µg/L nano-PS significantly reduced the microcosm pH. The extracellular enzyme activities of ß-glucosidase, leucine-aminopeptidase, and peroxidase were significantly promoted by nano-PS exposure for 5 days or 15 days. Total sporulation rate significantly decreased after the 15-day exposure to 1 and 100 µg/L nano-PS and significantly increased under 10 µg/L nano-PS. In contrast, nano-PS concentrations had no effects on fungal biomass. In addition, the reduced relative abundance of Geotrichum candidum lowered its contribution to leaf decomposition, resulting in a decreased litter decomposition rate of a 24.5-27.9 % after exposure. This suggests that 1-100 µg/L nano-PS inhibited leaf decomposition by inhibiting fungal reproduction and reducing the contribution of specific fungal species. In addition, the findings highlight the importance of exploring the potential mechanisms of the interaction between nanoplastics and fungal species.

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.

Fight or flee, a vital choice for Clostridioides difficile.

Clostridioides difficile is a leading cause of healthcare-associated infections, causing billions of economic losses every year. Its symptoms range from mild diarrhea to life-threatening damage to the colon. Transmission and recurrence of C. difficile infection (CDI) are mediated by the metabolically dormant spores, while the virulence of C. difficile is mainly due to the two large clostridial toxins, TcdA and TcdB. Producing toxins or forming spores are two different strategies for C. difficile to cope with harsh environmental conditions. It is of great significance to understand the molecular mechanisms for C. difficile to skew to either of the cellular processes. Here, we summarize the current understanding of the regulation and connections between toxin production and sporulation in C. difficile and further discuss the potential solutions for yet-to-be-answered questions.

Inactivation of guanylate kinase in Bacillus sp. TL7-3 cultivated under an optimized ratio of carbon and nitrogen sources influenced GTP regeneration capability and sporulation.

Bacillus sp. TL7-3 has potential as a dietary supplement to promote human and animal health. It produces spores that can survive in harsh environments. Thus, when supplemented with nutrients, these spores can withstand the acidic pH of the stomach and resume vegetative development in the gut when exposed to growth-promoting conditions. Spores are formed as a cellular defense mechanism when a culture experiences stress and process optimization to achieve high spore production in a typical batch process remains challenging. Existing literature on the manipulation of gene expression and enzyme activity during batch cultivation is limited. Studies on the growth patterns, morphological changes, and relevant gene expression have aided in enhancing spore production. The present study used the response surface methodology for medium optimization. The model suggested that yeast extract and NH4Cl were significant factors controlling spore production. A comparison between the high weight ratio of carbon and nitrogen (C:N) substrates (8.57:1) in the optimized and basal media (0.52:1) showed an 8.76-fold increase in the final spore concentration. The expression of major genes, including codY, spo0A, kinA, and spo0F, involved in the sporulation was compared when cultivating Bacillus sp. TL7-3 in media with varying C:N ratios. At high C:N ratios, spo0A, kinA, and spo0F were upregulated, whereas codY was downregulated. This led to decreased guanylate kinase activity, resulting in a low guanosine triphosphate concentration and inactivation of CodY, thereby reducing the repression of spo0A and CodY-repressed genes and stimulating sporulation.

Current and Ongoing Developments in Targeting Clostridioides difficile Infection and Recurrence.

Clostridioides difficile is a Gram-positive, spore-forming anaerobic bacterial pathogen that causes severe gastrointestinal infection in humans. This review provides background information on C. difficile infection and the pathogenesis and toxigenicity of C. difficile. The risk factors, causes, and the problem of recurrence of disease and current therapeutic treatments are also discussed. Recent therapeutic developments are reviewed including small molecules that inhibit toxin formation, disrupt the cell membrane, inhibit the sporulation process, and activate the host immune system in cells. Other treatments discussed include faecal microbiota treatment, antibody-based immunotherapies, probiotics, vaccines, and violet-blue light disinfection.

The Effect of Caco-2 Cells on Sporulation and Enterotoxin Expression by Foodborne Clostridium perfringens.

Clostridium perfringens enterotoxin (Cpe)-producing strains cause gastrointestinal infections in humans and account for the second-largest number of all foodborne outbreaks caused by bacterial toxins. The Cpe toxin is only produced during sporulation; this process might be affected when C. perfringens comes into contact with host cells. The current study determined how the cpe expression levels and spore formation changed over time during co-culture with Caco-2 cells (as a model of intestinal epithelial cells). In co-culture with Caco-2 cells, total C. perfringens cell counts first decreased and then remained more or less stable, whereas spore counts were stable over the whole incubation period. The cpe mRNA level in the co-culture with Caco-2 cells increased more rapidly than in the absence of Caco-2 cells (3.9-fold higher levels in coculture than in the absence of Caco-2 cells after 8 h of incubation). Finally, we found that cpe expression is inhibited by a cue released by Caco-2 cells (8.3-fold lower levels in the presence of supernatants of Caco-2 cells than in the absence of the supernatants after 10 h of incubation); as a consequence, the increased expression in co-culture with Caco-2 cells must be caused by a factor associated with the Caco-2 cells.

Membrane and organelle rearrangement during ascospore formation in budding yeast.

SUMMARYIn ascomycete fungi, sexual spores, termed ascospores, are formed after meiosis. Ascospore formation is an unusual cell division in which daughter cells are created within the cytoplasm of the mother cell by de novo generation of membranes that encapsulate each of the haploid chromosome sets created by meiosis. This review describes the molecular events underlying the creation, expansion, and closure of these membranes in the budding yeast, Saccharomyces cerevisiae. Recent advances in our understanding of the regulation of gene expression and the dynamic behavior of different membrane-bound organelles during this process are detailed. While less is known about ascospore formation in other systems, comparison to the distantly related fission yeast suggests that the molecular events will be broadly similar throughout the ascomycetes.

Endopeptidase activities of Clostridium botulinum toxins in the development of this bacterium.

By-products like CO2 and organic acids, produced during Clostridium botulinum growth, appear to inhibit its development and reduce ATP production. A decrease in ATP production creates an imbalance in the ATP/GTP ratio. GTP activates CodY, which regulates BoNT expression. This toxin is released into the extracellular medium. Its light chains act as a specific endopeptidase, targeting SNARE proteins. The specific amino acids released enter the cells and are metabolized by the Stickland reaction, resulting in the synthesis of ATP. This ATP might then be used by histidine kinases to activate Spo0A, the main regulator initiating sporulation, through phosphorylation.

High variation of virulence in Aphanomyces astaci strains lacks association with pathogenic traits and mtDNA haplogroups.

Introduced into Europe from North America 150 years ago alongside its native crayfish hosts, the invasive pathogen Aphanomyces astaci is considered one of the main causes of European crayfish population decline. For the past two centuries, this oomycete pathogen has been extensively studied, with the more recent efforts focused on containing and monitoring its spread across the continent. However, after the recent introduction of new strains, the newly-discovered diversity of A. astaci in North America and several years of coevolution with its European host, a new assessment of the traits linked to the pathogen's virulence is much needed. To fill this gap, we investigated the presence of phenotypic patterns (i.e., in vitro growth and sporulation rates) possibly associated with the pathogen's virulence (i.e., induced mortality in crayfish) in a collection of 14 A. astaci strains isolated both in North America and in Europe. The results highlighted a high variability in virulence, growth rate and motile spore production among the different strains, while the total-sporulation rate was more similar across strains. Surprisingly, growth and sporulation rates were not significantly correlated with virulence. Furthermore, none of the analysed parameters, including virulence, was significantly different among the major A. astaci haplogroups. These results indicate that each strain is defined by a characteristic combination of pathogenic features, specifically assembled for the environment and host faced by each strain. Thus, canonical mitochondrial markers, often used to infer the pathogen's virulence, are not accurate tools to deduce the phenotype of A. astaci strains. As the diversity of A. astaci strains in Europe is bound to increase due to translocations of new carrier crayfish species from North America, there is an urgent need to deepen our understanding of A. astaci's virulence variability and its ability to adapt to new hosts and environments.