Dormant bacterial spores encrypt a long-lasting transcriptional program to be executed during revival.

Sporulating bacteria can retreat into long-lasting dormant spores that preserve the capacity to germinate when propitious. However, how the revival transcriptional program is memorized for years remains elusive. We revealed that in dormant spores, core RNA polymerase (RNAP) resides in a central chromosomal domain, where it remains bound to a subset of intergenic promoter regions. These regions regulate genes encoding for most essential cellular functions, such as rRNAs and tRNAs. Upon awakening, RNAP recruits key transcriptional components, including sigma factor, and progresses to express the adjacent downstream genes. Mutants devoid of spore DNA-compacting proteins exhibit scattered RNAP localization and subsequently disordered firing of gene expression during germination. Accordingly, we propose that the spore chromosome is structured to preserve the transcriptional program by halting RNAP, prepared to execute transcription at the auspicious time. Such a mechanism may sustain long-term transcriptional programs in diverse organisms displaying a quiescent life form.

On the Mechanistic Basis of Killer Meiotic Drive in Fungi.

Spore killers are specific genetic elements in fungi that kill sexual spores that do not contain them. A range of studies in the last few years have provided the long-awaited first insights into the molecular mechanistic aspects of spore killing in different fungal models, including both yeast-forming and filamentous Ascomycota. Here we describe these recent advances, focusing on the wtf system in the fission yeast Schizosaccharomyces pombe; the Sk spore killers of Neurospora species; and two spore-killer systems in Podospora anserina, Spok and [Het-s]. The spore killers appear thus far mechanistically unrelated. They can involve large genomic rearrangements but most often rely on the action of just a single gene. Data gathered so far show that the protein domains involved in the killing and resistance processes differ among the systems and are not homologous. The emerging picture sketched by these studies is thus one of great mechanistic and evolutionary diversity of elements that cheat during meiosis and are thereby preferentially inherited over sexual generations.

The Spo13/Meikin pathway confines the onset of gamete differentiation to meiosis II in yeast.

Sexual reproduction requires genome haploidization by the two divisions of meiosis and a differentiation program to generate gametes. Here, we have investigated how sporulation, the yeast equivalent of gamete differentiation, is coordinated with progression through meiosis. Spore differentiation is initiated at metaphase II when a membrane-nucleating structure, called the meiotic plaque, is assembled at the centrosome. While all components of this structure accumulate already at entry into meiosis I, they cannot assemble because centrosomes are occupied by Spc72, the receptor of the γ-tubulin complex. Spc72 is removed from centrosomes by a pathway that depends on the polo-like kinase Cdc5 and the meiosis-specific kinase Ime2, which is unleashed by the degradation of Spo13/Meikin upon activation of the anaphase-promoting complex at anaphase I. Meiotic plaques are finally assembled upon reactivation of Cdk1 at entry into metaphase II. This unblocking-activation mechanism ensures that only single-copy genomes are packaged into spores and might serve as a paradigm for the regulation of other meiosis II-specific processes.

A dual-action antibiotic that kills Clostridioides difficile vegetative cells and inhibits spore germination.

Clostridioides difficile infection (CDI) is the most lethal of the five CDC urgent public health treats, resulting in 12,800 annual deaths in the United States alone [Antibiotic Resistance Threats in the United States, 2019 (2019), www.cdc.gov/DrugResistance/Biggest-Threats.html]. The high recurrence rate and the inability of antibiotics to treat such infections mandate discovery of new therapeutics. A major challenge with CDI is the production of spores, leading to multiple recurrences of infection in 25% of patients [C. P. Kelly, J. T. LaMont, N. Engl. J. Med. 359, 1932-1940 (2008)], with potentially lethal consequence. Herein, we describe the discovery of an oxadiazole as a bactericidal anti-C. difficile agent that inhibits both cell-wall peptidoglycan biosynthesis and spore germination. We document that the oxadiazole binds to the lytic transglycosylase SleC and the pseudoprotease CspC for prevention of spore germination. SleC degrades the cortex peptidoglycan, a critical step in the initiation of spore germination. CspC senses germinants and cogerminants. Binding to SleC is with higher affinity than that to CspC. Prevention of spore germination breaks the nefarious cycles of CDI recurrence in the face of the antibiotic challenge, which is a primary cause of therapeutic failure. The oxadiazole exhibits efficacy in a mouse model of recurrent CDI and holds promise in clinical treatment of CDI.

Proteome plasticity during Physcomitrium patens spore germination - from the desiccated phase to heterotrophic growth and reconstitution of photoautotrophy.

The establishment of moss spores is considered a milestone in plant evolution. They harbor protein networks underpinning desiccation tolerance and accumulation of storage compounds that can be found already in algae and that are also utilized in seeds and pollen. Furthermore, germinating spores must produce proteins that drive the transition through heterotrophic growth to the autotrophic plant. To get insight into the plasticity of this proteome, we investigated it at five timepoints of moss (Physcomitrium patens) spore germination and in protonemata and gametophores. The comparison to previously published Arabidopsis proteome data of seedling establishment showed that not only the proteomes of spores and seeds are functionally related, but also the proteomes of germinating spores and young seedlings. We observed similarities with regard to desiccation tolerance, lipid droplet proteome composition, control of dormancy, and ß-oxidation and the glyoxylate cycle. However, there were also striking differences. For example, spores lacked any obvious storage proteins. Furthermore, we did not detect homologs to the main triacylglycerol lipase in Arabidopsis seeds, SUGAR DEPENDENT1. Instead, we discovered a triacylglycerol lipase of the oil body lipase family and a lipoxygenase as being the overall most abundant proteins in spores. This finding indicates an alternative pathway for triacylglycerol degradation via oxylipin intermediates in the moss. The comparison of spores to Nicotiana tabacum pollen indicated similarities for example in regards to resistance to desiccation and hypoxia, but the overall developmental pattern did not align as in the case of seedling establishment and spore germination.

A DELAY OF GERMINATION 1 (DOG1)-like protein regulates spore germination in the moss Physcomitrium patens.

DELAY OF GERMINATION 1 is a key regulator of dormancy in flowering plants before seed germination. Bryophytes develop haploid spores with an analogous function to seeds. Here, we investigate whether DOG1 function during germination is conserved between bryophytes and flowering plants and analyse the underlying mechanism of DOG1 action in the moss Physcomitrium patens. Phylogenetic and in silico expression analyses were performed to identify and characterise DOG1 domain-containing genes in P. patens. Germination assays were performed to characterise a Ppdog1-like1 mutant, and replacement with AtDOG1 was carried out. Yeast two-hybrid assays were used to test the interaction of the PpDOG1-like protein with DELLA proteins from P. patens and A. thaliana. P. patens possesses nine DOG1 domain-containing genes. The DOG1-like protein PpDOG1-L1 (Pp3c3_9650) interacts with PpDELLAa and PpDELLAb and the A. thaliana DELLA protein AtRGA in yeast. Protein truncations revealed the DOG1 domain as necessary and sufficient for interaction with PpDELLA proteins. Spores of Ppdog1-l1 mutant germinate faster than wild type, but replacement with AtDOG1 reverses this effect. Our data demonstrate a role for the PpDOG1-LIKE1 protein in moss spore germination, possibly alongside PpDELLAs. This suggests a conserved DOG1 domain function in germination, albeit with differential adaptation of regulatory networks in seed and spore germination.

Regulation of late-acting operons by three transcription factors and a CRISPR-Cas component during Myxococcus xanthus development.

Upon starvation, rod-shaped Myxococcus xanthus bacteria form mounds and then differentiate into round, stress-resistant spores. Little is known about the regulation of late-acting operons important for spore formation. C-signaling has been proposed to activate FruA, which binds DNA cooperatively with MrpC to stimulate transcription of developmental genes. We report that this model can explain regulation of the fadIJ operon involved in spore metabolism, but not that of the spore coat biogenesis operons exoA-I, exoL-P, and nfsA-H. Rather, a mutation in fruA increased the transcript levels from these operons early in development, suggesting negative regulation by FruA, and a mutation in mrpC affected transcript levels from each operon differently. FruA bound to all four promoter regions in vitro, but strikingly each promoter region was unique in terms of whether or not MrpC and/or the DNA-binding domain of Nla6 bound, and in terms of cooperative binding. Furthermore, the DevI component of a CRISPR-Cas system is a negative regulator of all four operons, based on transcript measurements. Our results demonstrate complex regulation of sporulation genes by three transcription factors and a CRISPR-Cas component, which we propose produces spores suited to withstand starvation and environmental insults.

Clostridioides difficile Spore Formation and Germination: New Insights and Opportunities for Intervention.

Spore formation and germination are essential for the bacterial pathogen Clostridioides difficile to transmit infection. Despite the importance of these developmental processes to the infection cycle of C. difficile, the molecular mechanisms underlying how this obligate anaerobe forms infectious spores and how these spores germinate to initiate infection were largely unknown until recently. Work in the last decade has revealed that C. difficile uses a distinct mechanism for sensing and transducing germinant signals relative to previously characterized spore formers. The C. difficile spore assembly pathway also exhibits notable differences relative to Bacillus spp., where spore formation has been more extensively studied. For both these processes, factors that are conserved only in C. difficile or the related Peptostreptococcaceae family are employed, and even highly conserved spore proteins can have differential functions or requirements in C. difficile compared to other spore formers. This review summarizes our current understanding of the mechanisms controlling C. difficile spore formation and germination and describes strategies for inhibiting these processes to prevent C. difficile infection and disease recurrence.

Identification of a putative cell wall-hydrolyzing amidase involved in sporangiospore maturation in Actinoplanes missouriensis.

Actinoplanes missouriensis is a filamentous bacterium that differentiates into terminal sporangia, each containing a few hundred spores. Previously, we reported that a cell wall-hydrolyzing N-acetylglucosaminidase, GsmA, is required for the maturation process of sporangiospores in A. missouriensis; sporangia of the gsmA null mutant (ΔgsmA) strain released chains of 2-20 spores under sporangium dehiscence-inducing conditions. In this study, we identified and characterized a putative cell wall hydrolase (AsmA) that is also involved in sporangiospore maturation. AsmA was predicted to have a signal peptide for the general secretion pathway and an N-acetylmuramoyl-l-alanine amidase domain. The transcript level of asmA increased during the early stages of sporangium formation. The asmA null mutant (ΔasmA) strain showed phenotypes similar to those of the wild-type strain, but sporangia of the ΔgsmAΔasmA double mutant released longer spore chains than those from the ΔgsmA sporangia. Furthermore, a weak interaction between AsmA and GsmA was detected in a bacterial two-hybrid assay using Escherichia coli as the host. Based on these results, we propose that AsmA is an enzyme that hydrolyzes peptidoglycan at septum-forming sites to separate adjacent spores during sporangiospore maturation in cooperation with GsmA in A. missouriensis.IMPORTANCEActinoplanes missouriensis produces sporangiospores as dormant cells. The spores inside the sporangia are assumed to be formed from prespores generated by the compartmentalization of intrasporangium hyphae via septation. Previously, we identified GsmA as a cell wall hydrolase responsible for the separation of adjacent spores inside sporangia. However, we predicted that an additional cell wall hydrolase(s) is inevitably involved in the maturation process of sporangiospores because the sporangia of the gsmA null mutant strain released not only tandemly connected spore chains (2-20 spores) but also single spores. In this study, we successfully identified a putative cell wall hydrolase (AsmA) that is involved in sporangiospore maturation in A. missouriensis.

Co-evolution at protein-protein interfaces guides inference of stoichiometry of oligomeric protein complexes by de novo structure prediction.

The quaternary structure with specific stoichiometry is pivotal to the specific function of protein complexes. However, determining the structure of many protein complexes experimentally remains a major bottleneck. Structural bioinformatics approaches, such as the deep learning algorithm Alphafold2-multimer (AF2-multimer), leverage the co-evolution of amino acids and sequence-structure relationships for accurate de novo structure and contact prediction. Pseudo-likelihood maximization direct coupling analysis (plmDCA) has been used to detect co-evolving residue pairs by statistical modeling. Here, we provide evidence that combining both methods can be used for de novo prediction of the quaternary structure and stoichiometry of a protein complex. We achieve this by augmenting the existing AF2-multimer confidence metrics with an interpretable score to identify the complex with an optimal fraction of native contacts of co-evolving residue pairs at intermolecular interfaces. We use this strategy to predict the quaternary structure and non-trivial stoichiometries of Bacillus subtilis spore germination protein complexes with unknown structures. Co-evolution at intermolecular interfaces may therefore synergize with AI-based de novo quaternary structure prediction of structurally uncharacterized bacterial protein complexes.

The MAPK homolog, Smk1, promotes assembly of the glucan layer of the spore wall in S. cerevisiae.

Smk1 is a MAPK homolog in the yeast Saccharomyces cerevisiae that controls the postmeiotic program of spore wall assembly. During this program, haploid cells are surrounded by a layer of mannan and then a layer of glucan. These inner layers of the spore wall resemble the vegetative cell wall. Next, the outer layers consisting of chitin/chitosan and then dityrosine are assembled. The outer layers are spore-specific and provide protection against environmental stressors. Smk1 is required for the proper assembly of spore walls. However, the protective properties of the outer layers have limited our understanding of how Smk1 controls this morphogenetic program. Mutants lacking the chitin deacetylases, Cda1 and Cda2, form spores that lack the outer layers of the spore wall. In this study, cda1,2∆ cells were used to demonstrate that Smk1 promotes deposition of the glucan layer of the spore wall through the partially redundant glucan synthases Gsc2 and Fks3. Although Gsc2 is localized to sites of spore wall assembly in the wild type, it is mislocalized in the mother cell cytoplasm in the smk1∆ mutant. These findings suggest that Smk1 controls assembly of the spore wall by regulating the localization of Gsc2 during sporogenesis.

Multifactorial resistance of Bacillus subtilis spores to low-pressure plasma sterilization.

Common sterilization techniques for labile and sensitive materials have far-reaching applications in medical, pharmaceutical, and industrial fields. Heat inactivation, chemical treatment, and radiation are established methods to inactivate microorganisms, but pose a threat to humans and the environment and can damage susceptible materials or products. Recent studies have demonstrated that cold low-pressure plasma (LPP) treatment is an efficient alternative to common sterilization methods, as LPP's levels of radicals, ions, (V)UV-radiation, and exposure to an electromagnetic field can be modulated using different process gases, such as oxygen, nitrogen, argon, or synthetic (ambient) air. To further investigate the effects of LPP, spores of the Gram-positive model organism Bacillus subtilis were tested for their LPP susceptibility including wild-type spores and isogenic spores lacking DNA-repair mechanisms such as non-homologous end-joining (NHEJ) or abasic endonucleases, and protective proteins like α/ß-type small acid-soluble spore proteins (SASP), coat proteins, and catalase. These studies aimed to learn how spores resist LPP damage by examining the roles of key spore proteins and DNA-repair mechanisms. As expected, LPP treatment decreased spore survival, and survival after potential DNA damage generated by LPP involved efficient DNA repair following spore germination, spore DNA protection by α/ß-type SASP, and catalase breakdown of hydrogen peroxide that can generate oxygen radicals. Depending on the LPP composition and treatment time, LPP treatment offers another method to efficiently inactivate spore-forming bacteria.IMPORTANCESurface-associated contamination by endospore-forming bacteria poses a major challenge in sterilization, since the omnipresence of these highly resistant spores throughout nature makes contamination unavoidable, especially in unprocessed foods. Common bactericidal agents such as heat, UV and γ radiation, and toxic chemicals such as strong oxidizers: (i) are often not sufficient to completely inactivate spores; (ii) can pose risks to the applicant; or (iii) can cause unintended damage to the materials to be sterilized. Cold low-pressure plasma (LPP) has been proposed as an additional method for spore eradication. However, efficient use of LPP in decontamination requires understanding of spores' mechanisms of resistance to and protection against LPP.

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.

Discovery of a new volcanic soil material, "Akahoya," as an adsorbent for bacterial and viral pathogens and its application to environmental purification.

Akahoya is a volcanic soil rich in alumina, primarily deposited in Kyushu, Japan. We have found that Akahoya adsorbs bacteria in the water surrounding cattle grazing areas, suggesting a potential for environmental purification. This study investigated the spectrum of microorganisms adsorbed by Akahoya using a column filled with Akahoya through which a suspension of microorganisms was passed. Shirasu soil, another volcanic soil with a different chemical composition, was used as a control. Akahoya effectively adsorbed a diverse range of microorganisms including Escherichia coli, Campylobacter jejuni, Vibrio parahaemolyticus, Salmonella Enteritidis, Staphylococcus aureus, Clostridium perfringens, spores of Bacillus subtilis and Bacillus anthracis, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), murine norovirus, and avian influenza virus (H3N2), whereas Shirasu soil did not adsorb any of the organisms examined. Moreover, bacteria naturally present in river water, such as aerobic bacteria, total coliforms, and Enterobacteriaceae as indicators of river contamination, as well as E. coli added artificially to sterilized river water, were reduced to below the detection limit (<1 CFU/mL) after being passed through Akahoya. Additionally, the number of viable E. coli continued to decrease after contact with Akahoya for 1 month, suggesting bactericidal effects. Notably, the adsorption of E. coli to Akahoya was influenced by the concentration of phosphate and the pH of the suspension due to the interaction between the surface phosphorylation of organisms and Al2O3, the major chemical component of Akahoya. The present results demonstrate the remarkable ability of Akahoya to remove phosphate and microbes, suggesting that Akahoya could be used for water purification processes.IMPORTANCEAlthough a safe and sufficient water supply is essential for the maintenance of hygienic conditions, a major challenge is to develop a comprehensive effective, sustainable, and cost-effective technological approach for the treatment and purification of contaminated water. In this study, we demonstrated that a novel volcanic soil, Akahoya, which has unlimited availability, is a highly effective adsorbent for a wide range of bacterial and viral pathogens, suggesting its potential as a sustainable resource for this purpose. It was suggested that the adsorption of microorganisms on Akahoya was mediated by phosphate groups present on the surface structures of microorganisms, which bind to the alumina component of Akahoya according to the phosphate concentration and pH of the liquid phase. The present findings highlight the exceptional ability of Akahoya to eliminate or reduce phosphate and microorganisms effectively in water purification processes, thus contributing to the development of efficient and sustainable solutions for addressing water pollution challenges.

Construction nanobiotechnology approach for performance enhancement of microbially induced biomineralization (MIB) using a biopolymer encapsulated spore-based system.

The integration of green construction practices within the built environment has been significantly advanced by biotechnological innovations, among which microbially induced biomineralization (MIB), predominantly facilitated by various strains of spore-forming bacilli, emerges as a pivotal mechanism for the self-healing of concrete. However, the practical deployment of this technology faces challenges, notably the compromised viability of bacterial spores due to germination triggered by severe shear stress during concrete mixing. To address this limitation, a water-insoluble polymer (extracellular polymeric substance) produced by Cellulomonas flavigena was utilized to encapsulate and protect the spores. The encapsulation process was rigorously verified through physicochemical methodologies, including X-ray diffraction (XRD) analysis, which revealed alterations in the interlayer spacings of the extracellular polymeric substance (EPS) structure during the encapsulation process, indicating successful EPS coating of the spores. Furthermore, a proof of concept for the enhanced biomineralization capacity of EPS-coated spores was demonstrated. Standard analytical techniques confirmed the precipitation of calcite and vaterite among other minerals, underscoring the effectiveness of this novel approach. This breakthrough paves the way for the development of innovative, sustainable bioconcrete applications, aligning with broader environmental objectives and advancing the field of green construction technology.IMPORTANCEDevelopment of bioconcrete with self-healing capability through MIB constitutes an important sustainable construction biotechnology approach for restoration and repair of built environment. Like every promising technology, MIB also suffers from certain shortcomings in terms of compromised viability of the microbial cells after premature germination of the spores on exposure to shear stress caused during concrete mixing. In this study, these challenges were adequately addressed by successfully providing a protective coating of indigenously extracted EPS to the bacterial spores and elucidating the interactive mechanisms between them. The results showed stable encapsulation of the spores while providing mechanistic insights of the encapsulation phenomenon. The data also showed enhanced rate of biomineralization by encapsulated microbes when subjected to stress conditions.

Death caps (Amanita phalloides) frequently establish from sexual spores, but individuals can grow large and live for more than a decade in invaded forests.

Global change is reshaping Earth's biodiversity, but the changing distributions of nonpathogenic fungi remain largely undocumented, as do mechanisms enabling invasions. The ectomycorrhizal Amanita phalloides is native to Europe and invasive in North America. Using population genetics and genomics, we sought to describe the life history traits of this successfully invading symbiotic fungus. To test whether death caps spread underground using hyphae, or aboveground using sexual spores, we mapped and genotyped mushrooms from European and US sites. Larger genetic individuals (genets) would suggest spread mediated by vegetative growth, while many small genets would suggest dispersal mediated by spores. To test whether genets are ephemeral or persistent, we also sampled from populations over time. At nearly every site and across all time points, mushrooms resolve into small genets. Individuals frequently establish from sexual spores. But at one Californian site, a single individual measuring nearly 10 m across dominated. At two Californian sites, the same genetic individuals were discovered in 2004, 2014, and 2015, suggesting single individuals (both large and small) can reproduce repeatedly over relatively long timescales. A flexible life history strategy combining both mycelial growth and spore dispersal appears to underpin the invasion of this deadly perennial ectomycorrhizal fungus.

Transmission mode and dispersal traits correlate with host specificity in mammalian gut microbes.

Different host species associate with distinct gut microbes in mammals, a pattern sometimes referred to as phylosymbiosis. However, the processes shaping this host specificity are not well understood. One model proposes that barriers to microbial transmission promote specificity by limiting microbial dispersal between hosts. This model predicts that specificity levels measured across microbes is correlated to transmission mode (vertical vs. horizontal) and individual dispersal traits. Here, we leverage two large publicly available gut microbiota data sets (1490 samples from 195 host species) to test this prediction. We found that host specificity varies widely across bacteria (i.e., there are generalist and specialist bacteria) and depends on transmission mode and dispersal ability. Horizontally-like transmitted bacteria equipped with traits that facilitate switches between host (e.g., tolerance to oxygen) were found to be less specific (more generalist) than microbes without those traits, for example, vertically-like inherited bacteria that are intolerant to oxygen. Altogether, our findings are compatible with a model in which limited microbial dispersal abilities foster host specificity.

Systematics and evolutionary dynamics of insect-fern interactions in the specialized fern-spore feeding Cuprininae (Lepidoptera, Stathmopodidae).

Fern-spore-feeding (FSF) is rare and found in only four families of Lepidoptera. Stathmopodidae is the most speciose family that contains FSF species, and its subfamily Cuprininae exclusively specializes on FSF. However, three species of Stathmopodinae also specialize on FSF. To better understand the evolutionary history of FSF and, more generally, the significance of specialization on a peculiar host, a phylogenetic and taxonomic revision for this group is necessary. We reconstructed the most comprehensive molecular phylogeny, including one mitochondrial and four nuclear genes, of Stathmopodidae to date, including 137 samples representing 62 species, with a particular focus on the FSF subfamily, Cuprininae, including 33 species (41% of named species) from 6 of the 7 Cuprininae genera. Species from two other subfamilies, Stathmopodinae and Atkinsoniinae, were also included. We found that FSF evolved only once in Stathmopodidae and that the previous hypothesis of multiple origins of FSF was misled by inadequate taxonomy. Moreover, we showed that (1) speciation/extinction rates do not differ significantly between FSF and non-FSF groups and that (2) oligophage is the ancestral character state in Cuprininae. We further revealed that a faster rate of accumulating specialists over time, and thus a higher number of specialists, was achieved by a higher transition rate from oligophagages to specialists compared to the transition rate in the opposite direction. We finish by describing three new genera, Trigonodagen. nov., Petalagen. nov., and Pediformisgen. nov., and revalidating five genera: Cuprina, Calicotis, Thylacosceles, Actinoscelis, Thylacosceloides in Cuprininae, and we provide an updated taxonomic key to genera and a revised global checklist of Cuprininae.

Enhanced sporulation of B. licheniformis BF-002 through automatic co-feeding of carbon and nitrogen sources.

Bacillus licheniformis formulations are effective for environmental remediation, gut microbiota modulation, and soil improvement. An adequate spore quantity is crucial for the activity of B. licheniformis formulations. This study investigated the synergistic effects of carbon/nitrogen source consumption and concentration on B. licheniformis BF-002 cultivation, with the aim of developing an automatic co-feeding strategy to enhance spore production. Initial glucose (10 g/L) and amino nitrogen (1.5 g/L) concentrations promote cell growth, followed by reduced glucose (2.0 g/L) and amino nitrogen (0.5 g/L) concentrations for sustained spore generation. The spore quantity reached 2.59 × 1010 CFU/mL. An automatic co-feeding strategy was developed and implemented in 5 and 50 L cultivations, resulting in spore quantities of 2.35 × 1010 and 2.86 × 1010 CFU/mL, respectively, improving by 6.81% and 30.00% compared to that with a fixed glucose concentration (10.0 g/L). The culture broth obtained at both the 5 and 50 L scales was spray-dried, resulting in bacterial powder with cell viability rates of 85.94% and 82.68%, respectively. Even after exposure to harsh conditions involving high temperature and humidity, cell viability remained at 72.80% and 69.89%, respectively. Employing the automatic co-feeding strategy increased the transcription levels of the spore formation-related genes spo0A, spoIIGA, bofA, and spoIV by 7.42%, 8.46%, 8.87%, and 9.79%, respectively. The proposed strategy effectively promoted Bacillus growth and spore formation, thereby enhancing the quality of B. licheniformis formulations.

Synthesis and toxicological study of chitosan-pirul (Schinus molle L.) essential oil nanoparticles on Aspergillus flavus.

In recent years, the study of essential oils as antifungal alternatives and their encapsulation to increase their properties for greater effects has been tested. In this work, nanoparticles of chitosan-Schinus molle L. essential oil (CS-PEO-Np) with a size of 260 ± 31.1 nm were obtained by ionic gelation and evaluated in some growth phases of Aspergillus flavus, a toxigenic fungus. At a concentration of 250 µg/mL of CS-PEO-Np, the A. flavus mycelial growth was inhibited at 97.1% with respect to control, at 96 h of incubation; the germination and viability of spores were inhibited at 74.8 and 40%, respectively, after exposure to 500 µg/mL of these nanomaterials, at 12 h of incubation. The fluorescence images of stained spores with DAPI showed the affectations caused by nanoparticles in the cell membrane, vacuoles and vacuolar content, cell wall, and nucleic acids. For both nanoparticles, CS-Np and CS-PEO-Np, no mutagenic effect was observed in Salmonella Typhimurium; also, the phytotoxic assay showed low-to-moderate toxicity toward seeds, which was dependent on the nanoparticle's concentration. The acute toxicity of CS-PEO-Np to A. salina nauplii was considered low in comparison to CS-Np (control), which indicates that the incorporation of Schinus molle essential oil into nanoparticles of chitosan is a strategy to reduce the toxicity commonly associated with nanostructured materials. The nanoparticulated systems of CS-PEO-Np represent an effective and non-toxic alternative for the control of toxigenic fungi such as A. flavus by delaying the initial growth stage.