Design, synthesis and antimicrobial activity of novel berberine derivatives.

BACKGROUND: The threats to the safety of humans and the environment and the resistance of agricultural chemicals to plant pathogenic fungi and bacteria highlight an urgent need to find safe and efficient alternatives to chemical fungicides and bactericides. In this study, a series of Berberine (BBR) derivatives were designed, synthesized and evaluated for in vitro and in vivo antimicrobial activity against plant pathogenic fungi and bacteria. RESULTS: Bioassay results indicated that compounds A11, A14, A20, A21, A22, A25, A26, E1, E2, E3, Z1 and Z2 showed high inhibitory activity against Sclerotinia sclerotiorum and Botrytis cinerea. Especially, A25 showed a broad spectrum and the highest antifungal activity among these compounds. Its EC50 value against Botrytis cinerea was 1.34 µg mL-1. Compound E6 possessed high inhibitory activity against Xanthomonas oryzae and Xanthomonas Campestris, with MIC90 values of 3.12 µg mL-1 and 1.56 µg mL-1. A Topomer CoMFA model was generated for 3D-QSAR studies based on anti-B. cinerea effects, with high predictive accuracy, showed that the addition of an appropriate substituent group at the para-position of benzyl of BBR derivatives could effectively improve the anti-B. cinerea activity. In addition, compound A25 could significantly inhibit the spore germination of Botrytis cinerea at low concentration, and compound F4 exhibited remarkable curative and protective efficiencies on rice bacterial leaf blight. CONCLUSION: This study indicates that the BBR derivatives are hopeful for further exploration as the lead compound with novel antimicrobial agents. © 2024 Society of Chemical Industry.

Spore germination and lactic acid combined treatment: A new processing strategy for the shelf-life extension of instant wet noodles.

Bacillus amyloliquefaciens (BAM) was identified as the predominant spoilage bacteria in instant wet noodles (IWNs). The utilization of industrial acid treatment as a long shelf-life strategy resulted in reduced consumer acceptance due to the acidic taste of the products. This study proposed a processing strategy that integrated spore germination (SG) and lactic acid (LA) treatment to effectively reduce the spore survival rate and extend the shelf life of IWNs. L-histidine, d-glucose, and sodium chloride were highly efficient and safe germinants for BAM spores. In IWNs, compound germinants (1.0 % L-histidine, 0.5 % d-glucose, and 1.0 % sodium chloride) boosted the SG rate by 3.61 times. With synergistic LA treatment, the spore lethality increased by 34.41 % -41.68 %. Under the SG and reduced acid-heat conditions of pH 2.30-2.50, the mortality of spores could reach 92.00 %-93.17 %, which was 14.11 %-15.28 % higher than the industrial acid-heat condition of pH 2.10. DPA, ATP, and membrane potential showed that germinants reduced the spore membrane permeability and promoted the occurrence of spore membrane damage under acid-heat conditions. Moreover, this strategy significantly extended the shelf-life of IWNs by 3.00-5.50 times and controlled the pH ≥ 5.50. Additionally, it improved color, texture, and overall sensory evaluation. Accordingly, this strategy solved the contradiction between the long shelf-life of IWNs and the unacceptable acidification in industrial production.

Human in vivo assessment of the survival and germination of Heyndrickxia coagulans SNZ1969 spores delivered via gummy candies.

Confectionary products hold promise as unconventional food carriers for probiotic microorganisms. This study explored the delivery of Heyndrickxia coagulans SNZ1969, a spore-forming probiotic, using gummy candies. In this study, we prepared gummy candies containing bacterial spores with a viable count that remained stable during a 24-month shelf-life period, meeting the label claim of at least one billion CFUs per serving (24 g). Then, we carried out an intervention trial involving 24 healthy adults who consumed one serving per day for two weeks followed by an additional two weeks of follow-up. Fecal samples were collected and analyzed with a protocol that allowed the viable counts of SNZ1969, both in spore and vegetative forms. The obtained results revealed that bacterial spores germinated in all volunteers. SNZ1969 persistence in the gut was monitored for two weeks after the end of gummy candy consumption, indicating its potential for prolonged colonization. These findings highlight the potential of unconventional food carriers for probiotic delivery and suggest that spore-forming probiotics can be metabolically active in the human intestine. These findings provide information for the development of food products containing spore-forming probiotics and their potential benefits in promoting gastrointestinal health.

Oligomycin-producing Streptomyces sp. newly isolated from Swiss soils efficiently protect Arabidopsis thaliana against Botrytis cinerea.

Botrytis cinerea is a necrotrophic phytopathogen able to attack more than 200 different plant species causing strong yield losses worldwide. Many synthetic fungicides have been developed to control this disease, resulting in the rise of fungicide-resistance B. cinerea strains. The aim of this study was to identify Streptomyces strains showing antagonistic activity against B. cinerea to contribute to plant protection in an environmentally friendly way. We isolated 15 Actinomycete strains from 9 different Swiss soils. The culture filtrates of three isolates showing antifungal activity inhibited spore germination and delayed mycelial growth of B. cinerea. Infection experiments showed that Arabidopsis thaliana plants were more resistant to this pathogen after leaf treatment with the Streptomyces filtrates. Bioassay-guided isolation of the active compounds revealed the presence of germicidins A and B as well as of oligomycins A, B, and E. While germicidins were mostly inactive, oligomycin B reduced the mycelial growth of B. cinerea significantly. Moreover, all three oligomycins inhibited this fungus' spore germination, suggesting that these molecules might contribute to the Streptomyces's ability to protect plants against infection by the broad host-pathogen Botrytis cinerea. IMPORTANCE: This study reports the isolation of new Streptomyces strains with strong plant-protective potential mediated by their production of specialized metabolites. Using the broad host range pathogenic fungus Botrytis cinerea, we demonstrate that the cell-free filtrate of selected Streptomyces isolates efficiently inhibits different developmental stages of the fungus, including mycelial growth and the epidemiologically relevant spore germination. Beyond in vitro experiments, the strains and their metabolites also efficiently protected plants against the disease caused by this pathogen. This work further identifies oligomycins as active compounds involved in the observed antifungal activity of the strains. This work shows that we can harness the natural ability of soil-borne microbes and of their metabolites to efficiently fight other microbes responsible for significant crop losses. This opens the way to the development of environmentally friendly health protection measures for crops of agronomical relevance, based on these newly isolated strains or their metabolic extracts containing oligomycins.

Secofumitremorgins C and D, a pair of atropisomers from saltern-derived fungus Aspergillus fumigatus GXIMD00544.

A pair of atropisomers secofumitremorgins C (1a) and D (1b), together with fifteen known alkaloids (2-16), were isolated from a saltern-derived fungus Aspergillus fumigatus GXIMD00544. The structures of atropisomers 1a and 1b were elucidated by the detailed spectroscopic data, chemical reaction and quantum chemical calculations. Compounds 1 and 8 displayed antifungal spore germination effects against plant pathogenic fungus associated with sugarcane Fusarium sp. with inhibitory rates of 53% and 77% at the concentration of 100 µM, repectively. Atropisomers 1 also exhibited antifouling potential against Balanus amphitrite larval settlement with an inhibitory rate of 96% at the concentration of 100 µM.

Characterization of individual spores of two biological insecticides, Bacillus thuringiensis and Lysinibacillus sphaericus, in response to glutaraldehyde using single-cell optical approaches.

Bacillus thuringiensis (Bt) and Lysinibacillus sphaericus (Ls) are the most widely used microbial insecticides. Both encounter unfavorable environmental factors and pesticides in the field. Here, the responses of Bt and Ls spores to glutaraldehyde were characterized using Raman spectroscopy and differential interference contrast imaging at the single-cell level. Bt spores were more sensitive to glutaraldehyde than Ls spores under prolonged exposure: <1.0% of Bt spores were viable after 10 min of 0.5% (v/v) glutaraldehyde treatment, compared to ~ 20% of Ls spores. The Raman spectra of glutaraldehyde-treated Bt and Ls spores were almost identical to those of untreated spores; however, the germination process of individual spores was significantly altered. The time to onset of germination, the period of rapid Ca2+-2,6-pyridinedicarboxylic acid (CaDPA) release, and the period of cortex hydrolysis of treated Bt spores were significantly longer than those of untreated spores, with dodecylamine germination being particularly affected. Similarly, the germination of treated Ls spores was significantly prolonged, although the prolongation was less than that of Bt spores. Although the interiors of Bt and Ls spores were undamaged and CaDPA did not leak, proteins and structures involved in spore germination could be severely damaged, resulting in slower and significantly prolonged germination. This study provides insights into the impact of glutaraldehyde on bacterial spores at the single cell level and the variability in spore response to glutaraldehyde across species and populations.

Curcumin: An innovative approach for postharvest control of Alternaria alternata induced black rot in cherry tomatoes.

Curcumin, a natural bioactive compound derived from Curcuma longa, has been widely recognized for its antifungal properties. In this study, we investigated the effects of curcumin on the phytopathogenic fungus Alternaria alternata and its pathogenicity in cherry tomato fruit. The results demonstrated that curcumin treatment significantly inhibited mycelial growth and spore germination of A. alternata in a dose-dependent manner. Scanning electron microscopy revealed alterations in the morphology of A. alternata mycelia treated with curcumin. Furthermore, curcumin treatment led to an increase in malondialdehyde and hydrogen peroxide contents, indicating cell membrane damage in A. alternata. Moreover, curcumin exhibited a remarkable inhibitory effect on the incidence and lesion diameters of black rot caused by A. alternata in cherry tomato fruit. Gene expression analysis revealed upregulation of defense-related genes (POD, SOD, and CAT) in tomato fruit treated with curcumin. Additionally, curcumin treatment resulted in decreased activity of exocellular pathogenic enzymes (polygalacturonase, pectin lyase, and endo-1,4-ß-d-glucanase) in A. alternata. Overall, our findings highlight the potential of curcumin as an effective antifungal agent against A. alternata, providing insights into its inhibitory mechanisms on mycelial growth, spore germination, and pathogenicity in cherry tomato fruit.

Identification of Germinants and Expression of Germination Genes in Clostridium perfringens Strains Isolated from Diarrheic Animals.

In this study, we investigated the spore germination phenotype of Clostridium perfringens strains isolated from diarrheic animals (animal strains). The transcripts of germination-specific genes and their protein products were also measured. Our study found the following results: (i) animal strains spores germinated at a slower rate with AK (mixture of L-asparagine and KCl), L-cysteine, or L-lysine, but the extent of germination varied based on strains and germinants used; (ii) none of the amino acids (excluding L-cysteine and L-lysine) were identified as a universal germinant for spores of animal strains; (iii) animal strain spores germinated better at a pH range of 6.0-7.0; (iv) all tested germination-specific genes were expressed in animal strains; the levels of expression of major germinant receptor gene (gerKC) were higher and the cortex hydrolysis machinery genes (cspB and sleC) were lower in animal strains, compared to the food poisoning strain SM101; and (v) the levels of CspB and SleC were significantly lower in spores of animal strains compared to strain SM101, suggesting that these animal strains lack an efficient spore cortex hydrolysis machinery. In summary, our findings suggest that the poor or slow spore germination in C. perfringens animal strains might be due to incomplete spore cortex hydrolysis.

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.

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.

Thioflavin-T does not report on electrochemical potential and memory of dormant or germinating bacterial spores.

IMPORTANCE: Bacillus and Clostridium spores cause food spoilage and disease because of spores' dormancy and resistance to microbicides. However, when spores "come back to life" in germination, their resistance properties are lost. Thus, understanding the mechanisms of spore germination could facilitate the development of "germinate to eradicate" strategies. One germination feature is the memory of a pulsed germinant stimulus leading to greater germination following a second pulse. Recent observations of increases in spore binding of the potentiometric dye thioflavin-T early in their germination of spores led to the suggestion that increasing electrochemical potential is how spores "remember" germinant pulses. However, new work finds no increased thioflavin-T binding in the physiological germination of Coatless spores or of intact spores germinating with dodecylamine, even though spore memory is seen in both cases. Thus, using thioflavin-T uptake by germinating spores to assess the involvement of electrochemical potential in memory of germinant exposure, as suggested recently, is questionable.

Applying the first microcapsule-based self-healing microbial-induced calcium carbonate materials to prevent the migration of Pb ions.

Lead (Pb) accumulation can lead to serious threats to surrounding environments and damage to the liver and kidneys. In the past few years, microbial-induced carbonate precipitation (MICP) technology has been widely applied to achieve Pb immobilization due to its environmentally friendly nature. However, harsh pH conditions can cause the instability of the carbonate precipitation to degrade or dissolve, increasing the potential of Pb2+ migration into nearby environments. In this study, microcapsule-based self-healing microbial-induced calcium carbonate (MICC) materials were applied to prevent Pb migration. The highest sporulation rate of 95.8% was attained at 7 g/L yeast extract, 10 g/L NH4Cl, and 3.6 g/L Mn2+. In the germination phase, the microcapsule not only prevented the bacterial spores from being threatened by the acid treatment but secured their growth and reproduction. Micro analysis also revealed that cerussite, calcite, and aragonite minerals were present, while extracellular polymeric substances (EPSs) were identified via Fourier transform infrared spectroscopy (FTIR). These results confirm their involvement in combining Pb2+ and Ca2+. The immobilization efficiency of above 90% applied to MICC materials was attained, while it of below 5% applied to no MICC use was attained. The findings explore the potential of applying microcapsule-based self-healing MICC materials to prevent Pb ion migration when the calcium carbonate degrades under harsh pH conditions.

Interspecific competition affects spore germination and gametophore development of mosses.

Background: Interactions between moss species in their earliest growth stages have received little attention. To what extent interspecific competition or priority effects influence spore germination, protonemal development and gametophore emergence is unknown. We evaluated such effects in pairwise interaction between six common bryophyte species: Atrichum undulatum, Bryum argenteum, Ceratodon purpureus, Funaria hygrometrica, Hypnum cupressiforme, Leptobryum pyriforme. Methods: Interspecific interactions were assessed in vitro. Spores were sterilized and sown on agar plates in three treatments: 1) as single species cultures (controls), 2) as pairwise species cultures inoculated simultaneously, and 3) with a time lag of 20 days between species. Data on time needed for spore germination, germination rate, the time needed for gametophore differentiation, number of gametophores per germinated spore and average diameter of colonies were collected. We also performed spore germination tests in single-species cultures at the start and end of the study, as well as tests for density-dependency at spore germination and gametophore formation. Results: We observed strong pairwise interactive effects when sowing spores of different species simultaneously or with a delay of 20 days. The results indicate that spore germination is often inhibited by interspecific competition. The first species has an advantage as compared to the later colonizing species, i.e., an apparent priority effect. Interspecific interactions were also evident during gametophore development and included both inhibition and facilitation. Conclusion: We found pronounced differences in the relative performance of species in interaction with other species during spore germination and gametophore formation. Allelopathic effects are the most probable explanation for these observations. Our results under sterile lab conditions are likely to reflect processes that occur in the wild, governing biotic filtering and bryophyte community assembly during primary and secondary colonization.

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.

Enhancing the growth of thermophilic Bacillus licheniformis YYC4 by low-intensity fixed-frequency continuous ultrasound.

The effect of low-intensity fixed-frequency continuous ultrasound (LIFFCU) on the growth of Bacillus licheniformis YYC4 was investigated. The changes in morphology and activity of the organism, contributing to the growth were also explored. Compared with the control, a significant increase (48.95%) in the biomass of B. licheniformis YYC4 (at the logarithmic metaphase) was observed following the LIFFCU (28 kHz, 1.5 h and 120 W (equivalent to power density of 40 W/L)) treatment. SEM images showed that ultrasonication caused sonoporation, resulting in increased membrane permeability, evidenced by increase in cellular membrane potential, electrical conductivity of the culture, extracellular protein and nucleic acid, and intracellular Ca2+ content. Furthermore, LIFFCU action remarkably increased the extracellular protease activity, volatile components of the culture medium, microbial metabolic activity, and spore germination of the strain. Therefore, LIFFCU could be used to efficiently promote the growth of targeted microorganisms.

Lilium Gray Mold Suppression Conferred by the Host Antimicrobial Protein LsGRP1 Involves Main Pathogen-Targeted Manipulation of the Nonantimicrobial Region LsGRP1N.

Antimicrobial protein LsGRP1 protects Lilium from gray mold mainly caused by the destructive pathogen Botrytis elliptica; however, its nonantimicrobial region LsGRP1N conversely promotes spore germination of this fungus. By assaying the effects of LsGRP1N, LsGRP1, and the combination of LsGRP1N and the antimicrobial region LsGRP1C on fungal spore germination, hyphal growth, and Lilium gray mold development, LsGRP1N was found to improve the LsGRP1C sensitivity of B. elliptica and disease suppression by LsGRP1C. B. elliptica cell vitality assays indicated that LsGRP1N pretreatment uniquely enhanced the lethal efficiency of LsGRP1C compared to the control peptides. In addition, LsGRP1N-treated B. elliptica was demonstrated to lower infection-related gene expression and increase host-defense-eliciting activity, as indicated by reverse transcription quantitative polymerase chain reaction and histochemical-staining-based callose detection results, respectively. Therefore, LsGRP1N showed a novel mode of action for antimicrobial proteins by manipulating the main pathogen, which facilitated the development of target-specific and dormant microbe-eradicating antimicrobial agents.

Discovery of a Natural Hybrid Polyketide Produced by Endophytic Cladosporium sphaerospermum for Biocontrol of Phytopathogenic Fungus Botrytis cinerea.

The endophytic fungus Cladosporium sphaerospermum WBS017 exhibits broad-spectrum activity against plant pathogens, with particular effectiveness against Botrytis cinerea. Subsequently, a compound is isolated from strain WBS017 as the main active ingredient against B. cinerea using activity-guided separation and identified as hybrid polyketide (namely cladodionen, CLD) using UV, MS, NMR, etc. In vitro and in vivo antifungal activity tests demonstrate that CLD effectively inhibits the mycelial growth and spore germination, with an IC50 value of 1.13 and 0.095 mM, respectively, and exerts antifungal and fresh-keeping effects on both strawberry and tomato. Microscopy analysis reveals that the inhibitory effects of CLD on hyphae and spore germination are attributed to a decrease in structural stability of mycelia cells as well as the accumulation of reactive oxygen species (ROS). Furthermore, transcriptome analysis further indicates that spore germination is inhibited by suppressing the transcription levels of membrane or membrane-related genes, disturbing the balance of ROS metabolism, altering the primary metabolic pathways, genetic information processing, and cellular processes. Importantly, CLD demonstrates no significant toxicity on zebrafish embryos even at a concentration of 0.226 mM, indicating its potential as a safe biological-control agent. In summary, CLD would be a novel potential biological-control agent and can be considered as a promising fungicide to control B. cinerea.

Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions in a feedforward loop.

Clostridioides difficile infections begin when its metabolically dormant spores germinate in response to sensing bile acid germinants alongside amino acid and divalent cation co-germinants in the small intestine. While bile acid germinants are essential for C. difficile spore germination, it is currently unclear whether both co-germinant signals are required. One model proposes that divalent cations, particularly Ca2+, are essential for inducing germination, while another proposes that either co-germinant class can induce germination. The former model is based on the finding that spores defective in releasing large stores of internal Ca2+ in the form of calcium dipicolinic acid (CaDPA) cannot germinate when germination is induced with bile acid germinant and amino acid co-germinant alone. However, since the reduced optical density of CaDPA-less spores makes it difficult to accurately measure their germination, we developed a novel automated, time-lapse microscopy-based germination assay to analyze CaDPA mutant germination at the single-spore level. Using this assay, we found that CaDPA mutant spores germinate in the presence of amino acid co-germinant and bile acid germinant. Higher levels of amino acid co-germinants are nevertheless required to induce CaDPA mutant spores to germinate relative to WT spores because CaDPA released by WT spores during germination can function in a feedforward loop to potentiate the germination of other spores within the population. Collectively, these data indicate that Ca2+ is not essential for inducing C. difficile spore germination because amino acid and Ca2+ co-germinant signals are sensed by parallel signaling pathways. IMPORTANCE Clostridioides difficile spore germination is essential for this major nosocomial pathogen to initiate infection. C. difficile spores germinate in response to sensing bile acid germinant signals alongside co-germinant signals. There are two classes of co-germinant signals: Ca2+ and amino acids. Prior work suggested that Ca2+ is essential for C. difficile spore germination based on bulk population analyses of germinating CaDPA mutant spores. Since these assays rely on optical density to measure spore germination and the optical density of CaDPA mutant spores is reduced relative to WT spores, this bulk assay is limited in its capacity to analyze germination. To overcome this limitation, we developed an automated image analysis pipeline to monitor C. difficile spore germination using time-lapse microscopy. With this analysis pipeline, we demonstrate that, although Ca2+ is dispensable for inducing C. difficile spore germination, CaDPA can function in a feedforward loop to potentiate the germination of neighboring spores.

Genome-wide analysis of the GDSL esterase/lipase family genes in Physcomitrium patens and the involvement of GELP31 in spore germination.

In plants, the ability to produce hydrophobic substances that would provide protection from dehydration was required for the transition to land. This genome-wide investigation outlines the evolution of GDSL-type esterase/lipase (GELP) proteins in the moss Physcomitrium patens and suggests possible functions of some genes. GELP proteins play roles in the formation of hydrophobic polymers such as cutin and suberin that protect against dehydration and pathogen attack. GELP proteins are also implicated in processes such as pollen development and seed metabolism and germination. The P. patens GELP gene family comprises 48 genes and 14 pseudogenes. Phylogenetic analysis of all P. patens GELP sequences along with vascular plant GELP proteins with reported functions revealed that the P. patens genes clustered within previously identified A, B and C clades. A duplication model predicting the expansion of the GELP gene family within the P. patens lineage was constructed. Expression analysis combined with phylogenetic analysis suggested candidate genes for functions such as defence against pathogens, cutin metabolism, spore development and spore germination. The presence of relatively fewer GELP genes in P. patens may reduce the occurrence of functional redundancy that complicates the characterization of vascular plant GELP genes. Knockout lines of GELP31, which is highly expressed in sporophytes, were constructed. Gelp31 spores contained amorphous oil bodies and germinated late, suggesting (a) role(s) of GELP31 in lipid metabolism in spore development or germination. Future knockout studies of other candidate GELP genes will further elucidate the relationship between expansion of the family and the ability to withstand the harsh land environment.