Process Of Spore Germination Research Articles

Insights into the differential molecular response of non-germinated and germinated spores of Ameson portunus in vitro by comparative transcriptome analysis

Ameson portunus, the recently discovered causative agent of “toothpaste disease” of pond-cultured swimming crabs in China has caused enormous economic losses in aquaculture. Understanding the process of spore germination is helpful to elucidate the molecular mechanism of its invasion of host cells. Here, we obtained mature and germinating spores by isolation and purification and in vitro stimulation, respectively. Then, non-germinated and germinated spores were subjected to the comparative transcriptomic analysis to disclose differential molecular responses of these two stages. The highest germination rate, i.e., 71.45 %, was achieved in 0.01 mol/L KOH germination solution. There were 9,609 significantly differentially expressed genes (DEGs), with 685 up-regulated and 8,924 down-regulated DEGs. The up-regulated genes were significantly enriched in ribosome pathway, and the down-regulated genes were significantly enriched in various metabolic pathways, including carbohydrate metabolism, amino acid metabolism and other metabolism. The results suggested that spores require various carbohydrates and amino acids as energy to support their life activities during germination and synthesize large amounts of ribosomal proteins to provide sites for DNA replication, transcription, translation and protein synthesis of the spores of A. portunus within the host cells. Functional genes related to spore germination, such as protein phosphatase CheZ and aquaporin, were also analyzed. The analysis of transcriptome data and identification of functional genes will help to understand the process of spore germination and invasion.

Fungal spore swelling and germination are restricted by the macrophage phagolysosome

Many species of medically important fungi are prolific in the formation of asexual spores. Spores undergo a process of active swelling and cell wall remodelling before a germ tube is formed and filamentous growth ensues. Highly elongated germ tubes are known to be difficult to phagocytose and pose particular challenges for immune phagocytes. However, the significance of the earliest stages of spore germination during immune cell interactions has not been investigated and yet this is likely to be important for defence against sporogenous fungal pathogens. We show here that macrophages restrict the early phases of the spore germination process of Aspergillus fumigatus and Mucor circinelloides including the initial phase of spore swelling, spore germination and early polarised growth. Macrophages are therefore adept at retarding germination as well as subsequent vegetative growth which is likely to be critical for immune surveillance and protection against sporulating fungi.

Phosphorylation-mediated regulation of the Bacillus anthracis phosphoglycerate mutase by the Ser/Thr protein kinase PrkC

Bacillus anthracis Ser/Thr protein kinase PrkC is necessary for phenotypic memory and spore germination, and the loss of PrkC-dependent phosphorylation events affect the spore development. During sporulation, Bacillus sp. can store 3-Phosphoglycerate (3-PGA) that will be required at the onset of germination when ATP will be necessary. The Phosphoglycerate mutase (Pgm) catalyzes the isomerization of 2-PGA and 3-PGA and is important for spore germination as a key metabolic enzyme that maintains 3-PGA pool at later events. Therefore, regulation of Pgm is important for an efficient spore germination process and metabolic switching. While the increased expression of Pgm in B. anthracis decreases spore germination efficiency, it remains unexplored if PrkC could directly influence Pgm activity. Here, we report the phosphorylation and regulation of Pgm by PrkC and its impact on Pgm stability and catalytic activity. Mass spectrometry revealed Pgm phosphorylation on seven threonine residues. In silico mutational analysis highlighted the role of Thr459 residue towards metal and substrate binding. Altogether, we demonstrated that PrkC-mediated Pgm phosphorylation negatively regulates its activity that is essential to maintain Pgm in its apo-like isoform before germination. This study advances the role of Pgm regulation that represents an important switch for B. anthracis resumption of metabolism and spore germination.

Ger1 is a secreted aspartic acid protease essential for spore germination in Ustilago maydis.

Ustilago maydis expresses a number of proteases during its pathogenic lifecycle. Some of the proteases including both intracellular and extracellular ones have previously been shown to influence the virulence of the pathogen. However, any role of secreted proteases in the sporulation process of U. maydis have not been explored earlier. In this study we have investigated the biological function of one such secreted protease, Ger1 belonging to aspartic protease A1 family. An assessment of the real time expression of ger1 revealed an infection specific expression of the protein especially during late phases of infection. We also evaluated any contribution of the protein in the pathogenicity of the fungus. Our data revealed an involvement of Ger1 in the sporulation and spore germination processes of U. maydis. Ger1 also showed positive influence on the pathogenicity of the fungus and accordingly the ger1 deletion mutant exhibited reduced pathogenicity. The study also demonstrated the protease activity associated with Ger1 to be essential for its biological function. Fluorescence microscopy of maize plants infected with U. maydis cells expressing Ger1-mcherry-HA also revealed that Ger1 is efficiently secreted within maize apoplast.

A Short Review of Anti-Rust Fungi Peptides: Diversity and Bioassays

Pucciniales are fungal pathogens of plants that cause devastating rust diseases in agriculture. Chemically-synthesized pesticides help farmers to control rust epidemics, but governing bodies aim at limiting their use over the next decade. Defense peptides with antimicrobial activities may help to innovate a next generation of phytosanitary products for sustainable crop protection. This review comprehensively inventories the proteins or peptides exhibiting a biochemically-demonstrated antifungal activity toward Pucciniales (i.e., anti-rust proteins or peptides; hereafter ‘ARPs’), and also analyses the bioassays used to characterize them. In total, the review scrutinizes sixteen publications, which collectively report 35 ARPs. These studies used either in vitro or in planta bioassays, or a combination of both, to characterize ARPs; mostly by evaluating their ability to inhibit the spore germination process in vitro or to inhibit fungal growth and rust disease development in planta. Also, the manuscript shows that almost no mode of action against rust fungi was elucidated, although some might be inferred from studies performed on other fungi. This short review may serve as a knowledge and methodological basis to inform future studies addressing ARPs.

Bacillus subtilis spores in probiotic feed quantified via bacterial metabolite using planar chromatography

Interest in probiotics in animal production has increased due to the European ban on antibiotic growth promoters in 2006. Bacillus subtilis DSM 29784 (B. s. 29784) is one such probiotic feed additive used in poultry. Cell counting has been the most common tool for feed analysis, besides flow cytometry and quantitative polymerase chain reaction. However, quantification of the active probiotic in feed is challenging, since results are influenced by cultivation conditions, viable but non-culturable bacteria and the high contents of feed ingredients. This study presents the first quantitative analysis of a metabolite generated by B. s. 29784 spores in feed to draw conclusions on the amount of active dried spores in the feed. Thus, it is the first quantification of active probiotic bacteria at the trace level in feed based on metabolite production but not cell counting. To generate the calibration standards, solutions with different amounts of dried B. s. 29784 spores were cultured under the same conditions as the feed sample, ensuring independence from growth performance. Upstream cultivation, metabolite extraction and high-performance thin-layer chromatography analysis were proven to be highly reliable and reproducible. The repeatability of the method (RSD 1.9%) and the recovery (111% ± 21% in feed additive matrix, 96% ± 13% in ionized feed matrix) were excellent. The variations during cultivation occurred due to the complex spore germination process and presence of other microbes in the feed. This new procedure, detecting only those cells that produced the metabolite of interest, has several advantages as it takes into account bacterial viability, cultivation conditions, spore germination process, growth behavior and the influence of the nutrient-rich feed matrix. It truly reflects the activity of the probiotic in the feed product, allows side-by-side comparison of characteristic metabolite patterns and nutrient consumptions to understand the metabolism of dried spores in matrix.

Pre-infection Mechanisms on the Phylloplane: The First Biochemical Battlefield Between the Cacao Tree and Witches' Broom Pathogen

The leaf surface combines biochemical substances and pre-existing morphological structures, as well as the presence of microorganisms. This dynamic environment constitutes a plant's initial defense, as well as the first contact of phytopathogens during invasion. Spore germination starts on the phylloplane and is a fundamental process for fungal development, and hence the establishment of disease. In this review, we address the phylloplane's innate defense mechanisms and biochemical reactions involved in the early stage of phytopathogenic fungal development. The focus is present the pre-infection molecular and biochemical processes of the interaction between Theobroma cacao and Moniliophthora perniciosa, showing how the defense mechanisms of the phylloplane can act to inhibit proteins involved at the beginning of fungal spore germination. We conclude that the phylloplane of the cocoa resistant genotype to M. perniciosa has performed chemical compounds, pre-existing morphological structures and the presence of microorganisms that participate in the pre-infection defense of the plant. Also, the inhibition of proteins involved in the germination mechanism of M. perniciosa basidiospores by chemical and structural compounds present in the cocoa phylloplane may decrease the disease index. Therefore, understanding how the phylloplane defense acts in the fungal spore germination process is essential to develop pre-infection control strategies for cacao plants against witches' broom.

Reviving the view: evidence that macromolecule synthesis fuels bacterial spore germination.

The Gram positive bacterium Bacillus subtilis and its relatives are capable of forming a durable dormant long-lasting spore. Although spores can remain dormant for years, they possess the remarkable capacity to rapidly resume life and convert into actively growing cells. This cellular transition initiates with a most enigmatic irreversible event, termed germination, lasting only for a few minutes. Germination is typified by a morphological conversion that culminates in loss of spore resilient properties. Yet, the molecular events occurring during this brief critical phase are largely unknown. The current widely accepted view considers germination to occur without the need for any macromolecule synthesis; however, accumulating data from our laboratory and others, highlighted here, provide evidence that both transcription and translation occur during germination and are required for its execution. We further underline numerous overlooked studies, conducted mainly during the 1960s-1970s,reinforcing this notion. We propose to revisit the fascinating process of spore germination and redefine it as a pathway involving macromolecule synthesis. We expect our perspective to shed new light on the awakening process of a variety of spore-forming environmental, commensal, and pathogenic bacteria and possibly be applicable to additional organisms displaying a quiescent life form.

Heat Activation and Inactivation of Bacterial Spores: Is There an Overlap?

Heat activation at a sublethal temperature is widely applied to promote Bacillus species spore germination. This treatment also has the potential to be employed in food processing to eliminate undesired bacterial spores by enhancing their germination and then inactivating the less-heat-resistant germinated spores at a milder temperature. However, incorrect heat treatment could also generate heat damage in spores and lead to more heterogeneous spore germination. Here, the heat activation and heat damage profile of Bacillus subtilis spores was determined by testing spore germination and outgrowth at both population and single-spore levels. The heat treatments used were 40 to 80°C and for 0 to 300 min. The results were as follows. (i) Heat activation at 40 to 70°C promoted l-valine- and l-asparagine-glucose-fructose-potassium (AGFK)-induced germination in a time-dependent manner. (ii) The optimal heat activation temperatures for AGFK and l-valine germination via the GerB plus GerK or GerA germinant receptors were 65°C and 50 to 65°C, respectively. (iii) Heat inactivation of dormant spores appeared at 70°C, and the heat damage of molecules essential for germination and growth began at 70 and 65°C, respectively. (iv) Heat treatment at 75°C resulted in both activation of germination and damage to the germination apparatus, and 80°C treatment caused more pronounced heat damage. (v) For the spores that should withstand adverse environmental temperatures in nature, heat activation seemed functional for a subsequent optimal germination process, while heat damage affected both germination and outgrowth. IMPORTANCE Bacterial spores are thermal-stress-resistant structures that can thus survive food preservation strategies and revive through the process of spore germination. The more heat resistant spores are, the more heterogeneous their germination upon the addition of germinants. Upon germination, spores can cause food spoilage and food intoxication. Here, we provide new information on both heat activation and inactivation regimes and their effects on the (heterogeneity of) spore germination.

Rapid determination of spore germinability of Clostridium perfringens based on microscopic hyperspectral imaging technology and chemometrics

The Gram-positive, anaerobic, spore-forming bacterium, Clostridium perfringens (C. perfringens) causes a variety of diseases in humans and other animals. Spore germination is thought to be the first stage of infection by C. perfringens. AGFK, a mixture of l-asparagine, d-glucose, d-fructose, and potassium ions, is an effective nutrient germinant. The objective of this study was to investigate the effects of different AGFK concentrations (0, 50, 100, 200 mM/mL) on C. perfringens spore germination. This paper proposes a novel rapid method for the measurement of spore germinability based on microscopic hyperspectral imaging technology (HSIT). The spore germination rate (Srate), the OD600% and Ca2+-DPA% of C. perfringens were determined by chemical methods under different concentrations of AGFK. The results showed that spores have a maximum germination rate of 94.59% after 80 min with 100 mM/mL AGFK. Microscopic HSIT revealed that the spectral and spatial characteristics of spores varied during the spore germination process. Multivariate analyses (GA-siPLS and GA-PLS) and the gray symbiotic matrix (GLCM) were used to extract highly correlated spectral and spatial descriptors from the time-series data from microscopic HSIT, respectively. Single spectral, spatial signals and data fusion of spectral and spatial information were then used to predict the Srate, the OD600% and Ca2+-DPA % by GA-PLS, respectively. The result show that the Srate calibration was built by GA-PLS using data fusion variables and yielded acceptable results (Rc = 0.96, RMSEC = 0.64, Rcv = 0.93, RMSEP = 0.87, Rp = 0.94). The OD600% optimal model was built by GA-PLS using image variables and yielded acceptable results (Rc = 0.93, RMSEC = 19.36, Rcv = 0.91, RMSEP = 24.36, Rp = 0.89). For Ca2+-DPA %, the model based on the fusion of spectral and imaging data was optimal. The Ca2+-DPA % calibration yielded acceptable results (Rc = 0.95, RMSEC = 49.83, Rcv = 0.93, RMSEP = 58.98, Rp = 0.92). This work demonstrates the potential of microscopic HSIT for the non-destructive detection of spore germinability. The data fusion models also significantly improved the prediction of spore germinability. In conclusion, microscopic HSIT exhibits considerable promise for nondestructive diagnostics of spore germination.

Construction and application of actin fluorescent marker in Verticillium dahliae Kleb.

Actin filaments play an important role in fungal life processes such as growth, development and cytokinesis. The expression vector pSULPH-Lifeact-mCherry of fluorescent mCherry-labeled actin was transferred into Verticillium dahliae Kleb. wild type V592 by the genetic transformation system mediated by Agrobacterium tumefaciens to obtain the stable fluorescent labeled actin strain V592/Lifeact-mCherry. Then we detected its biological phenotype and the dynamic changes of actin fluorescence during the process of spore germination, mycelial growth and development. There was no significant difference in the colony morphology, colonial growth rate, sporulation and germination rate between the fluorescent labeled actin strain and the wild type. The actin fluorescence signal was observed at the tip of the conidia and hyphae and the septum clearly. Actin participated in the formation of the contractile actomyosin ring (CAR) during cytokinesis by observing the dynamic behavior of the actin in the process of hyphal septum formation. The fluorescent labeled actin strain can be used to study the dynamics of actin in fungal development to provide theoretical and practical support for further study of the mechanism of actin in fungal development and pathogenesis.

The Ser/Thr protein kinase PrkC imprints phenotypic memory in Bacillus anthracis spores by phosphorylating the glycolytic enzyme enolase

Bacillus anthracis is the causative agent of anthrax in humans, bovine, and other animals. B. anthracis pathogenesis requires differentiation of dormant spores into vegetative cells. The spores inherit cellular components as phenotypic memory from the parent cell, and this memory plays a critical role in facilitating the spores' revival. Because metabolism initiates at the beginning of spore germination, here we metabolically reprogrammed B. anthracis cells to understand the role of glycolytic enzymes in this process. We show that increased expression of enolase (Eno) in the sporulating mother cell decreases germination efficiency. Eno is phosphorylated by the conserved Ser/Thr protein kinase PrkC which decreases the catalytic activity of Eno. We found that phosphorylation also regulates Eno expression and localization, thereby controlling the overall spore germination process. Using MS analysis, we identified the sites of phosphorylation in Eno, and substitution(s) of selected phosphorylation sites helped establish the functional correlation between phosphorylation and Eno activity. We propose that PrkC-mediated regulation of Eno may help sporulating B. anthracis cells in adapting to nutrient deprivation. In summary, to the best of our knowledge, our study provides the first evidence that in sporulating B. anthracis, PrkC imprints phenotypic memory that facilitates the germination process.

An alternative biocontrol agent of soil-borne phytopathogens: A new antifungal compound produced by a plant growth promoting bacterium isolated from North Algeria

Bacteria isolated from different environments can be exploited for biocontrol purposes by the identification of the molecules involved in the antifungal activity. The present study was aimed at investigating antifungal protein compounds purified from a previously identified plant growth promoting bacterium, Pseudomonas protegens N isolated from agricultural land in northern Algeria. Therefore, a novel protein was purified by chromatographic and ultrafiltration steps and its antifungal activity together with growth-inhibition mechanism was evaluated against different fungi by plate-based assays. In addition, stereomicroscopy and transmission electron microscopy (TEM) was performed to explore the inhibition activity of the compound on spore germination processes. The protein, showing a molecular mass of about 100 kDa under native conditions, was revealed to be in the surface-membrane fraction and displayed an efficient activity against a variety of phytopathogenic fungi, being Alternaria the best target towards which it exhibited a marked fungicidal action and inhibition of spore germination. Moreover, the compound was able to significantly decrease fungal infection on tomato fruits producing also morphological aberrations on conidia. The obtained results suggested that the isolated compound could represent a promising agent for eco-friendly management of plant pathogens in agriculture.

Positions 299 and 302 of the GerAA subunit are important for function of the GerA spore germination receptor in Bacillus subtilis.

Bacillus subtilis, as a model spore-forming Gram-positive bacterium, has been extensively used for spore germination research. Within this field, nutrient-dependent germination with specific germinant receptors (GerA, responding to L-alanine or L-valine; GerB and GerK, acting together to start spore germination process in response to AGFK) has been the most studied. There are three different variants of the GerAA subunit (299T/302S, 299A/302P, 299A/302S) of the GerA germination receptor present in B. subtilis subs. subtilis laboratory strains. According to our research, the 299A/302P one, unlike the others, interferes with the spore’s ability to germinate in L-alanine as assessed by the measurement of DPA release upon stimulation with the germinant. Multiple genetic manipulations described in this work followed by spore germination tests, together with secondary structure predictions led us to the following conclusions. First, position 302 of GerAA protein is crucial in terms of GerA germination receptor functionality; a proline residue at this position renders the GerA receptor non-functional, most probably due to a change in the protein secondary structure. Second, the 302P GerAA variant has most probably an impaired affinity to other components of GerA receptor. Together, these may explain the loss of GerA receptor’s function. Analysis of the GerAA protein should get us closer to understanding the mechanism of GerA receptor function.

Canonical germinant receptor is dispensable for spore germination in Clostridium botulinum group II strain NCTC 11219

Clostridium botulinum is an anaerobic sporeforming bacterium that is notorious for producing a potent neurotoxin. Spores of C. botulinum can survive mild food processing treatments and subsequently germinate, multiply, produce toxin and cause botulism. Control of spore germination and outgrowth is therefore essential for the safety of mildly processed foods. However, little is known about the process of spore germination in group II C. botulinum (gIICb), which are a major concern in chilled foods because they are psychrotrophic. The classical model of spore germination states that germination is triggered by the binding of a germinant molecule to a cognate germinant receptor. Remarkably, unlike many other sporeformers, gIICb has only one predicted canonical germinant receptor although it responds to multiple germinants. Therefore, we deleted the gerBAC locus that encodes this germinant receptor to determine its role in germination. Surprisingly, the deletion did not affect germination by any of the nutrient germinants, nor by the non-nutrient dodecylamine. We conclude that one or more other, so far unidentified, germinant receptors must be responsible for nutrient induced germination in gIICb. Furthermore, the gerBAC locus was strongly conserved with intact open reading frames in 159 gIICb genomes, suggesting that it has nevertheless an important function.

Spore germination and early gametophyte development of Platycerium wandae (Polypodiaceae) from Papua, Indonesia

Abstract. Praptosuwiryo TNg. 2017. Spore germination and early gametophyte development of Platycerium wandae (Polypodiaceae) from Papua, Indonesia. Biodiversitas 18: 175-182. Studies on gametophyte development in ferns are very important, as the data generated is often useful for supporting taxa delimitation in ferns and fern-allies. This data is also very important for understanding the ecology, reproductive biology, evolution and distribution of ferns. The study reported here aimed to develop a deeper understand of morphogenesis in the fern Platycerium wandae Racib. by investigating the process of spore germination and early gametophyte development of the species on natural media. Fresh spores of P. wandae were germinated in a mixed media consisting of minced roots of Cyathea contaminans and charcoaled rice hulls (1: 1) under green house condition. Spores of P. wandae are monolete, ellipsoid, non-chlorophyllous, dark brown, and lacking perine. Spores germinated between 7 to 14 days after sowing (DAS). Spore germination of P. wandae is of the Vittaria-type and the prothallial development is of the Aspidium-type, characterized by early development of unicelullar trichomes (30-40 DAS). Only unisexual gametophytes were observed at 60-80 DAS. New data is provided concerning the morphogenesis of P. wandae from Papua, Indonesia, on natural media. The findings are relevant to reproductive biology, would contribute to establish an efficient ex situ propagation strategies for the conservation of epiphyitic ferns and to facilitate further in situ studies of gametophyte ecology.
 Keywords: Gametophyte development, Platycerium wandae, spore germination, staghorn fern

The Cooperative and Interdependent Roles of GerA, GerK, and Ynd in Germination of Bacillus licheniformis Spores.

When nutrients are scarce, Bacillus species form metabolically dormant and extremely resistant spores that enable survival over long periods of time under conditions not permitting growth. The presence of specific nutrients triggers spore germination through interaction with germinant receptors located in the spore's inner membrane. Bacillus licheniformis is a biotechnologically important species, but it is also associated with food spoilage and food-borne disease. The B. licheniformis ATCC 14580/DSM13 genome exhibits three gerA family operons (gerA, gerK, and ynd) encoding germinant receptors. We show that spores of B. licheniformis germinate efficiently in response to a range of different single l-amino acid germinants, in addition to a weak germination response seen with d-glucose. Mutational analyses revealed that the GerA and Ynd germination receptors function cooperatively in triggering an efficient germination response with single l-amino acid germinants, whereas the GerK germination receptor is essential for germination with d-glucose. Mutant spores expressing only GerA and GerK or only Ynd and GerK show reduced or severely impaired germination responses, respectively, with single l-amino acid germinants. Neither GerA nor Ynd could function alone in stimulating spore germination. Together, these results functionally characterize the germination receptor operons present in B. licheniformis We demonstrate the overlapping germinant recognition patterns of the GerA and Ynd germination receptors and the cooperative functionalities between GerA, Ynd, and GerK in inducing germination. To ensure safe food production and durable foods, there is an obvious need for more knowledge on spore-forming bacteria. It is the process of spore germination that ultimately leads to food spoilage and food poisoning. Bacillus licheniformis is a biotechnologically important species that is also associated with food spoilage and food-borne disease. Despite its importance, the mechanisms of spore germination are poorly characterized in this species. This study provides novel knowledge on germination of B. licheniformis spores. We characterize the germinant recognition profiles of the three germinant receptors present in B. licheniformis spores and demonstrate that the GerA germinant receptor cooperates with the Ynd and GerK germinant receptors to enable an effective germination response to l-amino acids. We also demonstrate that GerK is required for germination in response to the single germinant glucose. This study demonstrates the complex interactions between germinant receptors necessary for efficient germination of B. licheniformis spores.

Addition of abscisic acid increases the production of chitin deacetylase by Colletotrichum gloeosporioides in submerged culture

The activity of chitin deacetylase from Colletotrichum gloeosporioides was studied by the addition of phytohormones (gibberellic acid, indole acetic acid, and abscisic acid) and amino sugars (glucosamine and N-acetyl glucosamine) in culture media. Abscisic acid exerted a positive and significant effect on enzyme production with 9.5-fold higher activity (1.05Umgprotein−1) than the control (0.11Umgprotein−1). Subsequently, this phytohormone was used in batch culture with higher chitin deacetylase activity being found at acidic pH (3.5) than at neutral pH (7). Furthermore, the highest activity was determined at the acceleration growth phase. The chitin deacetylase production was ascribed to the lag phase within the spore germination process and germ tube elongation instead of during the formation of appressoria, as evidenced by the scanning electronic microscopy results. Therefore, more inoculum and medium containing abscisic acid were added to the fed-batch culture, resulting in a significant increase in chitin deacetylase activity (3.64Umgprotein−1). The addition of abscisic acid led to changes in the acetylation degree of chitin extracted from the cell wall of C. gloeosporioides, with lower degree of acetylation (DA of 75.6%) than that determined with the culture without abscisic acid (DA of 90.6%).

Spore Germination.

Despite being resistant to a variety of environmental insults, the bacterial endospore can sense the presence of small molecules and respond by germinating, losing the specialized structures of the dormant spore, and resuming active metabolism, before outgrowing into vegetative cells. Our current level of understanding of the spore germination process in bacilli and clostridia is reviewed, with particular emphasis on the germinant receptors characterized in Bacillus subtilis, Bacillus cereus, and Bacillus anthracis. The recent evidence for a local clustering of receptors in a "germinosome" would begin to explain how signals from different receptors could be integrated. The SpoVA proteins, involved in the uptake of Ca2+-dipicolinic acid into the forespore during sporulation, are also responsible for its release during germination. Lytic enzymes SleB and CwlJ, found in bacilli and some clostridia, hydrolyze the spore cortex: other clostridia use SleC for this purpose. With genome sequencing has come the appreciation that there is considerable diversity in the setting for the germination machinery between bacilli and clostridia.

Transcriptome analysis of Bacillus thuringiensis spore life, germination and cell outgrowth in a vegetable-based food model

Toxigenic species belonging to Bacillus cereus sensu lato, including Bacillus thuringiensis, cause foodborne outbreaks thanks to their capacity to survive as spores and to grow in food matrixes. The goal of this work was to assess by means of a genome-wide transcriptional assay, in the food isolate B. thuringiensis UC10070, the gene expression behind the process of spore germination and consequent outgrowth in a vegetable-based food model. Scanning electron microscopy and Energy Dispersive X-ray microanalysis were applied to select the key steps of B. thuringiensis UC10070 cell cycle to be analyzed with DNA-microarrays. At only 40 min from heat activation, germination started rapidly and in less than two hours spores transformed in active growing cells. A total of 1646 genes were found to be differentially expressed and modulated during the entire B. cereus life cycle in the food model, with most of the significant genes belonging to transport, transcriptional regulation and protein synthesis, cell wall and motility and DNA repair groups. Gene expression studies revealed that toxin-coding genes nheC, cytK and hblC were found to be expressed in vegetative cells growing in the food model.