Sporulating Cells Research Articles

The Bacillus subtilis germinant receptor GerA triggers premature germination in response to morphological defects during sporulation.

During sporulation in Bacillus subtilis, germinant receptors assemble in the inner membrane of the developing spore. In response to specific nutrients, these receptors trigger germination and outgrowth. In a transposon-sequencing screen, we serendipitously discovered that loss of function mutations in the gerA receptor partially suppress the phenotypes of > 25 sporulation mutants. Most of these mutants have modest defects in the assembly of the spore protective layers that are exacerbated in the presence of a functional GerA receptor. Several lines of evidence indicate that these mutants inappropriately trigger the activation of GerA during sporulation resulting in premature germination. These findings led us to discover that up to 8% of wild-type sporulating cells trigger premature germination during differentiation in a GerA-dependent manner. This phenomenon was observed in domesticated and undomesticated wild-type strains sporulating in liquid and on solid media. Our data indicate that the GerA receptor is poised on a knife's edge during spore development. We propose that this sensitized state ensures a rapid response to nutrient availability and also elicits premature germination of spores with improperly assembled protective layers resulting in the elimination of even mildly defective individuals from the population.

Use of predictive model to describe sporicidal and cell viability efficacy of betel leaf (Piper betle L.) essential oil on Aspergillus flavus and Penicillium expansum and its antifungal activity in raw apple juice

The present study describes the effect of betel leaf (Piper betle L.) essential oil (BLEO) on spore inactivation and cell viability of Aspergillus flavus and Penicillium expansum using predictive model and its antifungal activity in raw apple juice. Survival curve of the spores under minimum fungicidal concentration of BLEO was modelled by a modified Weibull model: logN=logN0−(t/δ)β. The forward and side-scatter parameters of flow cytometer suggested differences in size and complexity of spores. Propidium iodide was used as fluorescent stain to distinguish between viable and non-viable spores. Scanning electron microscopic images revealed morphological alterations of treated spores. The cell viability effect of BLEO on mycelial matrix of A. flavus and P. expansum was measured using tetrazolium salt MTT and the inhibition was fitted to dose-response curves. Mycelial biomass of a selected mould in raw apple juice treated with BLEO based microemulsion (BLEO-ME) having different concentration of BLEO was determined based on ergosterol content. BLEO-ME exhibited fungal growth inhibition in raw apple juice, which suggests potential of BLEO as antifungal agent in food system.

Inactivation of Bacterial Spores and Vegetative Bacterial Cells by Interaction with ZnO-Fe<sub>2</sub>O<sub>3</sub> Nanoparticles and UV Radiation

ZnO-Fe 2 O 3 nanoparticles (ZnO-Fe NPs) were synthesized and characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and dynamic light scattering (DLS). The generation of chemical reactive hydroxyl radicals ( • OH) was measured spectrophotometrically (UV-Vis) by monitoring of p -nitrosodimethylaniline ( p NDA) bleaching. Inactivation of E. coli and B. subtilis spores in the presence of different concentrations of ZnO-Fe NPs, under UV 365nm or visible radiation, was evaluated. We observed the best results under visible light, of which inactivation of E. coli of about 90% was accomplished in 30 minutes, while B. subtilis inactivation close to 90% was achieved in 120 minutes. These results indicate that the prepared photocatalytic systems are promising for improving water quality by reducing the viability of water-borne microorganisms, including bacterial spores.

Inactivation of Bacterial Spores and Vegetative Bacterial Cells by Interaction with ZnO-Fe<sub>2</sub>O<sub>3</sub> Nanoparticles and UV Radiation

ZnO-Fe₂O₃nanoparticles (ZnO-Fe NPs) were synthesized and characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and dynamic light scattering (DLS). The generation of chemical reactive hydroxyl radicals (^•OH) was measured spectrophotometrically (UV-Vis) by monitoring of <em>p</em>-nitrosodimethylaniline (<em>p</em>NDA) bleaching. Inactivation of <em>E. coli</em> and <em>B. subtilis</em> spores in the presence of different concentrations of ZnO-Fe NPs, under UV_365nm or visible radiation, was evaluated. We observed the best results under visible light, of which inactivation of <em>E. coli</em> of about 90% was accomplished in 30 minutes, while <em>B. subtilis </em>inactivation close to 90% was achieved in 120 minutes. These results indicate that the prepared photocatalytic systems are promising for improving water quality by reducing the viability of water-borne microorganisms, including bacterial spores.

Destruction of biological particles using non-thermal plasma.

Mechanism of inactivation of bio-particles exposed to non-thermal plasma (NTP), namely, dielectric barrier discharge (DBD), and plasma jet (PJ), has been studied using E. coli, B. subtilis spore, S. cerevisiae and bacteriophages. States of different biological components were monitored during the course of inactivation. Analysis of green fluorescent protein, GFP, introduced into E. coli. or B. subtiles spore cells proved that radicals generated by NTP penetrate into microbes, destroying the cell membrane and finally damage the genes. We have evaluated the damage of the bacteriophages. Bacteriophage λ having double stranded DNA was exposed to DBD, then DNA was purified and subjected to in vitro DNA packaging reactions. The re-packaged phages consist of the DNA from discharged phages and brand-new coat proteins were proved to be active, indicating that the damage of coat proteins is responsible for inactivation. M13 phages having single stranded DNA were also examined with the same manner. In this case, damage to the DNA was as severe as that of the coat proteins. For practical applications, DBD showed very intense sterilization ability for B. Subtilis spore with the D-value of less than 10 s. This result indicates a possibility of application of NTP for quick sterilization.

Bioconversion of glycerol to poly(HB-co-HV) copolymer in an inexpensive medium by a Bacillus megaterium strain isolated from marine sediments

A Bacillus megaterium strain, as an alternative to produce biopolymers from an industrial waste in a low cost medium, is studied. A culture medium composed by salt solution, trace elements, glycerol and urea was used. The growth of B. megaterium BBST4 and the production of PHA achieved by different concentrations of glycerol and urea, were analyzed at various C/N ratios. The effect of temperature, the incubation time and the addition of yeast extract, on the growth, PHA production and sporulation, were determined. The strain showed better growth and PHA production (30% w/w) after 48h of incubation at 30°C in a SWG medium with 3% of glycerol, 0.08% of urea, and 0.005% of yeast extract. Under these conditions no sporulating cells were observed. The obtained PHA was characterized by FTIR, NMR, DSC, SEC and GC–MS. It was determined that the biopolymer produced by B. megaterium BBST4 is a copolymer, composed of 3-hydroxybutyrate (92.2mol%) and 3-hydroxyvalerate (7.8mol%). This copolymer offer industrial and biomedical applications.

Chemical and Stress Resistances of Clostridium difficile Spores and Vegetative Cells.

Clostridium difficile is a Gram-positive, sporogenic and anaerobic bacterium that causes a potentially fatal colitis. C. difficile enters the body as dormant spores that germinate in the colon to form vegetative cells that secrete toxins and cause the symptoms of infection. During transit through the intestine, some vegetative cells transform into spores, which are more resistant to killing by environmental insults than the vegetative cells. Understanding the inherent resistance properties of the vegetative and spore forms of C. difficile is imperative for the development of methods to target and destroy the bacterium. The objective of this study was to define the chemical and environmental resistance properties of C. difficile vegetative cells and spores. We examined vegetative cell and spore tolerances of three C. difficile strains, including 630Δerm, a 012 ribotype and a derivative of a past epidemic strain; R20291, a 027 ribotype and current epidemic strain; and 5325, a clinical isolate that is a 078 ribotype. All isolates were tested for tolerance to ethanol, oxygen, hydrogen peroxide, butanol, chloroform, heat and sodium hypochlorite (household bleach). Our results indicate that 630Δerm vegetative cells (630 spo0A) are more resistant to oxidative stress than those of R20291 (R20291 spo0A) and 5325 (5325 spo0A). In addition, 5325 spo0A vegetative cells exhibited greater resistance to organic solvents. In contrast, 630Δerm spores were more sensitive than R20291 or 5325 spores to butanol. Spores from all three strains exhibited high levels of resistance to ethanol, hydrogen peroxide, chloroform and heat, although R20291 spores were more resistant to temperatures in the range of 60–75°C. Finally, household bleach served as the only chemical reagent tested that consistently reduced C. difficile vegetative cells and spores of all tested strains. These findings establish conditions that result in vegetative cell and spore elimination and illustrate the resistance of C. difficile to common decontamination methods. These results further demonstrate that the vegetative cells and spores of various C. difficile strains have different resistance properties that may impact decontamination of surfaces and hands.

Aag Hypoxanthine-DNA Glycosylase Is Synthesized in the Forespore Compartment and Involved in Counteracting the Genotoxic and Mutagenic Effects of Hypoxanthine and Alkylated Bases in DNA during Bacillus subtilis Sporulation.

In this study, evidence is presented revealing that aag, encoding a DNA glycosylase implicated in processing of hypoxanthine and alkylated DNA bases, exhibits a forespore-specific pattern of gene expression during B. subtilis sporulation. Consistent with this spatiotemporal mode of expression, Aag was found to protect the sporulating cells of this microorganism from the noxious and mutagenic effects of base deamination and alkylation.

Identifying the role of reactive oxygen species (ROSs) in Fusarium solani spores inactivation.

The inactivation mechanism of photocatalytic disinfectants on bacteria is well known. In contrast, the potential inactivation of fungal spores by visible-light induced photocatalysis has been recognized, but the inactivation mechanism is poorly understood. We hypothesize that photocatalytically generated reactive oxygen species (ROSs) are directly involved in this mechanism. To test this hypothesis, we identified the roles of ROSs in the inactivation of Fusarium solani spores. As the photocatalysts, we doped TiO2 with 3 typical dopants, forming Ag/TiO2, N/TiO2 and Er3+:YAlO3/TiO2. The Ag/TiO2 photocatalysis was dominated by H2O2, with the longest lifetime among the investigated ROSs. Ag/TiO2 photocatalysis yielded almost 100 % inactivation efficiency and preserved the cell-wall shape of the spores, thus minimizing the biomolecule leakage. Er3+:YAlO3/TiO2 was dominated by h+ ROSs, yielding an inactivation efficiency of 91 %; however, the severe leakage released large numbers of molecular bio-products. Severe damage to the cell walls by the h+ species was confirmed in micrograph observations. Subsequent to cell wall breakage, the Er3+:YAlO3/TiO2 nanoparticles entered the spore cells and directly oxidized the intracellular material. The N/TiO2 photocatalysis, with •O2− dominated ROSs, delivered intermediate performance. In conclusion, photocatalysts that generate H2O2-dominated ROSs are most preferred for spore inactivation.

Nonthermal plasma treatment of Aspergillus spp. spores on hazelnuts in an atmospheric pressure fluidized bed plasma system: Impact of process parameters and surveillance of the residual viability of spores

In this study, the impact of fluidized bed reactor diameters and plasma forming gases on inactivation efficiency of the Atmospheric Pressure Fluidized Bed Plasma (APFBP) system for aflatoxigenic spores of Aspergillus flavus and Aspergillus parasiticus on hazelnuts were investigated. Hazelnuts were artificially contaminated with A. flavus and A. parasiticus and then treated with dry air or nitrogen plasma for up to 5 min in two different fluidizing bed reactors of APFBP system at various plasma parameters. The decontamination effect of APFBP on Aspergillus spp. spores increased with the applied reference voltage and the frequency. The killing effect of plasma on the spores decreased as the diameter of the fluidized bed reactor increased. The fungicidal effects on A. flavus (4.17 log) and A. parasiticus (4.09 log) were found for air plasma treatment after 5 min. Due to the formation of active plasma species in the presence of oxygen, the air plasma generated at APFBP system was more effective than nitrogen plasma on decontamination of Aspergillus spp. spores, as expected. The total inactivation of the natural background microbiota of hazelnuts was obtained within maximum 2 min APFBP treatment. The aflatoxigenic spores that remained on hazelnuts after plasma process were considered as damaged cells, because they could not continue growing during storage at 25 °C for 30 days. The damage caused by APFBP treatment on Aspergillus spp. spore cells was demonstrated by using scanning electron microscopy.

Isolation of insect parasitic fungi Corrdyceps spp. rich in bioactive beauvericin from Pu Mat National Park, Nghe An

Insect parasitic fungi Cordyceps sp. B6 was isolated as single spores from fungi on the host insect pupae of Lepidoptera, collected at Pu Mat National Park, Nghe An, Vietnam. Cordyceps B6 strain was identified by the method of molecular biology, in which the DNA of the strain was extracted, cleaned and used as a template to amplify the gene segment using primers for ITS1 and ITS2. ITS gene segment of the strain was sequenced and compared to sequences in the GeneBank database using BLAST (NCBI). With the similarity of 99% compared to the corresponding gene fragment of Cordyceps takaomontana, Cordyceps B6 strain was identified as C. takaomontana. C. takaomontana B6 was best activated with liquid medium CT1 containing glucose 20 g/l, yeast extract 5 g / l, peptone 10 g/l, KH2PO4 1 g/l, 0.5 g MgSO4/l . In this medium, the density could reach 5 x 106 spores cells/ml after 7 days of culture. When surface fermentation performed in appropriate conditions (25 °C, pH= 7, light intensity 200 lux, humidity 80%), the fresh weight of 18.72 g/vial of spore cells and 2.09 mg of active beauvericin per gram of freeze-dry weight were obtained after 45 days of culture. With a high concentration of active beauvericin, C. takaomontana B6 could be considered as a potential source for development of functional food and raw materials for the pharmaceutical industry.

Ultrastructure Variability of the Exosporium Layer of Clostridium difficile Spores from Sporulating Cultures and Biofilms.

The anaerobic sporeformer Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea in developed and developing countries. The metabolically dormant spore form is considered the morphotype responsible for transmission, infection, and persistence, and the outermost exosporium layer is likely to play a major role in spore-host interactions during recurrent infections, contributing to the persistence of the spore in the host. A recent study (M. Pizarro-Guajardo, P. Calderón-Romero, P. Castro-Córdova, P. Mora-Uribe, and D. Paredes-Sabja, Appl Environ Microbiol 82:2202-2209, 2016, http://dx.doi.org/10.1128/AEM.03410-15) demonstrated by transmission electron microscopy the presence of two ultrastructural morphotypes of the exosporium layer in spores formed from the same sporulating culture. However, whether these distinct morphotypes appeared due to purification techniques and whether they appeared during biofilm development remain unclear. In this communication, we demonstrate through transmission electron microscopy that these two exosporium morphotypes are formed under sporulation conditions and are also present in spores formed during biofilm development. In summary, this work provides definitive evidence that in a population of sporulating cells, spores with a thick outermost exosporium layer and spores with a thin outermost exosporium layer are formed. Clostridium difficile spores are recognized as the morphotype of persistence and transmission of C. difficile infections. Spores of C. difficile are intrinsically resistant to all known antibiotic therapies. Development of spore-based removal strategies requires a detailed knowledge of the spore surface for proper antigen selection. In this context, in this work we provide definitive evidence that two types of spores, those with a thick outermost exosporium layer and those with a thin outermost exosporium layer, are formed in the same C. difficile sporulating culture or during biofilm development.

Beyond animals and plants: dynamic maternal effects in the fungus Neurospora crassa.

Maternal effects are widely documented in animals and plants, but not in fungi or other eukaryotes. A principal cause of maternal effects is asymmetrical parental investment in a zygote, creating greater maternal vs. paternal influence on offspring phenotypes. Asymmetrical investments are not limited to animals and plants, but are also prevalent in fungi and groups including apicomplexans, dinoflagellates and red algae. Evidence suggesting maternal effects among fungi is sparse and anecdotal. In an experiment designed to test for maternal effects across sexual reproduction in the model fungus Neurospora crassa, we measured offspring phenotypes from crosses of all possible pairs of 22 individuals. Crosses encompassed reciprocals of 11 mating-type 'A' and 11 mating-type 'a' wild strains. After controlling for the genetic and geographic distances between strains in any individual cross, we found strong evidence for maternal control of perithecia (sporocarp) production, as well as maternal effects on spore numbers and spore germination. However, both parents exert equal influence on the percentage of spores that are pigmented and size of pigmented spores. We propose a model linking the stage-specific presence or absence of maternal effects to cellular developmental processes: effects appear to be mediated primarily through the maternal cytoplasm, and, after spore cell walls form, maternal influence on spore development is limited. Maternal effects in fungi, thus far largely ignored, are likely to shape species' evolution and ecologies. Moreover, the association of anisogamy and maternal effects in a fungus suggests maternal effects may also influence the biology of other anisogamous eukaryotes.

Complex polar machinery required for proper chromosome segregation in vegetative and sporulating cells of Bacillus subtilis.

SummaryChromosome segregation is an essential process of cell multiplication. In prokaryotes, segregation starts with the newly replicated sister origins of replication, oriCs, which move apart to defined positions in the cell. We have developed a genetic screen to identify mutants defective in placement of oriC during spore development in the Gram‐positive bacterium Bacillus subtilis. In addition to the previously identified proteins Soj and DivIVA, our screen identified several new factors involved in polar recruitment of oriC: a reported regulator of competence ComN, and the regulators of division site selection MinD and MinJ. Previous work implicated Soj as an important regulator of oriC positioning in the cell. Our results suggest a model in which the DivIVA‐interacting proteins ComN and MinJ recruit MinD to the cell pole, and that these proteins work upstream of Soj to enable oriC placement. We show that these proteins form a polar complex, which acts in parallel with but distinct from the sporulation‐specific RacA pathway of oriC placement, and also functions during vegetative growth. Our study further shows that MinD has two distinct cell cycle roles, in cell division and chromosome segregation, and highlights that cell probably use multiple parallel mechanisms to ensure accurate chromosome segregation.

Two distinct pathways lead Bacillus thuringiensis to commit to sporulation in biofilm

The spore-forming bacterium Bacillus thuringiensis is an efficient biofilm producer, responsible for persistent contamination of industrial food processing systems. B. thuringiensis biofilms are highly heterogeneous bacterial structures in which three distinct cell types controlled by quorum sensing regulators were identified: PlcR-controlled virulent cells, NprR-dependent necrotrophic cells and cells committed to sporulation, a differentiation process controlled by Rap phosphatases and Spo0A-P. Interestingly, a cell lineage study revealed that, in LB medium or in insect larvae, only necrotrophic cells became spores. Here we analyzed cellular differentiation undertaken by cells growing in biofilm in a medium optimized for sporulation. No virulent cells were identified; surprisingly, two distinct routes could lead to differentiation as a spore in this growth condition: the NprR-dependent route, followed by the majority of cells, and the newly identified NprR-independent route, which is followed by 20% of sporulating cells.

A pilot study to assess bacterial and toxin reduction in patients with Clostridium difficile infection given fidaxomicin or vancomycin.

BackgroundTo assess the effect of fidaxomicin and vancomycin on Clostridium difficile toxins and correlation with clinical and microbiologic outcomes.MethodsHospitalized patients with C. difficile infection were randomly assigned a 10-day course of fidaxomicin or vancomycin. Stool samples collected at baseline (day 0), mid-therapy (days 3–5), end of therapy (days 10–13) and follow-up (days 19–38) were assessed for quantity of toxins A and B as well as spore and vegetative cells counts. Correlation of toxins concentrations with microbiologic and clinical findings were evaluated.ResultsAmong 34 patients 12 had detectable toxin concentrations at baseline seven were randomized to fidaxomicin and five to vancomycin. Overall both fidaxomicin and vancomycin resulted in drop of both toxins concentrations by midpoint of therapy. The drop in toxin A concentrations was maintained up to the follow-up period with fidaxomicin but not with vancomycin even in patients who developed recurrence. Patients who developed recurrence in the fidaxomicin group had lower concentrations of toxin B versus the recurrence patient of vancomycin group. Presence of vegetative cells and spores was significantly linked with high toxin A (P = 0.003 and <0.001 respectively) and toxin B (P = 0.007 and <0.001 respectively) concentrations across time points. Toxin B concentrations but not A significantly correlated with stool consistency (P < 0.001) and frequency (P = 0.05).ConclusionsFidaxomicin was associated with sustained reduction of both toxins up to 30 days post therapy versus vancomycin. Multiple clinical or microbiologic observations were correlated with toxin A or B concentrations.

Probing phenotypic growth in expanding Bacillus subtilis biofilms.

We develop an optical imaging technique for spatially and temporally tracking biofilm growth and the distribution of the main phenotypes of a Bacillus subtilis strain with a triple-fluorescent reporter for motility, matrix production, and sporulation. We develop a calibration procedure for determining the biofilm thickness from the transmission images, which is based on Beer-Lambert's law and involves cross-sectioning of biofilms. To obtain the phenotype distribution, we assume a linear relationship between the number of cells and their fluorescence and determine the best combination of calibration coefficients that matches the total number of cells for all three phenotypes and with the total number of cells from the transmission images. Based on this analysis, we resolve the composition of the biofilm in terms of motile, matrix-producing, sporulating cells and low-fluorescent materials which includes matrix and cells that are dead or have low fluorescent gene expression. We take advantage of the circular growth to make kymograph plots of all three phenotypes and the dominant phenotype in terms of radial distance and time. To visualize the nonlocal character of biofilm growth, we also make kymographs using the local colonization time. Our technique is suitable for real-time, noninvasive, quantitative studies of the growth and phenotype distribution of biofilms which are either exposed to different conditions such as biocides, nutrient depletion, dehydration, or waste accumulation.

The RecA-Dependent SOS Response Is Active and Required for Processing of DNA Damage during Bacillus subtilis Sporulation.

The expression of and role played by RecA in protecting sporulating cells of Bacillus subtilis from DNA damage has been determined. Results showed that the DNA-alkylating agent Mitomycin-C (M-C) activated expression of a PrecA-gfpmut3a fusion in both sporulating cells’ mother cell and forespore compartments. The expression levels of a recA-lacZ fusion were significantly lower in sporulating than in growing cells. However, M-C induced levels of ß-galactosidase from a recA-lacZ fusion ~6- and 3-fold in the mother cell and forespore compartments of B. subtilis sporangia, respectively. Disruption of recA slowed sporulation and sensitized sporulating cells to M-C and UV-C radiation, and the M-C and UV-C sensitivity of sporangia lacking the transcriptional repair-coupling factor Mfd was significantly increased by loss of RecA. We postulate that when DNA damage is encountered during sporulation, RecA activates the SOS response thus providing sporangia with the repair machinery to process DNA lesions that may compromise the spatio-temporal expression of genes that are essential for efficient spore formation.

Characterization of SCO4439, a D-alanyl-D-alanine carboxypeptidase involved in spore cell wall maturation, resistance, and germination in Streptomyces coelicolor.

This work contributes to the understanding of cell wall modifications during sporulation and germination in Streptomyces by assessing the biological function and biochemical properties of SCO4439, a D-alanyl-D-alanine carboxypeptidase (DD-CPase) constitutively expressed during development. SCO4439 harbors a DD-CPase domain and a putative transcriptional regulator domain, separated by a putative transmembrane region. The recombinant protein shows that DD-CPase activity is inhibited by penicillin G. The spores of the SCO4439::Tn5062 mutant are affected in their resistance to heat and acid and showed a dramatic increase in swelling during germination. The mycelium of the SCO4439::Tn5062 mutant is more sensitive to glycopeptide antibiotics (vancomycin and teicoplanin). The DD-CPase domain and the hydrophobic transmembrane region are highly conserved in Streptomyces, and both are essential for complementing the wild type phenotypes in the mutant. A model for the biological mechanism behind the observed phenotypes is proposed, in which SCO4439 DD-CPase releases D-Ala from peptidoglycan (PG) precursors, thereby reducing the substrate pool for PG crosslinking (transpeptidation). PG crosslinking regulates spore physical resistance and germination, and modulates mycelium resistance to glycopeptides. This study is the first demonstration of the role of a DD-CPase in the maturation of the spore cell wall.

PpASCL, the Physcomitrella patens Anther-Specific Chalcone Synthase-Like Enzyme Implicated in Sporopollenin Biosynthesis, Is Needed for Integrity of the Moss Spore Wall and Spore Viability.

Sporopollenin is the main constituent of the exine layer of spore and pollen walls. The anther-specific chalcone synthase-like (ASCL) enzyme of Physcomitrella patens, PpASCL, has previously been implicated in the biosynthesis of sporopollenin, the main constituent of exine and perine, the two outermost layers of the moss spore cell wall. We made targeted knockouts of the corresponding gene, PpASCL, and phenotypically characterized ascl sporophytes and spores at different developmental stages. Ascl plants developed normally until late in sporophytic development, when the spores produced were structurally aberrant and inviable. The development of the ascl spore cell wall appeared to be arrested early in microspore development, resulting in small, collapsed spores with altered surface morphology. The typical stratification of the spore cell wall was absent with only an abnormal perine recognisable above an amorphous layer possibly representing remnants of compromised intine and/or exine. Equivalent resistance of the spore walls of ascl mutants and the control strain to acetolysis suggests the presence of chemically inert, defective sporopollenin in the mutants. Anatomical abnormalities of late-stage ascl sporophytes include a persistent large columella and an air space incompletely filled with spores. Our results indicate that the evolutionarily conserved PpASCL gene is needed for proper construction of the spore wall and for normal maturation and viability of moss spores.