Dormant Spores Research Articles

Altruistic feeding and cell-cell signaling during bacterial differentiation actively enhance phenotypic heterogeneity.

Starvation triggers bacterial spore formation, a committed differentiation program that transforms a vegetative cell into a dormant spore. Cells in a population enter sporulation nonuniformly to secure against the possibility that favorable growth conditions, which put sporulation-committed cells at a disadvantage, may resume. This heterogeneous behavior is initiated by a passive mechanism: stochastic activation of a master transcriptional regulator. Here, we identify a cell-cell communication pathway containing the proteins ShfA (YabQ) and ShfP (YvnB) that actively promotes phenotypic heterogeneity, wherein Bacillus subtilis cells that start sporulating early use a calcineurin-like phosphoesterase to release glycerol, which simultaneously acts as a signaling molecule and a nutrient to delay nonsporulating cells from entering sporulation. This produced a more diverse population that was better poised to exploit a sudden influx of nutrients compared to those generating heterogeneity via stochastic gene expression alone. Although conflict systems are prevalent among microbes, genetically encoded cooperative behavior in unicellular organisms can evidently also boost inclusive fitness.

Complex sporulation-specific expression of transcription termination factor Rho highlights its involvement in Bacillus subtilis cell differentiation

Termination factor Rho, responsible for the main factor-dependent pathway of transcription termination and the major inhibitor of antisense transcription, is an emerging regulator of various physiological processes in microorganisms. In Gram-positive bacterium Bacillus subtilis, Rho is involved in the control of cell adaptation to starvation and, in particular, in the control of sporulation, a complex differentiation program leading to formation of a highly resistant dormant spore. While the initiation of sporulation requires a decrease in Rho protein levels during the transition to stationary phase, the mechanisms regulating the expression of rho gene throughout the cell cycle remain largely unknown. Here we show that a drop in the activity of the vegetative SigA-dependent rho promoter causes the inhibition of rho expression in stationary phase. However, after the initiation of sporulation, rho gene is specifically reactivated in two compartments of the sporulating cell using distinct mechanisms. In the mother cell, rho expression occurs by read-through transcription initiated at the SigH-dependent promoter of the distal spo0F gene. In the forespore, rho gene is transcribed from the intrinsic promoter recognized by the alternative sigma factor SigF. These regulatory elements ensure the activity of Rho during sporulation, which appears important for the proper formation of spores. We provide experimental evidence that disruption of the spatiotemporal expression of rho during sporulation affects the resistance properties of spores, their morphology, and the ability to return to vegetative growth under favorable growth conditions.

Multimodal Molecular Imaging Reveals a Novel Membrane Component in Sporangia of the Rare Actinomycete Actinoplanes missouriensis.

The bacterium Actinoplanes missouriensis belongs to the genus Actinoplanes, a prolific source of useful natural products. This microbe forms globular structures called sporangia, which contain many dormant spores. Recent studies using transmission electron microscopy have shown that the A. missouriensis sporangium membrane has an unprecedented three-layer structure, but its molecular components remain unclear. Here, we present multimodal (spontaneous Raman scattering, coherent anti-Stokes Raman scattering, second harmonic generation, sum frequency generation, and third-order sum frequency generation) label-free molecular imaging of intact A. missouriensis sporangia. Spontaneous Raman imaging assisted with multivariate curve resolution-alternating least-squares analysis revealed a novel component in the sporangium membrane that exhibits unique Raman bands at 1550 and 1615 cm-1 in addition to those characteristic of lipids. A plausible candidate for this component is an unsaturated carbonyl compound with an aliphatic moiety derived from fatty acid. Furthermore, second harmonic generation imaging revealed that a layer(s) of the sporangium membrane containing this unknown component has an ordered, noncentrosymmetric structure like fibrillar proteins and amylopectin. Our results suggest that the sporangium membrane is a new type of biological membrane, not only in terms of architecture but also in terms of components. We demonstrate that multimodal molecular imaging with Raman scattering as the core technology will provide a promising platform for interrogating the chemical components, whether known or unknown, of diverse biological structures produced by microbes.

Roles for the canonical polarity machinery in the de novo establishment of polarity in budding yeast spores.

Budding in the yeast Saccharomyces cerevisiae occurs at a single site pre-determined by cortical landmarks deposited during prior budding. During mating between haploid cells in the lab, external pheromone cues override the cortical landmarks to drive polarization and cell fusion. By contrast, in haploid gametes (called spores) produced by meiosis, a pre-determined polarity site drives initial polarized morphogenesis independent of mating partner location. Spore membranes are made de novo so existing cortical landmarks were unknown, as were the mechanisms by which the spore polarity site is made and how it works. We find that the landmark canonically required for distal budding, Bud8, stably marks the spore polarity site along with Bud5, a GEF for the GTPase Rsr1 that canonically links cortical landmarks to the conserved Cdc42 polarity machinery. Cdc42 and other GTPase regulators arrive at the site during its biogenesis, after spore membrane closure but apparently at the site where membrane synthesis began, and then these factors leave, pointing to a discrete "functionalization" step. Filamentous actin may be required for initial establishment of the site, but thereafter Bud8 accumulates independent of actin filaments. These results suggest a distinct polarization mechanism that may provide insights into gamete polarization in other organisms.

Cryo-ET reveals the in situ architecture of the polar tube invasion apparatus from microsporidian parasites.

Microsporidia are divergent fungal pathogens that employ a harpoon-like apparatus called the polar tube (PT) to invade host cells. The PT architecture and its association with neighboring organelles remain poorly understood. Here, we use cryo-electron tomography to investigate the structural cell biology of the PT in dormant spores from the human-infecting microsporidian species, Encephalitozoon intestinalis . Segmentation and subtomogram averaging of the PT reveal at least four layers: two protein-based layers surrounded by a membrane, and filled with a dense core. Regularly spaced protein filaments form the structural skeleton of the PT. Combining cryo-electron tomography with cellular modeling, we propose a model for the 3-dimensional organization of the polaroplast, an organelle that is continuous with the membrane layer that envelops the PT. Our results reveal the ultrastructure of the microsporidian invasion apparatus in situ , laying the foundation for understanding infection mechanisms.

Cytoplasmic fluidization contributes to breaking spore dormancy in fission yeast

The cytoplasm is a complex, crowded environment that influences myriad cellular processes including protein folding and metabolic reactions. Recent studies have suggested that changes in the biophysical properties of the cytoplasm play a key role in cellular homeostasis and adaptation. However, it still remains unclear how cells control their cytoplasmic properties in response to environmental cues. Here, we used fission yeast spores as a model system of dormant cells to elucidate the mechanisms underlying regulation of the cytoplasmic properties. By tracking fluorescent tracer particles, we found that particle mobility decreased in spores compared to vegetative cells and rapidly increased at the onset of dormancy breaking upon glucose addition. This cytoplasmic fluidization depended on glucose-sensing via the cyclic adenosine monophosphate-protein kinase A pathway. PKA activation led to trehalose degradation through trehalase Ntp1, thereby increasing particle mobility as the amount of trehalose decreased. In contrast, the rapid cytoplasmic fluidization did not require de novo protein synthesis, cytoskeletal dynamics, or cell volume increase. Furthermore, the measurement of diffusion coefficients with tracer particles of different sizes suggests that the spore cytoplasm impedes the movement of larger protein complexes (40 to 150 nm) such as ribosomes, while allowing free diffusion of smaller molecules (~3 nm) such as second messengers and signaling proteins. Our experiments have thus uncovered a series of signaling events that enable cells to quickly fluidize the cytoplasm at the onset of dormancy breaking.

The inhibitory effects of monolauroyl-galactosylglycerol against spores of Bacillus cereus and its potential application in dairy

Bacillus cereus is a typical spore-forming strain that can transform into dormant spores to resist unfavorable growth conditions. Among the microorganisms that cause spoilage of dairy products, B. cereus accounts for a large proportion. In this study, we evaluated the inhibitory activity of monolauroyl-galactosylglycerol (MLGG) on B. cereus spores and explored its inhibitory mechanism. The minimum inhibitory spore concentration (MISC) and the minimum sporicidal concentration (MSC) of MLGG against B. cereus spores were 0.313 mg/mL and 2.5 mg/mL, respectively. Scanning electron microscopy was used to observe the changes in spore morphology after MLGG treatment. It was found that low concentrations of MLGG caused irregular spore shape, with a few spores slightly ruptured and high concentrations of MLGG increased the degree of spore rupture, resulting in significant leakage of spore contents. By measuring the effect of MLGG on spore structure, it could be determined that MLGG damaged the coat of the spores and weakened the resistance of the spores, but did not affect the activity of cortical lyases. Therefore, spores could not resume growth even with the addition of exogenous lytic enzymes. After propidium iodide (PI) staining of the spores, flow cytometry analysis revealed that the positive area gradually increased, indicating that the permeability of the inner membrane of the spores was disrupted, leading to dipicolinic acid (DPA) release. The amount of DPA release was proportional to the MLGG concentration, and the growth rate of the spores decreased under hypertonic conditions. These findings indicated that MLGG inactivated spores primarily by damaging the inner membrane of the spores.

Orthotic Thermoplastic Demonstrates a Similar Contamination Potential with Bacillus Bacteria Recovered from Thermoplastic Radiation Therapy Patient Masks

Thermoplastics used to construct a variety of patient medical devices can become contaminated by harmful bacteria. We investigated whether two different Bacillus species recovered from patient radiation therapy thermoplastic masks could similarly contaminate thermoplastic material used to construct patient orthoses (splints). Bacillus bacteria form dormant spores, which have been shown to enhance its attachment to thermoplastics. Bacterial attachment and recovery were examined using an orthotic thermoplastic with an anti-stick coating being compared to uncoated material used in radiation therapy applications. Triplicate sample squares were seeded with a saline suspension of either B. cereus (MAB03F) or B. megaterium (DAB01F) containing a similar number of spores. Squares were subsequently sampled at 1 h, 1 week, 2 weeks, 4 weeks, and 8 weeks. The number of recovered bacteria was counted. Differences in material hydrophobicity were determined by water contact angle analysis. Both Bacillus species attached to each material within 1 h, and their spores were recovered at 8 weeks. However, a decreasing trend in adhesion, over time, was noted to the coated material with an opposite increasing trend in the uncoated material. Decreased Bacillus species spore adhesion to coated material with a lower hydrophobicity suggests a greater potential for spore transfer to patients wearing contaminated orthoses.

A Dual-Mode Fluorescent Nanoprobe for the Detection and Visual Screening of Pathogenic Bacterial Spores.

Bacterial vegetative cells turn into metabolically dormant spores in certain environmental situations. Once suitable conditions trigger the germination of spores belonging to the pathogenic bacterial category, public safety and environmental hygiene will be threatened, and lives will even be endangered when encountering fatal ones. Instant identification of pathogenic bacterial spores remains a challenging task, since most current approaches belonging to complicated biological methods unsuitable for onsite sensing or emerging alternative chemical techniques are still inseparable from professional instruments. Here we developed a polychromatic fluorescent nanoprobe for ratiometric detection and visual inspection of the pathogenic bacterial spore biomarker, dipicolinic acid (DPA), realizing rapidly accurate screening of pathogenic bacterial spores such as Bacillus anthracis spores. The nanoprobe is made of aminoclay-coated silicon nanoparticles and functionalized with europium ions, exhibiting selective and sensitive response toward DPA and Bacillus subtilis spores (simulants for Bacillus anthracis spores) with excellent linearity. The proposed sensing strategy allowing spore determination of as few as 0.3 × 105 CFU/mL within 10 s was further applied to real environmental sample detection with good accuracy and reliability. Visual quantitative determination can be achieved by analyzing the RGB values of the corresponding test solution color via a color recognition APP on a smartphone. Different test samples can be photographed at the same time, hence the efficient accomplishment of examining bulk samples within minutes. Potentially employed in various on-site sensing occasions, this strategy may develop into a powerful means for distinguishing hazardous pathogens to facilitate timely and proper actions of dealing with multifarious security issues.

Determination of cyclic adenosine phosphate and protein content in dormant chlamydospore and nondormant chlamydospore of Arthrobotrys flagrans.

Arthrobotrys flagrans, a nematode-eating fungus, is an effective component of animal parasitic nematode biocontrol agents. In the dried formulation, the majority of spores are in an endogenous dormant state. This study focuses on dormant chlamydospore and nondormant chlamydospore of A. flagrans to investigate the differences in cyclic adenosine monophosphate (cAMP) and protein content between the two types of spores. cAMP and soluble proteins were extracted from the nondormant chlamydospore and dormant chlamydospore of two isolates of A. flagrans. The cAMP Direct Immunoassay Kit and Bradford protein concentration assay kit (Coomassie brilliant blue method) were used to detect the cAMP and protein content in two types of spores. Results showed that the content of cAMP in dormant spores of both isolates was significantly higher than that in nondormant spores (p < 0.05). The protein content of dormant spores in DH055 bacteria was significantly higher than that of nondormant spores (p < 0.05). In addition, the protein content of dormant spores of the SDH035 strain was slightly higher than that of nondormant spores, but the difference was not significant (p > 0.05). The results obtained in this study provide evidence for the biochemical mechanism of chlamydospore dormancy or the germination of the nematophagous fungus A. flagrans.

Altruistic feeding and cell-cell signaling during bacterial differentiation actively enhance phenotypic heterogeneity.

Starvation triggers bacterial spore formation, a committed differentiation program that transforms a vegetative cell into a dormant spore. Cells in a population enter sporulation non-uniformly to secure against the possibility that favorable growth conditions, which puts sporulation-committed cells at a disadvantage, may resume. This heterogeneous behavior is initiated by a passive mechanism: stochastic activation of a master transcriptional regulator. Here, we identify a cell-cell communication pathway that actively promotes phenotypic heterogeneity, wherein Bacillus subtilis cells that start sporulating early utilize a calcineurin-like phosphoesterase to release glycerol, which simultaneously acts as a signaling molecule and a nutrient to delay non-sporulating cells from entering sporulation. This produced a more diverse population that was better poised to exploit a sudden influx of nutrients compared to those generating heterogeneity via stochastic gene expression alone. Although conflict systems are prevalent among microbes, genetically encoded cooperative behavior in unicellular organisms can evidently also boost inclusive fitness.

Hyperspectral imaging and deep learning for detection and quantification of germination in Bacillus cereus spores

SummaryGermination of Bacillus cereus spores followed by growth and replication of the vegetative cells in food can result in food poisoning and therefore significant economic and health impacts. This study explores a novel approach to detect and differentiate spores and germinated B. cereus cells using hyperspectral imaging (HSI) in combination with machine learning using three different germination triggers. HSI could successfully differentiate between dormant spores, germinated cells and structural controls (non‐spores). The spectral data in the visible‐near‐infrared range are sensitive to unique structural and chemical characteristics specific to spores, setting them apart from their vegetative counterparts and non‐biological controls. This non‐destructive and robust approach shows significant potential for detection and assessment of the physiological state (dormant or germinated). Therefore, HSI is a potential method for the detection of germination in B. cereus spores and merits further research and validation.

Energetics of the microsporidian polar tube invasion machinery

Microsporidia are eukaryotic, obligate intracellular parasites that infect a wide range of hosts, leading to health and economic burdens worldwide. Microsporidia use an unusual invasion organelle called the polar tube (PT), which is ejected from a dormant spore at ultra-fast speeds, to infect host cells. The mechanics of PT ejection are impressive. Anncaliia algerae microsporidia spores (3–4 μm in size) shoot out a 100-nm-wide PT at a speed of 300 μm/s, creating a shear rate of 3000 s-1. The infectious cargo, which contains two nuclei, is shot through this narrow tube for a distance of ∼60–140 μm (Jaroenlak et al, 2020) and into the host cell. Considering the large hydraulic resistance in an extremely thin tube and the low-Reynolds-number nature of the process, it is not known how microsporidia can achieve this ultrafast event. In this study, we use Serial Block-Face Scanning Electron Microscopy to capture 3-dimensional snapshots of A. algerae spores in different states of the PT ejection process. Grounded in these data, we propose a theoretical framework starting with a systematic exploration of possible topological connectivity amongst organelles, and assess the energy requirements of the resulting models. We perform PT firing experiments in media of varying viscosity, and use the results to rank our proposed hypotheses based on their predicted energy requirement. We also present a possible mechanism for cargo translocation, and quantitatively compare our predictions to experimental observations. Our study provides a comprehensive biophysical analysis of the energy dissipation of microsporidian infection process and demonstrates the extreme limits of cellular hydraulics.

Energetics of the microsporidian polar tube invasion machinery.

Microsporidia are eukaryotic, obligate intracellular parasites that infect a wide range of hosts, leading to health and economic burdens worldwide. Microsporidia use an unusual invasion organelle called the polar tube (PT), which is ejected from a dormant spore at ultra-fast speeds, to infect host cells. The mechanics of PT ejection are impressive. Anncaliia algerae microsporidia spores (3-4 μm in size) shoot out a 100-nm-wide PT at a speed of 300 μm/s, creating a shear rate of 3000 s-1. The infectious cargo, which contains two nuclei, is shot through this narrow tube for a distance of ∼60-140 μm (Jaroenlak et al, 2020) and into the host cell. Considering the large hydraulic resistance in an extremely thin tube and the low-Reynolds-number nature of the process, it is not known how microsporidia can achieve this ultrafast event. In this study, we use Serial Block-Face Scanning Electron Microscopy to capture 3-dimensional snapshots of A. algerae spores in different states of the PT ejection process. Grounded in these data, we propose a theoretical framework starting with a systematic exploration of possible topological connectivity amongst organelles, and assess the energy requirements of the resulting models. We perform PT firing experiments in media of varying viscosity, and use the results to rank our proposed hypotheses based on their predicted energy requirement. We also present a possible mechanism for cargo translocation, and quantitatively compare our predictions to experimental observations. Our study provides a comprehensive biophysical analysis of the energy dissipation of microsporidian infection process and demonstrates the extreme limits of cellular hydraulics.

The pH-responsive SmrR-SmrT system modulates C. difficile antimicrobial resistance, spore formation, and toxin production.

Clostridioides difficile is an anaerobic gastrointestinal pathogen that spreads through the environment as dormant spores. To survive, replicate, and sporulate in the host intestine, C. difficile must adapt to a variety of conditions in its environment, including changes in pH, the availability of metabolites, host immune factors, and a diverse array of other species. Prior studies showed that changes in intestinal conditions, such as pH, can affect C. difficile toxin production, spore formation, and cell survival. However, little is understood about the specific genes and pathways that facilitate environmental adaptation and lead to changes in C. difficile cell outcomes. In this study, we investigated two genes, CD2505 and CD2506, that are differentially regulated by pH to determine if they impact C. difficile growth and sporulation. Using deletion mutants, we examined the effects of both genes (herein smrR and smrT) on sporulation frequency, toxin production, and antimicrobial resistance. We determined that SmrR is a repressor of smrRT that responds to pH and suppresses sporulation and toxin production through regulation of the SmrT transporter. Further, we showed that SmrT confers resistance to erythromycin and lincomycin, establishing a connection between the regulation of sporulation and antimicrobial resistance.IMPORTANCEClostridioides difficile is a mammalian pathogen that colonizes the large intestine and produces toxins that lead to severe diarrheal disease. C. difficile is a major threat to public health due to its intrinsic resistance to antimicrobials and its ability to form dormant spores that are easily spread from host to host. In this study, we examined the contribution of two genes, smrR and smrT, on sporulation, toxin production, and antimicrobial resistance. Our results indicate that SmrR represses smrT expression, while production of SmrT increases spore and toxin production, as well as resistance to antibiotics.

Fight or flee, a vital choice for Clostridioides difficile.

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

Elucidating Bacterial Spore Dynamics through Lanthanide-Enhanced Live Imaging.

Identifying and distinguishing dormant and active bacterial spores are vital for biosecurity, food safety, and space exploration. Yet, there is a lack of simple, quick, and nondestructive methods to achieve this. The common Schaeffer-Fulton method is both sample-destructive and requires significant operator involvement. In this study, we employed lanthanide-beta-diketonate complexes to directly observe both dormant and germinated single spores. Staining is instantaneous and requires minimal sample processing. The complex stains areas outside the core of dormant spores, leaving the core hollow and nonfluorescent. However, upon germination, the complex enters the core, making it brightly fluorescent. This difference was noted in five bacterial species including Bacillus, Clostridium, and Clostridioides. Various lanthanides and beta-diketonates can be mixed to form a range of spore-visualizing complexes. Due to their low toxicity, these complexes allow for live imaging of single germinating spores. We demonstrate low-cost imaging using a USB microscope as well as imaging of spores in milk matrices. This method provides a valuable tool for studying bacterial spores.

Spore germination: Two ion channels are better than one.

Germination is the process by which spores emerge from dormancy. Although spores can remain dormant for decades, the study of germination is an active field of research. In this issue of Genes & Development, Gao and colleagues (pp. 31-45) address a perplexing question: How can a dormant spore initiate germination in response to environmental cues? Three distinct complexes are involved: GerA, a germinant-gated ion channel; 5AF/FigP, a second ion channel required for amplification; and SpoVA, a channel for dipicolinic acid (DPA). DPA release is followed by rehydration of the spore core, thus allowing the resumption of metabolic activity.

Differential expression and gene correlation analyses reveal core CAZymes in Fusarium oxysporum f.sp. lycopersici exposed to mutagen and heat stress

AbstractFusarium oxysporum f.sp. lycopersici is the casual organism of tomato wilt disease which is a devastating plant pathogen responsible for annual loss of this fruit crop. An important step in fungal growth and development is germination, that is transformation of dormant spores into a growing hypha. For this, a set of special type of enzymes known as CAZymes are required for construction and remodelling of polysaccharides and penetration into the host. This necessitates to understand the underlying mechanism of hyphal growth and the crucial CAZymes involved in the process. Therefore, a comparative transcriptomic study of F. oxysporum f.sp. lycopersici using publically available RNA‐seq data under two perturbed conditions was taken up to find out the core differentially expressed genes (DEGs) followed by CAZyme annotation and their co‐expression. Among these two perturbations, 514 core DEGs were found, from which 32 CAZymes were annotated, with majority of them being glycosyl hydrolases and glycosyl transferases. Most of these CAzymes were also found to be downregulated in both the stresses. Further, among these annotated DEGs, the interaction network revealed the association of CAZymes with zinc finger transcription factors, enzymes associated with fatty acid biosynthesis, and sulphur and citrate metabolism. Hence, in the present study, the key or core CAZymes and their association with other enzymes were revealed in the impaired fungus which are otherwise crucial for growth in normal conditions.

Functional characterization of Nosema bombycis (microsporidia) trehalase 3.

Nosema bombycis, an obligate intracellular parasite, is a single-celled eukaryote known to infect various tissues of silkworms, leading to the manifestation of pebrine. Trehalase, a glycosidase responsible for catalyzing the hydrolysis of trehalose into two glucose molecules, assumes a crucial role in thermal stress tolerance, dehydration, desiccation stress, and asexual development. Despite its recognized importance in these processes, the specific role of trehalase in N. bombycis remains uncertain. This investigation focused on exploring the functions of trehalase 3 in N. bombycis (NbTre3). Immunofluorescence analysis of mature (dormant) spores indicated that NbTre3 primarily localizes to the spore membrane or spore wall, suggesting a potential involvement in spore germination. Reverse transcription-quantitative polymerase chain reaction results indicated that the transcriptional level of NbTre3 peaked at 6h post N. bombycis infection, potentially contributing to energy storage for proliferation. Throughout the life cycle of N. bombycis within the host cell, NbTre3 was detected in sporoplasm during the proliferative stage rather than the sporulation stage. RNA interference experiments revealed a substantial decrease in the relative transcriptional level of NbTre3, accompanied by a certain reduction in the relative transcriptional level of Nb16S rRNA. These outcomes suggest that NbTre3 may play a role in the proliferation of N. bombycis. The application of the His pull-down technique identified 28 proteins interacting with NbTre3, predominantly originating from the host silkworm. This finding implies that NbTre3 may participate in the metabolism of the host cell, potentially utilizing the host cell's energy resources.