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Germination of Bacillus spores is induced by the interaction of specific nutrient molecules with germinant receptors (GRs) localized in the spore's inner membrane. GRs typically consist of three subunits referred to as A, B, and C, although functions of individual subunits are not known. Here we present the crystal structure of the N-terminal domain (NTD) of the A subunit of the Bacillus megaterium GerK3 GR, revealing two distinct globular subdomains bisected by a cleft, a fold with strong homology to substratebinding proteins in bacterial ABC transporters. Molecular docking, chemical shift perturbation measurement, and mutagenesis coupled with spore germination analyses support a proposed model that the interface between the two subdomains in the NTD of GR A subunits serves as the germinant binding site and plays a critical role in spore germination. Our findings provide a conceptual framework for understanding the germinant recruitment mechanism by which GRs trigger spore germination. [ABSTRACT FROM AUTHOR]

Supercritical carbon dioxide (scCO2) is an attractive substitute for conventional organic solvents due to its unique transport and thermodynamic properties, its renewability and labile nature, and its high solubility for compounds such as alcohols, ketones, and aldehydes. However, biological systems that use scCO2 are mainly limited to in vitro processes due to its strong inhibition of cell viability and growth. To solve this problem, we used a bioprospecting approach to isolate a microbial strain with the natural ability to grow while exposed to scCO2. Enrichment culture and serial passaging of deep subsurface fluids from the McElmo Dome scCO2 reservoir in aqueous media under scCO2 headspace enabled the isolation of spore-forming strain Bacillus megaterium SR7. Sequencing and analysis of the complete 5.51 Mbp genome and physiological characterization revealed the capacity for facultative anaerobic metabolism, including fermentative growth on a diverse range of organic substrates. Supplementation of growth medium with L-alanine for chemical induction of spore germination significantly improved growth frequencies and biomass accumulation under scCO2 headspace. Detection of endogenous fermentative compounds in cultures grown under scCO2 represents the first observation of bioproduct generation and accumulation under this condition. Culturing development and metabolic characterization of B. megaterium SR7 represent initial advancements in the effort toward enabling exploitation of scCO2 as a sustainable solvent for in vivo bioprocessing.

Fungal infestation causes deterioration of stored food grains. Most fungal species produce secondary metabolites like aflatoxins which are highly toxic to animals and humans. Aspergillus flavus has been found to be the predominant contaminant in stored wheat grains collected from the godowns of Food Corporation of India, West Bengal. The present study focuses on the development of sensitive polyclonal antibodies (PAbs) for molecular immunological detection of dominant toxigenic fungus. Pure A. flavus isolate was cultured on coconut agar media and its spores were harvested and inactivated by 4% formaldehyde. The inactivated spores were injected into a rabbit along with Freund's complete/incomplete adjuvant for the development of PAbs. Specificity of the raised antibodies in rabbit serum was examined by enzyme-linked immunosorbent assay (ELISA) using spore proteins as antigen obtained by bead beating method. Out of several proteins (ranging from 10 to 200 kDa present in spore, only two prominent proteins of around 76 kDa and 100 kDa were detected by western blot analysis using raised polyclonal antiserum. The PAbs were purified with protein A column followed by spore proteins conjugated CNBr activated sepharose column for its use in the detection of fungal antigens. This highly purified raised antibody can be used for the development of rapid, sensitive, and accurate techniques (such as dot blot/ELISA) for the detection of toxigenic fungi present in stored wheat grains. [ABSTRACT FROM AUTHOR]

Supercritical carbon dioxide (scCO2) is an attractive substitute for conventional organic solvents due to its unique transport and thermodynamic properties, its renewability and labile nature, and its high solubility for compounds such as alcohols, ketones, and aldehydes. However, biological systems that use scCO2 are mainly limited to in vitro processes due to its strong inhibition of cell viability and growth. To solve this problem, we used a bioprospecting approach to isolate a microbial strain with the natural ability to grow while exposed to scCO2. Enrichment culture and serial passaging of deep subsurface fluids from the McElmo Dome scCO2 reservoir in aqueous media under scCO2 headspace enabled the isolation of spore-forming strain Bacillus megaterium SR7. Sequencing and analysis of the complete 5.51 Mbp genome and physiological characterization revealed the capacity for facultative anaerobic metabolism, including fermentative growth on a diverse range of organic substrates. Supplementation of growth medium with L-alanine for chemical induction of spore germination significantly improved growth frequencies and biomass accumulation under scCO2 headspace. Detection of endogenous fermentative compounds in cultures grown under scCO2 represents the first observation of bioproduct generation and accumulation under this condition. Culturing development and metabolic characterization of B. megaterium SR7 represent initial advancements in the effort toward enabling exploitation of scCO2 as a sustainable solvent for in vivo bioprocessing. [ABSTRACT FROM AUTHOR]

Bacterial spores are resistant to physical and chemical insults, which makes them a major concern for public health and industry. Spores help bacteria to survive extreme environmental conditions that vegetative cells cannot tolerate. Spore resistance and dormancy are important properties for applications in medicine, veterinary health, food safety, crop protection and other domains. The resistance of bacterial spores results from a protective multilayered structure and from the unique composition of the spore core. The mechanisms of sporulation and germination, the first stage after breaking of dormancy, and organization of spore structure have been extensively studied in Bacillus species. This review aims to illustrate how far the structure, composition and properties of spores are shaped by the environmental conditions in which spores form. We look at the physiological and molecular mechanisms underpinning how sporulation media and environment deeply affect spore yield, spore properties like resistance to wet heat and physical and chemical agents, germination and further growth. For example, spore core water content decreases as sporulation temperature increases, and resistance to wet heat increases. Controlling the fate of Bacillus spores is pivotal to controlling bacterial risks and process efficiencies in, for example, the food industry, and better control hinges on better understanding how sporulation conditions influence spore properties. [ABSTRACT FROM AUTHOR]

Clostridium perfringens type A isolates carrying a chromosomal enterotoxin ( cpe ) gene (C- cpe ) are generally linked to food poisoning, while isolates carrying cpe on a plasmid (P- cpe ) are associated with non-food-borne gastrointestinal diseases. Both C- cpe and P- cpe isolates can form metabolically dormant spores, which through germination process return to actively growing cells to cause diseases. In our previous study, we showed that only 3 out of 20 amino acids (aa) in phosphate buffer (pH 7.0) triggered germination of spores of P- cpe isolates (P- cpe spores). We now found that 14 out of 20 individual aa tested induced germination of P- cpe spores in the presence of bicarbonate buffer (pH 7.0). However, no significant spore germination was observed with bicarbonate (pH 7.0) alone, indicating that aa and bicarbonate are co-germinants for P- cpe spores. P- cpe strain F4969 gerKC spores did not germinate, and gerAA spores germinated extremely poorly as compared to wild-type and gerKA spores with aa-bicarbonate (pH 7.0) co-germinants. The germination defects in gerKC and gerAA spores were partially restored by complementing gerKC or gerAA spores with wild-type gerKC or gerAA , respectively. Collectively, this study identified aa-bicarbonate as a novel nutrient germinant for P- cpe spores and provided evidence that GerKC and GerAA play major roles in aa-bicarbonate induced germination. [ABSTRACT FROM AUTHOR]

Spore awakening is a series of actions that starts with purely physical processes and continues via the launching of gene expression and metabolic activities, eventually achieving a vegetative phase of growth. In spore-forming microorganisms, the germination process is controlled by intra- and inter-species communication. However, in the Streptomyces clade, which is capable of developing a plethora of valuable compounds, the chemical signals produced during germination have not been systematically studied before. Our previously published data revealed that several secondary metabolite biosynthetic genes are expressed during germination. Therefore, we focus here on the secondary metabolite production during this developmental stage. Using high-performance liquid chromatography-mass spectrometry, we found that the sesquiterpenoid antibiotic albaflavenone, the polyketide germicidin A, and chalcone are produced during germination of the model streptomycete, S. coelicolor. Interestingly, the last two compounds revealed an inhibitory effect on the germination process. The secondary metabolites originating from the early stage of microbial growth may coordinate the development of the producer (quorum sensing) and/or play a role in competitive microflora repression (quorum quenching) in their nature environments. [ABSTRACT FROM AUTHOR]

Clostridium difficile infection (CDI) is an important hospital-acquired infection resulting from the germination of spores in the intestine as a consequence of antibiotic-mediated dysbiosis of the gut microbiota. Key to this is CotE, a protein displayed on the spore surface and carrying 2 functional elements, an N-terminal peroxiredoxin and a C-terminal chitinase domain. Using isogenic mutants, we show in vitro and ex vivo that CotE enables binding of spores to mucus by direct interaction with mucin and contributes to its degradation. In animal models of CDI, we show that when CotE is absent, both colonization and virulence were markedly reduced. We demonstrate here that the attachment of spores to the intestine is essential in the development of CDI. Spores are usually regarded as biochemically dormant, but our findings demonstrate that rather than being simply agents of transmission and dissemination, spores directly contribute to the establishment and promotion of disease. [ABSTRACT FROM AUTHOR]

The complex development undergone by Streptomyces encompasses transitions from vegetative mycelial forms to reproductive aerial hyphae that differentiate into chains of single-celled spores. Whereas their mycelial life - connected with spore formation and antibiotic production - is deeply investigated, spore germination as the counterpoint in their life cycle has received much less attention. Still, germination represents a system of transformation from metabolic zero point to a new living lap. There are several aspects of germination that may attract our attention: (1) Dormant spores are strikingly well-prepared for the future metabolic restart; they possess stable transcriptome, hydrolytic enzymes, chaperones, and other required macromolecules stabilized in a trehalose milieu; (2) Germination itself is a specific sequence of events leading to a complete morphological remodeling that include spore swelling, cell wall reconstruction, and eventually germ tube emergences; (3) Still not fully unveiled are the strategies that enable the process, including a single cell's signal transduction and gene expression control, as well as intercellular communication and the probability of germination across the whole population. This review summarizes our current knowledge about the germination process in Streptomyces, while focusing on the aforementioned points. [ABSTRACT FROM AUTHOR]

Bacillus and Clostridium species form spores, which pose a challenge to the food industry due to their ubiquitous nature and extreme resistance. Pressurization at <300 MPa triggers spore germination by activating germination receptors (GRs), while pressurization at >300 MPa likely triggers germination by opening dipicolinic acid (DPA) channels present in the inner membrane of the spores. In this work, we expose spores of Bacillus licheniformis, a species associated with food spoilage and occasionally with food poisoning, to high pressure (HP) for holding times of up to 2 h. By using mutant spores lacking one or several GRs, we dissect the roles of the GerA, Ynd, and GerK GRs in moderately HP (mHP; 150 MPa)-induced s pore germination. We show that Ynd alone is sufficient for efficient mHP-induced spore germination. GerK also triggers germination with mHP, although at a reduced germination rate compared to that of Ynd. GerA stimulates mHP-induced germination but only in the presence of either the intact GerK or Ynd GR. These results suggests that the effectiveness of the individual GRs in mHP-induced germination differs from their effectiveness in nutrient-induced germination, where GerA plays an essential role. In contrast to Bacillus subtilis spores, treatment with very HP (vHP) of 550 MPa at 37°C did not promote effective germination of B. licheniformis spores. However, treatment with vHP in combination with elevated temperatures (60°C) gave a synergistic effect on spore germination and inactivation. Together, these results provide novel insights into how HP affects B. licheniformis spore germination and inactivation and the role of individual GRs in this process. IMPORTANCE Bacterial spores are inherently resistant to food-processing regimes, such as high-temperature short-time pasteurization, and may therefore compromise food durability and safety. The induction of spore germination facilitates subsequent inactivation by gentler processing conditions that maintain the sensory and nutritional qualities of the food. High-pressure (HP) processing is a nonthermal food-processing technology used to eliminate microbes from food. The application of this technology for spore eradication in the food industry requires a better understanding of how HP affects the spores of different bacterial species. The present study provides novel insights into how HP affects Bacillus licheniformis spores, a species associated with food spoilage and occasionally food poisoning. We describe the roles of different germination receptors in HP-induced germination and the effects of two different pressure levels on the germination and inactivation of spores. This study will potentially contribute to the effort to implement HP technology for spore inactivation in the food industry. [ABSTRACT FROM AUTHOR]

The changing epidemiology of Clostridium difficile infection over the past decades presents a significant challenge in the management of C. difficile associated diseases. The gastrointestinal tract microbiota provides colonization resistance against C. difficile, and growing evidence suggests that gut microbial derived secondary bile acids (SBAs) play a role. We hypothesized that the C. difficile life cycle; spore germination and outgrowth, growth, and toxin production, of strains that vary by age and ribotype will differ in their sensitivity to SBAs. C. difficile strains R20291 and CD196 (ribotype 027), M68 and CF5 (017), 630 (012), BI9 (001) and M120 (078) were used to define taurocholate (TCA) mediated spore germination and outgrowth, growth, and toxin activity in the absence and presence of gut microbial derived SBAs (deoxycholate, isodeoxycholate, lithocholate, isolithocholate, ursodeoxycholate, ω -muricholate, and hyodeoxycholate) found in the human and mouse large intestine. C. difficile strains varied in their rates of germination, growth kinetics, and toxin activity without the addition of SBAs. C. difficile M120, a highly divergent strain, had robust germination, growth, but significantly lower toxin activity compared to other strains. Many SBAs were able to inhibit TCA mediated spore germination and outgrowth, growth, and toxin activity in a dose dependent manner, but the level of inhibition and resistance varied across all strains and ribotypes. This study illustrates how clinically relevant C. difficile strains can have different responses when exposed to SBAs present in the gastrointestinal tract. [ABSTRACT FROM AUTHOR]

Background Spore-forming bacteria are a major cause of food spoilage and food poisoning. Spores that resist physical and chemical treatments used in the food industry may germinate and multiply. Spore germination, outgrowth and growth constitute a complex and highly heterogeneous process. Scope and approach Various techniques and methods can be used to observe the germination, outgrowth and early multiplication process of spore-forming bacteria and/or to quantify the impact of environmental conditions on its progress over time within a spore population. These techniques can be classified by different criteria: (i) the scale of analysis, from populations or cells to molecules, and (ii) the number of analyzed objects (cells) and (iii) the potential of the method to describe and/or quantify the impact of lethal or sub-lethal treatments or environmental conditions. Such treatments are applied to a spore population or a single spore and take into account parameters at the cellular level (growth capacity, morphological properties) to molecular level (proteomics, transcriptomics, spore molecular composition). Key findings and conclusion A better understanding and quantification of the germination, outgrowth and growth process require the implementation of several complementary methods. Methods providing information at single and population levels, as well as at molecular and cellular levels, are essential to assess and control the fate of spore-forming bacteria development in food systems. [ABSTRACT FROM AUTHOR]

Standard antibiotic-based strategies for the treatment of Clostridium difficile infections disrupt indigenous microbiota and commonly fail to eradicate bacterial spores, two key factors that allow recurrence of infection. As an alternative approach to controlling C. difficile infection, a series of bile acid derivatives have been prepared that inhibit taurocholate-induced spore germination. These analogues have been evaluated in a highly virulent NAP1 strain using optical density and phase-contrast microscopy assays. Heterocycle substitutions at C24 were well-tolerated and several tetrazole-containing derivatives were highly potent inhibitors in both assays, with complete inhibition of spore germination observed at 10-25 μM. To limit intestinal absorption, C7-sulfated analogues designed to avoid active and passive transport pathways were prepared. One of these derivatives, compound 21b, was found to be a potent inhibitor of C. difficile spore germination and poorly permeable in a Caco-2 model of intestinal epithelial absorption, suggesting that it is likely to be gut-restricted. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate rosemary (Rosmarinus officinalis L.) growing wild in Hammam Dalaa (Algeria) for total phenolic content, free radical scavenging activity (FRSA) and test the effect of its extracts on the growth of the green mold of citrus, Penicillium digitatum, under in vitro conditions. The results obtained using the Folin-Ciocalteu method showed a high content of the extract in polyphenols reaching 129 mg gallic acid equivalents/g and relatively low content of flavonoids (38mg of quercetin equivalents/g dry extract). In the DPPH assay the methanol extract exhibited a FRSA close to those of the synthetic antioxidants, tested as positive controls, and higher compared to that of the EO, methanol extract and BHT. The in vitro antifungal assay by means of different methods showed a clear inhibitory effect of the rosemary essential oil and methanol extract on P. digitatum. The inhibitory effect registered at the 6th day of incubation at 25°C was between 5 to 79% when the essential oil was applied as fumigant at concentrations ranging from 10 to 50 μl, and was between 13 and 50% when applied by contact bioassay at levels from 1000 to 3500 μl/l. Spore germination was strongly affected by the oil applied by disk diffusion method. The methanol extract exhibited inhibition exceeding 50% of the fungus mycelial growth but at relatively high concentration 0.8g/l. These results support the studies on rosemary as a promising source of preservatives. [ABSTRACT FROM AUTHOR]

Spore inoculum quality in filamentous bioprocesses is a critical parameter associated with viable spore concentration (1) and spore germination (2). It influences pellet morphology and, consequently, process performance. The state-of-the-art method to measure viable spore concentration is tedious, associated with significant inherent bias, and not applicable in real-time. Therefore, it is not usable as process analytical technology (PAT). Spore germination has so far been monitored using image analysis, which is hampered by complex medium background often observed in filamentous bioprocesses. The method presented here is based on the combination of viability staining and large-particle flow cytometry which enables measurements in real-time and hence aims to be applicable as a PAT tool. It is compatible with the complex media background and allows the quantification of metabolically active spores and the monitoring of spore germination. A distinction of germinated spores and not germinated spores was based on logistic regression, using multiparameteric data from flow cytometry. In a first step, a significant correlation between colony-forming unit (CFU) counts and viable spore concentration (1) in an industrially relevant model bioprocess was found. Spore germination (2) was followed over the initial process phase with close temporal resolution. The validation of the method showed an error below 5 %. Differences in spore germination for various spore inocula ages and spore inoculum concentrations were monitored. The real-time applicability of the method suggests the implementation as a PAT tool in filamentous bioprocesses. [ABSTRACT FROM AUTHOR]

Penicillium camemberti is a technologically relevant fungus used to manufacture mold-ripened cheeses. This fungal species produces many volatile organic compounds (VOCs) including ammonia, methyl-ketones, alcohols and esters. Although it is now well known that VOCs can act as signaling molecules, nothing is known about their involvement in P. camemberti lifecycle. In this study, spore germination was shown to be self-regulated by quorum sensing in P. camemberti . This phenomenon, also called “crowding effect”, is population-dependent ( i.e. observed at high population densities). After determining the volatile nature of the compounds involved in this process, 1-octanol was identified as the main compound produced at high-spore density using GC-MS. Its inhibitory effect was confirmed in vitro and 3 mM 1-octanol totally inhibited spore germination while 100 μM only transiently inhibited spore germination. This is the first time that self-inhibition of spore germination is demonstrated in P. camemberti. The obtained results provide interesting perspectives for better control of mold-ripened cheese processes. [ABSTRACT FROM AUTHOR]

Armadillo-related proteins regulate development throughout eukaryotic kingdoms. In the flowering plant Arabidopsis thaliana, Armadillo-related ARABIDILLO proteins promote multicellular root branching. ARABIDILLO homologues exist throughout land plants, including early-diverging species lacking true roots, suggesting that early-evolving ARABIDILLOs had additional biological roles., Here we investigated, using molecular genetics, the conservation and diversification of ARABIDILLO protein function in plants separated by c. 450 million years of evolution., We demonstrate that ARABIDILLO homologues in the moss Physcomitrella patens regulate a previously undiscovered inhibitory effect of abscisic acid ( ABA) on spore germination. Furthermore, we show that A. thaliana ARABIDILLOs function similarly during seed germination. Early-diverging ARABIDILLO homologues from both P. patens and the lycophyte Selaginella moellendorffii can substitute for ARABIDILLO function during A. thaliana root development and seed germination., We conclude that (1) ABA was co-opted early in plant evolution to regulate functionally analogous processes in spore- and seed-producing plants and (2) plant ARABIDILLO germination functions were co-opted early into both gametophyte and sporophyte, with a specific rooting function evolving later in the land plant lineage. [ABSTRACT FROM AUTHOR]

Dispersal is a key step in land plant life cycles, usually via formation of spores or seeds. Regulation of spore- or seed-germination allows control over the timing of transition from one generation to the next, enabling plant dispersal. A combination of environmental and genetic factors determines when seed germination occurs. Endogenous hormones mediate this decision in response to the environment. Less is known about how spore germination is controlled in earlier-evolving nonseed plants., Here, we present an in-depth analysis of the environmental and hormonal regulation of spore germination in the model bryophyte Physcomitrella patens ( Aphanoregma patens)., Our data suggest that the environmental signals regulating germination are conserved, but also that downstream hormone integration pathways mediating these responses in seeds were acquired after the evolution of the bryophyte lineage. Moreover, the role of abscisic acid and diterpenes (gibberellins) in germination assumed much greater importance as land plant evolution progressed., We conclude that the endogenous hormone signalling networks mediating germination in response to the environment may have evolved independently in spores and seeds. This paves the way for future research about how the mechanisms of plant dispersal on land evolved. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Heat resistant endospore formation plays an important role in Clostridium perfringens-associated foodborne illnesses. The spores allow the bacterium to survive heating during normal cooking processes followed by germination and outgrowth of the bacterium in contaminated foods. To identify proteins associated with germination and other spore functions, comparative spore membrane proteome analysis of C. perfringens dormant and germinated spores from strain SM101, using gel-based protein separation and liquid chromatography coupled with MALDI-TOF/TOF mass spectrometry, was performed. A total of 494 proteins were identified, and 117 of them were predicted to be integral membrane or membrane-associated proteins. Among these membrane proteins, 16 and 26 were detected only in dormant and germinated spores, respectively. One protein detected only in germinated spore membranes was the enzyme cyanophycinase, a protease that cleaves the polymer cyanophycin, which is composed of L-arginine-poly(L-aspartic acid), to β-Asp-Arg. Genes encoding cyanophycinase and cyanophycin synthetase have been observed in many species of Clostridium, but their role has not been defined. To determine the function of cyanophycin in C. perfringens, a mutation was introduced in the cphA gene, encoding cyanophycin synthetase. In comparison to the parent strain, SM101, the spores of the mutant strain retained wild type levels of heat resistance, but fewer were made and the spores were smaller in size, suggesting cyanophycin synthesis plays a role in spore assembly. Although cyanophycin could not be extracted from sporulating C. perfringens cells, an Escherichia coli strain expressing the cphA gene made copious amounts of cyanophycin, confirming cphA encodes a cyanophycin synthetase. [ABSTRACT FROM AUTHOR]

A mixed starter, composed of high-quality Rhodiola rosea Fuqu, was developed. Compared with normal Fuqu, Rhodiola rosea Fuqu demonstrated higher spore numbers, spore germination rates, protease activity, liquefying amylase activity and total acid and amino acid nitrogen. Specifically, the spore number was 1.36 times that of regular Fuqu. Protease activity, liquefying amylase activity, total acid and amino acid nitrogen were 0.475 g/100 mL, 104.16 U/g dry Qu, 35.67 mmol/L and 26.08 g/100 mL, respectively. These values were 21.48, 7.41, 6.07 and 67.82% higher than the regular Fuqu. The Rhodiola rosea soy sauce indices were also better than those for the control. The DPPH scavenging abilities of R. rosea soy sauce, control soy sauce, market soy sauce 1 and market soy sauce 2 were 74.25, 44.01, 34.73 and 11.38%, respectively. The total phenolic content in these four samples was 387.38, 309.23, 212.85 and 202.60 mg GAE/g, respectively. As observed with the DPPH and total phenolic assay, the R. rosea soy sauce showed the highest antioxidant capacity. A simple, rapid, and sensitive determination method of salidroside in R. rosea Fuqu and R. rosea soy sauce using HPLC-ESI-MS/MS was developed and validated with multiple reaction monitoring in the negative mode. [ABSTRACT FROM AUTHOR]

The spore-forming obligate anaerobe Clostridium difficile is a leading cause of antibiotic-associated diarrhea around the world. In order for C. difficile to cause infection, its metabolically dormant spores must germinate in the gastrointestinal tract. During germination, spores degrade their protective cortex peptidoglycan layers, release dipicolinic acid (DPA), and hydrate their cores. In C. difficile, cortex hydrolysis is necessary for DPA release, whereas in Bacillus subtilis, DPA release is necessary for cortex hydrolysis. Given this difference, we tested whether DPA synthesis and/or release was required for C. difficile spore germination by constructing mutations in either spoVAC or dpaAB, which encode an ion channel predicted to transport DPA into the forespore and the enzyme complex predicted to synthesize DPA, respectively. C. difficile spoVAC and dpaAB mutant spores lacked DPA but could be stably purified and were more hydrated than wild-type spores; in contrast, B. subtilis spoVAC and dpaAB mutant spores were unstable. Although C. difficile spoVAC and dpaAB mutant spores exhibited wild-type germination responses, they were more readily killed by wet heat. Cortex hydrolysis was not affected by this treatment, indicating that wet heat inhibits a stage downstream of this event. Interestingly, C. difficile spoVAC mutant spores were significantly more sensitive to heat treatment than dpaAB mutant spores, indicating that SpoVAC plays additional roles in conferring heat resistance. Taken together, our results demonstrate that SpoVAC and DPA synthetase control C. difficile spore resistance and reveal differential requirements for these proteins among the Firmicutes. [ABSTRACT FROM AUTHOR]

Bacterial spores are widespread in the environment and can contaminate milk. Spores are resistant to thermal conditions and your germination reduces milk shelf-life because the aerobic bacteria that are sporulated produce proteases and lipases. The aim of this study was identify Paenibacillus sp., the spoilage microbiota, arising from the germination of spores in raw milk and your spoilage potential. Twenty different milk samples were treated at 80°C/12min and plated to isolate spore-forming bacteria. These strains were picked in milk agar and tributyrin agar for verification of their potential proteolytic and lipolytic activities, respectively. Amplification and sequencing of the 16S rRNA gene of the strains for identification by similarity to the DNA sequences deposited in GenBank was performed. One hundred and thirty-seven isolates were obtained, of which 40 (29.2%) showed spoilage activity for milk. Of these, three (7.5%) were identified as strains of Paenibacillus sp., and all were lipolytic. Paenibacillus sp. have been identified as primarily responsible for the spoilage of pasteurized milk with a long shelf-life in other countries. To increase the shelf-life of Brazilian pasteurized milk, it is important to identify the sporulated microbes to determine their origin and to control the contamination of milk by vegetative forms such as spores. [ABSTRACT FROM AUTHOR]

The efficiency of high-pressure (HP) treatment to eliminate vegetative bacterial cells is synergistically increased by many natural antimicrobials, but the effects on spores are poorly described. Here we report the effect of eleven plant essential oils on the nutrient- and HP-induced germination of spores of a group VI psychrotolerant Bacillus cereus strain. Ten oils partially inhibited nutrient-induced germination. These oils also inhibited HP-induced germination, but some inhibited only germination at moderate (200 MPa) pressure and others only at very high (600 MPa) pressure. Inhibition of spore germination by essential oils may have an adverse effect on the effectiveness of spore inactivation by HP at moderate temperatures, and this should be taken into account when designing combined processes. Essential oil from carrot seed did not inhibit nutrient or HP germination although it showed growth inhibitory properties, and essential oils with these properties may therefore open interesting perspectives in combination treatments with HP. Industrial relevance HP treatment is an alternative processing technique that preserves a better balance of food quality and microbiological safety as compared to thermal processing. While most vegetative bacteria are efficiently inactivated by HP, inactivation of spores is inefficient. At moderate temperature, spore inactivation proceeds in a two-step process in which spores first germinate and are subsequently inactivated. The combination with natural antimicrobials is a promising approach to enhance the efficiency of HP processing because it exerts a synergistic effect on inactivation of vegetative bacteria. However, the current work is one of the first to document the effect of essential oils on the HP-induced germination of spores. [ABSTRACT FROM AUTHOR]

This study investigates the effect of sonic stimulation on Bacillus endospore germination. Germinating endospores in a microtiter plate were exposed to audible sound wave generated by an array of piezoelectric transducers. In situ germination kinetics was measured by terbium-dipicolinate fluorescence assay, optical density measurement and phase contrast microscopy. Fluorescence results revealed that sonic stimulation (5 kHz at 90 dB) promoted the germination speed by 43.7% ± 11.3% and final germination level by 61.7% ± 11.9% of Bacillus atrophaeus. This acoustic energy absorbed by endospores is postulated to change membrane permeability and increase enzyme activities; thereby, expediting the germination process. This also raises the likelihood of dormant endospores undergoing germination because of a rapid release of unidentified chemical mediators for quorum sensing. On the other hand, acoustic effect was not observed in B. subtilis endospores. This may be attributed to the different spore aspect ratio, 1.43 ± 0.05 for B. atrophaeus and 2.02 ± 0.08 for B. subtilis, which results in a difference in specific absorption rates towards audible sound waves. Our results demonstrate the modulation of endospore germination by an external field to shed light on germination mechanism and cell-wave interaction. [ABSTRACT FROM AUTHOR]

Germination of Bacillus spores is triggered by the interaction of germinant molecules with specialized receptor proteins localized to the spore inner membrane. Germinant receptors (GRs) are comprised typically of three interacting protein subunits, each of which is essential for receptor function. At least some GRs appear to have a fourth component, referred to as a D-subunit protein. A number of D-subunit proteins were shown previously to be capable of modulating the activity of associated GRs. Here, we investigate the topology and structure-function relationships of the Bacillus megaterium QM B1551 GerUD protein, which is associated with the GerU GR. The presented data demonstrate that GerUD can be subjected to relatively extensive structural modifications while retaining function. Indeed, the presence of either of the two transmembrane spanning domains is sufficient to modulate an efficient GerU-mediated germinative response. The precise function of D-subunit proteins has yet to be established, although they may act as molecular chaperones within the spore inner-membrane environment. [ABSTRACT FROM AUTHOR]

Clostridium difficile is a Gram-positive spore-forming pathogen and a leading cause of nosocomial diarrhea. C. difficile infections are transmitted when ingested spores germinate in the gastrointestinal tract and transform into vegetative cells. Germination begins when the germinant receptor CspC detects bile salts in the gut. CspC is a subtilisin-like serine pseudoprotease that activates the related CspB serine protease through an unknown mechanism. Activated CspB cleaves the pro-SleC zymogen, which allows the activated SleC cortex hydrolase to degrade the protective cortex layer. While these regulators are essential for C. difficile spores to outgrow and form toxin-secreting vegetative cells, the mechanisms controlling their function have only been partially characterized. In this study, we identify the lipoprotein GerS as a novel regulator of C. difficile spore germination using targeted mutagenesis. A gerS mutant has a severe germination defect and fails to degrade cortex even though it processes SleC at wildtype levels. Using complementation analyses, we demonstrate that GerS secretion, but not lipidation, is necessary for GerS to activate SleC. Importantly, loss of GerS attenuates the virulence of C. difficile in a hamster model of infection. Since GerS appears to be conserved exclusively in related Peptostreptococcaeace family members, our results contribute to a growing body of work indicating that C. difficile has evolved distinct mechanisms for controlling the exit from dormancy relative to B. subtilis and other spore-forming organisms. [ABSTRACT FROM AUTHOR]

The inhibitory activities of nisin or/and potassium sorbate on spores and vegetative cells of Bacillus sporothermodurans LTIS27, which are known to be a contaminant of dairy products and to be extremely heat-resistant, were investigated. First, the tested concentrations of nisin or potassium sorbate inhibited vegetative cell growth; with the minimum inhibitory concentrations were 5 × 10 3 IU/ml and 2% (w/v), respectively. Then, the behaviour of vegetative cells and spores in presence of sub-lethal concentrations of nisin (50 UI/ml) or/and potassium sorbate (0.2%), in milk at 37 °C for 5 days, were evaluated. In the absence of inhibitors, strain grew and sporulated at the end of the exponential phase. Nisin (50 UI/ml) was able to inhibit spore outgrowth but didn't affect their germination. It induced an immediate and transitory reduction (1.6log (10) after 1 h and 2.8log (10) after 6 h of incubation) of vegetative cell growth which reappeared between 10 h and 24 h. Potassium sorbate (0.2%) had a durable bacteriostatic effect (1.1log (10) after 6 h), on vegetative cells, followed by a slower regrowth. It was able to inhibit both germination and outgrowth of spores. Association of nisin and potassium sorbate, at sub-lethal concentrations, showed a synergistic effect and resulted in a total inhibition of cells growth after 5 days. The results illustrate the efficacy of nisin and potassium sorbate in combination, and the commercial potential of applying such treatment to decontaminate any product that has a problem with persistence of bacterial spores. [ABSTRACT FROM AUTHOR]

When exposed to nutrient or nonnutrient germinants, individual Bacillus spores can return to life through germination followed by outgrowth. Laser tweezers, Raman spectroscopy, and either differential interference contrast or phase-contrast microscopy were used to analyze the slow dipicolinic acid (DPA) leakage (normally ~20% of spore DPA) from individual spores that takes place prior to the lag time, Tlag, when spores begin rapid release of remaining DPA. Major conclusions from this work with Bacillus subtilis spores were as follows: (i) slow DPA leakage from wild-type spores germinating with nutrients did not begin immediately after nutrient exposure but only at a later heterogeneous time T1; (ii) the period of slow DPA leakage (ΔTleakage = Tlag -- T1) was heterolag geneous among individual spores, although the amount of DPA released in this period was relatively constant; (iii) increases in germination temperature significantly decreased T1 times but increased values of ΔTleakage; (iv) upon germination with L-valine for 10 min followed by addition of D-alanine to block further germination, all germinated spores had T1 times of less than 10 min, suggesting that T 1 the time when spores become committed to germinate; (v) elevated levels of Spo VA proteins involved in DPA movement in spore germination decreased T1 Tlag times but not the amount of DPA released in ΔTleakage; (vi) lack of the cortex-lytic enzyme CwlJ increased DPA leakage during germination due to longer ΔTleakage times in which more DPA was released; and (vii) there was slow DPA leakage early in germination of B. subtilis spores by the nonnutrients CaDPA and dodecylamine and in nutrient germination of Bacillus cereus and Bacillus megaterium spores. Overall, these findings have identified and characterized a new early event in Bacillus spore germination. [ABSTRACT FROM AUTHOR]

Dormancy is a common strategy adopted by bacterial cells as a means of surviving adverse environmental conditions. For Streptomyces bacteria, this involves developing chains of dormant exospores that extend away from the colony surface. Both spore formation and subsequent spore germination are tightly controlled processes, and while significant progress has been made in understanding the underlying regulatory and enzymatic bases for these, there are still significant gaps in our understanding. One class of proteins with a potential role in spore-associated processes are the so-called resuscitation-promoting factors, or Rpfs, which in other actinobacteria are needed to restore active growth to dormant cell populations. The model species Streptomyces coelicolor encodes five Rpf proteins (RpfA to RfpE), and here we show that these proteins have overlapping functions during growth. Collectively, the S. coelicolor Rpfs promote spore germination and are critical for growth under nutrient-limiting conditions. Previous studies have revealed structural similarities between the Rpf domain and lysozyme, and our in vitro biochemical assays revealed various levels of peptidoglycan cleavage capabilities for each of these five Streptomyces enzymes. Peptidoglycan remodeling by enzymes such as these must be stringently governed so as to retain the structural integrity of the cell wall. Our results suggest that one of the Rpfs, RpfB, is subject to a unique mode of enzymatic autoregulation, mediated by a domain of previously unknown function (DUF348) located within the N terminus of the protein; removal of this domain led to significantly enhanced peptidoglycan cleavage. [ABSTRACT FROM AUTHOR]

The survival of bacterial spores after heat treatment and the subsequent germination and outgrowth in a food product can lead to spoilage of the food product and economical losses. Prediction of time-temperature conditions that lead to sufficient inactivation requires access to detailed spore thermal inactivation kinetics of relevant model strains. In this study, the thermal inactivation kinetics of spores of fourteen strains belonging to the Bacillus subtilis group were determined in detail, using both batch heating in capillary tubes and continuous flow heating in a micro heater. The inactivation data were fitted using a log linear model. Based on the spore heat resistance data, two distinct groups ( p < 0.001) within the B. subtilis group could be identified. One group of strains had spores with an average D 120 °C of 0.33 s, while the spores of the other group displayed significantly higher heat resistances, with an average D 120 °C of 45.7 s. When comparing spore inactivation data obtained using batch- and continuous flow heating, the z -values were significantly different, hence extrapolation from one system to the other was not justified. This study clearly shows that heat resistances of spores from different strains in the B. subtilis group can vary greatly. Strains can be separated into two groups, to which different spore heat inactivation kinetics apply. [ABSTRACT FROM AUTHOR]

Resistance characteristics of bacterial endospores towards various environmental stresses such as chemicals and heat are in part attributed to their coat proteins. Heat resistance is developed in a late stage of sporulation and during maturation of released spores. Using our gel-free proteomic approach and LC-FT-ICR-MS/MS analysis we have monitored the efficiency of the tryptic digestion of proteins in the coat during spore maturation over a period of eight days, using metabolically 15 N labeled mature spores as reference. The results showed that during spore maturation the loss of digestion efficiency of outer coat and crust proteins synchronized with the increase in heat resistance. This implicates that spore maturation involves chemical cross-linking of outer coat and crust layer proteins leaving the inner coat layer proteins unmodified. It appears that digestion efficiencies of spore surface proteins can be linked to their location within the coat and crust layers. We also attempted to study a possible link between spore maturation and the observed heterogeneity in spore germination. [ABSTRACT FROM AUTHOR]

Tea is one of the most widely consumed beverages in the world and known for its antimicrobial activity against many microorganisms. Preliminary studies have shown that tea polyphenols can inhibit the growth of a wide range of Gram-positive bacteria. However, the effect of these compounds on germination and outgrowth of bacterial spores is unclear. Spore-forming bacteria are an aggravating problem for the food industry due to spore formation and their subsequent returning to vegetative state during food storage, thus posing spoilage and food safety challenges. Here we analysed the effect of tea compounds: gallic acid, gallocatechin gallate, Teavigo (>90% epigallocatechin gallate), and theaflavin 3,3′-digallate on spore germination and outgrowth and subsequent growth of vegetative cells of Bacillus subtilis . To quantitatively analyse the effect of these compounds, live cell images were tracked from single phase-bright spores up to microcolony formation and analysed with the automated image analysis tool “SporeTracker”. In general, the tested compounds had a significant effect on most stages of germination and outgrowth. However, germination efficiency (ability of spores to become phase-dark) was not affected. Gallic acid most strongly reduced the ability to grow out. Additionally, all compounds, in particular theaflavin 3,3′-digallate, clearly affected the growth of emerging vegetative cells. [ABSTRACT FROM AUTHOR]

High hydrostatic pressure (HHP) is the most-widely adopted novel non-thermal technology for the commercial pasteurization of foods. However, HHP-induced inactivation of bacterial spores remains a challenge due to spore resistance to the treatment limits of currently available industrial HHP units (i.e. ∼650 MPa and 50 °C). Several reports have demonstrated that high pressure can modulate the germination machinery of bacterial spores, rendering them susceptible to subsequent inactivation treatments. Unfortunately, high pressure-induced germination is a unique phenomenon for spores of the genus Bacillus but not of Clostridium. Alternative strategies to inactivate bacterial spores at commercially available pressure and temperature levels include promoting the germination step by inclusion of known germinants into the food formulation to increase the lethality of HHP treatments on bacterial spores. The aim of this review is to provide an overview of the molecular basis involved in pressure-triggered germination of bacterial spores and of novel strategies to inactivate bacterial spores with HHP treatments. [ABSTRACT FROM AUTHOR]

The loss of cell culturability (spore counts), spore germination and subsequent vegetative growth of Bacillus subtilis were evaluated after pulsed light (PL) treatment. The effect of PL on those mechanisms was dependent on the total fluence applied, decreasing the spore counts when increasing total fluence (0.3-12 J/cm). Neither spore germination nor vegetative growth was affected after exposure to low total fluence (0.3 J/cm). A fluence between 1 and 2 J/cm did not change the rate of germination, but significantly delayed bacterial growth. The lag period before growth increased with the total fluence. For higher total fluence (5.5 J/cm), the rate of spore germination was considerably reduced. When B. subtilis spores were subjected to 12 J/cm, no cultivable cells were detected (the maximum detectable level of cell inactivation was reached), being both spore germination and vegetative growth prevented for at least 48 h. For PL treatments in which germination occurred, a temporary increase in the resistance of B. subtilis cells to PL was found in the early phase of germination except for the lowest fluence (0.5 J/cm). After this period, cells became more sensitive, having similar resistance to PL during exponential and stationary phase of growth. PL sensitized B. subtilis cells (spore or vegetative forms) to posterior thermal treatments, demonstrating a synergistic effect of heat and PL which points out the potential application of such combination of treatments for microbial inactivation. [ABSTRACT FROM AUTHOR]

Germination of Bacillus subtilis spores is normally initiated when nutrients from the environment interact with germinant receptors (GRs) in the spores' inner membrane (IM), in which most of the lipids are immobile. GRs and another germination protein, GerD, colocalize in the IM of dormant spores in a small focus termed the "germinosome," and this colocalization or focus formation is dependent upon GerD, which is also essential for rapid GR-dependent spore germination. To determine the fate of the germinosome and germination proteins during spore germination and outgrowth, we employed differential interference microscopy and epifluorescence microscopy to track germinating spores with fluorescent fusions to germination proteins and used Western blot analyses to measure germination protein levels. We found that after initiation of spore germination, the germinosome foci ultimately changed into larger disperse patterns, with≥75% of spore populations displaying this pattern in spores germinated for 1 h, although>80% of spores germinated for 30 min retained the germinosome foci. Western blot analysis revealed that levels of GR proteins and the SpoVA proteins essential for dipicolinic acid release changed minimally during this period, although GerD levels decreased~50% within 15 min in germinated spores. Since the dispersion of the germinosome during germination was slower than the decrease in GerD levels, either germinosome stability is not compromised by~2-fold decreases in GerD levels or other factors, such as restoration of rapid IM lipid mobility, are also significant in germinosome dispersion as spore germination proceeds. [ABSTRACT FROM AUTHOR]

Proper expression of the dev operon is important for normal development of Myxococcus xanthus. When starved, these bacteria coordinate their gliding movements to build mounds that become fruiting bodies as some cells differentiate into spores. Mutations in the devTRS genes impair sporulation. Expression of the operon occurs within nascent fruiting bodies and depends in part on C signaling. Here, we report that expression of the dev operon, like that of several other C-signal-dependent genes, is subject to combinatorial control by the transcription factors MrpC2 and FruA. A DNA fragment upstream of the dev promoter was bound by a protein in an extract containing MrpC2, protecting the region spanning positions+77 to+54. Mutations in this region impaired binding of purified MrpC2 and abolished developmental expression of reporter fusions. The association of MrpC2 and/or its longer form, MrpC, with the dev promoter region depended on FruA in vivo, based on chromatin immunoprecipitation analysis, and purified FruA appeared to bind cooperatively with MrpC2 to DNA just upstream of the dev promoter in vitro. We conclude that cooperative binding of the two proteins to this promoter-proximal site is crucial for dev expression. 5= deletion analysis implied a second upstream positive regulatory site, which corresponded to a site of weak cooperative binding of MrpC2 and FruA and boosted dev expression 24 h into development. This site is unique among the C-signal-dependent genes studied so far. Deletion of this site in the M. xanthus chromosome did not impair sporulation under laboratory conditions. [ABSTRACT FROM AUTHOR]

The spore-forming bacterial pathogen C lostridium difficile is a leading cause of health-care-associated diarrhea worldwide. Although C . difficile spore formation is essential for disease transmission, the regulatory pathways that control this developmental process have only been partially characterized. In the well-studied spore-former B acillus subtilis, the highly conserved σ E, SpoIIID and σ K regulatory proteins control gene expression in the mother cell to ensure proper spore formation. To define the precise requirement for SpoIIID and σ K during C . difficile sporulation, we analyzed spoIIID and sigK mutants using heterologous expression systems and RNA- Seq transcriptional profiling. These analyses revealed that expression of sigK from a SpoIIID-independent promoter largely bypasses the need for SpoIIID to produce heat-resistant spores. We also observed that σ K is active upon translation, suggesting that SpoIIID primarily functions to activate sigK. SpoIIID nevertheless plays auxiliary roles during sporulation, as it enhances levels of the exosporium morphogenetic protein CdeC in a σ K-dependent manner. Analyses of purified spores further revealed that SpoIIID and σ K control the adherence of the CotB coat protein to C . difficile spores, indicating that these proteins regulate multiple stages of spore formation. Collectively, these results highlight that diverse mechanisms control spore formation in the Firmicutes. [ABSTRACT FROM AUTHOR]

A microfluidic device for studies on the germination of bacterial spores (e.g. Bacillus subtilis) based on non-specific interactions on the nanoscale is presented. A decrease in the population of spores during germination followed by the appearance of transition forms and an increase in the number of vegetative cells can be registered directly and simultaneously by using the microfluidic device, which is equipped with a conductive polymer layer (polyaniline) in the form of a nano-network. The lab-on-a-chip-type device, operating in a continuous flow regime, allows monitoring of germination of bacterial spores and analysis of the process in detail. The procedure is fast and accurate enough for quantitative real-time monitoring of the main steps of germination, including final transformation of the spores into vegetative cells. All of this is done without the use of biomarkers or any bio-specific materials, such as enzymes, antibodies and aptamers, and is simply based on an analysis of physicochemical interactions on the nanoscale level. [ABSTRACT FROM AUTHOR]

Background: Bacterial spore germination is a developmental process during which all required metabolic pathways are restored to transfer cells from their dormant state into vegetative growth. Streptomyces are soil dwelling filamentous bacteria with complex life cycle, studied mostly for they ability to synthesize secondary metabolites including antibiotics. Methods: Here, we present a systematic approach that analyzes gene expression data obtained from 13 time points taken over 5.5 h of Streptomyces germination. Genes whose expression was significantly enhanced/diminished during the time-course were identified, and classified to metabolic and regulatory pathways. Results: The classification into metabolic pathways revealed timing of the activation of specific pathways during the course of germination. The analysis also identified remarkable changes in the expression of specific sigma factors over the course of germination. Based on our knowledge of the targets of these factors, we speculate on their possible roles during germination. Among the factors whose expression was enhanced during the initial part of germination, SigE is though to manage cell wall reconstruction, SigR controls protein reaggregation, and others (SigH, SigB, SigI, SigJ) control osmotic and oxidative stress responses. Conclusions: From the results, we conclude that most of the metabolic pathway mRNAs required for the initial phases of germination were synthesized during the sporulation process and stably conserved in the spore. After rehydration in growth medium, the stored mRNAs are being degraded and resynthesized during first hour. From the analysis of sigma factors we conclude that conditions favoring germination evoke stress-like cell responses. [ABSTRACT FROM AUTHOR]

Aims: To evaluate five commercially available sporicidal wipes and two disinfecting wipes for their ability to inactivate Bacillus atrophaeus spores deposited onto various material surfaces. Methods and Results: Decontamination efficacy of the wipes was initially tested on glass Petri dishes (150 mm diameter). Following exposure for a specified time of contact, survival of the spores was assessed by quantification of the remaining viable spores, both on the coupon surface and on the towelette itself, with efficacy quantified in terms of mean log reduction. Based on these data, five wipes were down-selected for evaluation on a larger scale, using 36 × 36 cm coupons of five different material types. Conclusions: Results suggest that sodium hypochlorite-based sporicidal wipes were most effective, having completely inactivated the Bacillus spores on the glass Petri dish and several materials. Additionally, results demonstrate that the manufacturer-prescribed contact times for Clostridium difficile achieved a 6 log10 reduction of B. atrophaeus spores. Moreover, commercially available disinfecting wipes were not able to kill Bacillus spores as evaluated. Significance and Impact of the Study: These data show the potential of sporicidal wipes for decontamination of small, contained areas of biological contamination and may help on-scene coordinators develop remediation plans following a biological terrorism event. [ABSTRACT FROM AUTHOR]

Aims: Decontamination and remediation of a site contaminated by the accidental or intentional release of fully virulent Bacillus anthracis spores are difficult, costly and potentially damaging to the environment. Development of novel decontamination strategies that have minimal environmental impacts remains a high priority. Although ungerminated spores are amongst the most resilient organisms known, once exposed to germinants, the germinating spores, in some cases, become susceptible to antimicrobial environments. We evaluated the concept that once germinated, B. anthracis spores would be less hazardous and significantly easier to remediate than ungerminated dormant spores. Methods and Results: Through in vitro germination and sensitivity assays, we demonstrated that upon germination, B. anthracis Ames spores and Bacillus thuringiensis Al Hakam spores (serving as a surrogate for B. anthracis) become susceptible to environmental stressors. The majority of these germinated B. anthracis and B. thuringiensis spores were nonviable after exposure to a defined minimal germination-inducing solution for prolonged periods of time. Additionally, we examined the impact of potential secondary disinfectant strategies including bleach, hydrogen peroxide, formaldehyde and artificial UV-A, UV-B and UV-C radiation, employed after a 60-min germinationinduction step. Each secondary disinfectant employs a unique mechanism of killing; as a result, germination-induction strategies are better suited for some secondary disinfectants than others. Conclusions: These results provide evidence that the deployment of an optimal combination strategy of germination-induction/secondary disinfection may be a promising aspect of wide-area decontamination following a B. anthracis contamination event. Significance and Impact of the Study: By inducing spores to germinate, our data confirm that the resulting cells exhibit sensitivities that can be leveraged when paired with certain decontamination measures. This increased susceptibility could be exploited to devise more efficient and safe decontamination measures and may obviate the need for more stringent methods that are currently in place. [ABSTRACT FROM AUTHOR]

Bacillus endospores have a wide variety of important medical and industrial applications. This is an overview of the fundamental aspects of the life cycle, spore structure and factors that influence the spore resistance of spore-forming Bacillus . Bacillus atrophaeus was used as reference microorganism for this review because their spores are widely used to study spore resistance and morphology. Understanding the mechanisms involved in the cell cycle and spore survival is important for developing strategies for spore killing; producing highly resistant spores for biodefense, food and pharmaceutical applications; and developing new bioactive molecules and methods for spore surface display. [ABSTRACT FROM AUTHOR]

This study reports the inhibitory effect of single walled carbon nanotubes (SWCNTs) on biofilm formation fromBacillus anthracisspores. Although the presence of 50 to 100 μg ml−1of SWCNTs in the suspension increased spore attachment in the wells of 96-well plates, the presence of 200 μg ml−1of SWCNTs in the germination solution decreased the germination percentage of the attached spores by 93.14%, completely inhibiting subsequent biofilm formation. The inhibition kinetics of 50 μg ml−1SWCNTs on biofilm formation showed that this concentration inhibited biofilm formation by 81.2% after incubation for 48 h. SWCNT treatment in the earlier stages of biofilm formation was more effective compared to treatment at later stages. Mature biofilms were highly resistant to SWCNT treatment. [ABSTRACT FROM AUTHOR]

The infectious agent of the disease anthrax is the spore of Bacillus anthracis. Bacterial spores are extremely resistant to environmental stresses, which greatly hinders spore decontamination efforts. The spore cortex, a thick layer of modified peptidoglycan, contributes to spore dormancy and resistance by maintaining the low water content of the spore core. The cortex is degraded by germination-specific lytic enzymes (GSLEs) during spore germination, rendering the cells vulnerable to common disinfection techniques. This study investigates the relationship between SleB, a GSLE in B. anthracis, and YpeB, a protein necessary for SleB stability and function. The results indicate that ΔsleB and ΔypeB spores exhibit similar germination phenotypes and that the two proteins have a strict codependency for their incorporation into the dormant spore. In the absence of its partner protein, SleB or YpeB is proteolytically degraded soon after expression during sporulation, rather than escaping the developing spore. The three PepSY domains of YpeB were examined for their roles in the interaction with SleB. YpeB truncation mutants illustrate the necessity of a region beyond the first PepSY domain for SleB stability. Furthermore, site-directed mutagenesis of highly conserved residues within the PepSY domains resulted in germination defects corresponding to reduced levels of both SleB and YpeB in the mutant spores. These results identify residues involved in the stability of both proteins and reiterate their codependent relationship. It is hoped that the study of GSLEs and interacting proteins will lead to the use of GSLEs as targets for efficient activation of spore germination and facilitation of spore cleanup. [ABSTRACT FROM AUTHOR]

Clostridium difficile is a Gram-positive, spore-forming obligate anaerobe and a major nosocomial pathogen of worldwide concern. Owing to its strict anaerobic requirements, the infectious and transmissible morphotype is the dormant spore. In susceptible patients, C. difficile spores germinate in the colon to form the vegetative cells that initiate Clostridium difficile infections (CDI). During CDI, C. difficile induces a sporulation pathway that produces more spores; these spores are responsible for the persistence of C. difficile in patients and horizontal transmission between hospitalized patients. Although important to the C. difficile lifecycle, the C. difficile spore proteome is poorly conserved when compared to members of the Bacillus genus. Further, recent studies have revealed significant differences between C. difficile and Bacillus subtilis at the level of sporulation, germination, and spore coat and exosporium morphogenesis. In this review, the regulation of the sporulation and germination pathways and the morphogenesis of the spore coat and exosporium will be discussed. [ABSTRACT FROM AUTHOR]

Spores are popular as direct-fed microbials, though little is known about their mode of action. Hence, the first objective of the present study was to evaluate the in vitro germination and growth rate of Bacillus subtilis spores. Approximately 90% of B. subtilis spores germinate within 60 min in the presence of feed in vitro. The second objective was to determine the distribution of these spores throughout different anatomical segments of the gastrointestinal tract (GIT) in a chicken model. For in vivo evaluation of persistence and dissemination, spores were administered to day-of-hatch broiler chicks either as a single gavage dose or constantly in the feed. During 2 independent experiments, chicks were housed in isolation chambers and fed sterile corn-soy-based diets. In these experiments one group of chickens was supplemented with 106 spores/g of feed, whereas a second group was gavaged with a single dose of 106 spores per chick on day of hatch. In both experiments, crop, ileum, and cecae were sampled from 5 chicks at 24, 48, 72, 96, and 120 h. Viable B. subtilis spores were determined by plate count method after heat treatment (75℃ for 10 min). The number of recovered spores was constant through 120 h in each of the enteric regions from chickens receiving spores supplemented in the feed. However, the number of recovered B. subtilis spores was consistently about 105 spores per gram of digesta, which is about a 1-logio reduction of the feed inclusion rate, suggesting approximately a 90% germination rate in the GIT when fed. On the other hand, recovered B. subtilis spores from chicks that received a single gavage dose decreased with time, with only approximately 10² spores per gram of sample by 120 h. This confirms that B. subtilis spores are transiently present in the GIT of chickens, but the persistence of vegetative cells is presently unknown. For persistent benefit, continuous administration of effective B. subtilis direct-fed microbials as vegetative cells or spores is advisable. [ABSTRACT FROM AUTHOR]

A critical event during spore germination is the release of Ca- DPA (calcium in complex with dipicolinic acid). The mechanism of release of Ca- DPA through the inner membrane of the spore is not clear, but proteins encoded by the B acillus subtilis spoVA operon are involved in the process. We cloned and expressed the spoVAC gene in E scherichia coli and characterized the SpoVAC protein. We show that SpoVAC protects E . coli against osmotic downshift, suggesting that it might act as a mechanosensitive channel. Purified SpoVAC was reconstituted in unilamellar lipid vesicles to determine the gating mechanism and pore properties of the protein. By means of a fluorescence-dequenching assay, we show that SpoVAC is activated upon insertion into the membrane of the amphiphiles lyso PC and dodecylamine. Patch clamp experiments on E . coli giant spheroplast as well as giant unilamellar vesicles ( GUVs) containing SpoVAC show that the protein forms transient pores with main conductance values of about 0.15 and 0.1 nS respectively. Overall, our data indicate that SpoVAC acts as a mechanosensitive channel and has properties that would allow the release of Ca- DPA and amino acids during germination of the spore. [ABSTRACT FROM AUTHOR]

Abstract: Spore germination in Bacillus species represents an excellent model system with which to study the molecular mechanisms underlying the nutritional control of growth and development. Binding of specific chemical nutrients to their cognate receptors located in the spore inner membrane triggers the germination process that leads to a resumption of metabolism in spore outgrowth. Recent studies suggest that the inner membrane GerD lipoprotein plays a critical role in the receptor-mediated activation of downstream germination events. The 121-residue core polypeptide of GerD (GerD60-180) from Geobacillus stearothermophilus forms a stable α-helical trimer in aqueous solution. The 2.3-Å-resolution crystal structure of the trimer reveals a neatly twisted superhelical rope, with unusual supercoiling induced by parallel triple-helix interactions. The overall geometry comprises three interleaved hydrophobic screws of interacting helices linked by short turns that have not been seen before. Using complementation analysis in a series of Bacillus subtilis gerD mutants, we demonstrated that alterations in the GerD trimer structure have profound effects on nutrient germination. This important structure–function relationship of trimeric GerD is supported by our identification of a dominant negative gerD mutation in B. subtilis. These results and those of others lead us to propose that GerD mediates clustering of germination proteins in the inner membrane of dormant spores and thus promotes the rapid and cooperative germination response to nutrients. [Copyright &y& Elsevier]