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Peter Setlow - One of the best experts on this subject based on the ideXlab platform.
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Bacterial Spore mrna what s up with that
Frontiers in Microbiology, 2020Co-Authors: Peter Setlow, Graham ChristieAbstract:Bacteria belonging to the orders Bacillales and Clostridiales form Spores in response to nutrient starvation. From a simplified morphological perspective, the Spore can be considered as comprising a central protoplast or core, that is, enveloped sequentially by an inner membrane (IM), a peptidoglycan cortex, an outer membrane, and a proteinaceous coat. All of these structures are characterized by unique morphological and/or structural features, which collectively confer metabolic dormancy and properties of environmental resistance to the quiescent Spore. These properties are maintained until the Spore is stimulated to germinate, outgrow and form a new vegetative cell. Spore germination comprises a series of partially overlapping biochemical and biophysical events - efflux of ions from the core, rehydration and IM reorganization, disassembly of cortex and coat - all of which appear to take place in the absence of de novo ATP and protein synthesis. If the latter points are correct, why then do Spores of all species examined to date contain a diverse range of mRNA molecules deposited within the Spore core? Are some of these molecules "functional," serving as translationally active units that are required for efficient Spore germination and outgrowth, or are they just remnants from sporulation whose sole purpose is to provide a reservoir of ribonucleotides for the newly outgrowing cell? What is the fate of these molecules during Spore senescence, and indeed, are conditions within the Spore core likely to provide any opportunity for changes in the transcriptional profile of the Spore during dormancy? This review encompasses a historical perspective of Spore ribonucleotide biology, from the earliest biochemical led analyses - some of which in hindsight have proved to be remarkably prescient - through the transcriptomic era at the turn of this century, to the latest next generation sequencing derived insights. We provide an overview of the key literature to facilitate reasoned responses to the aforementioned questions, and many others, prior to concluding by identifying the major outstanding issues in this crucial area of Spore biology.
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modeling Bacterial Spore germination effects of humectants on high pressure and nutrient germination
2020Co-Authors: Christopher J. Doona, Florence E. Feeherry, Peter Setlow, Kenneth KustinAbstract:Abstract The germination kinetics of Bacillus subtilis, Bacillus cereus, and Bacillus megaterium Spores were comprehensively studied using nutrients, dodecylamine, exogenous dipicolinic acid (DPA), and the nonthermal technology of High Pressure Processing (HPP) as germinants at water activities aw = 0.995–0.698 and DPA loss measured for Spore populations and single Spores. Primary findings were: i) germinant receptor (GR)-dependent germination was less sensitive to changes in aw than were other germination mechanisms; ii) HPP germination was less sensitive to changes in aw than was germination by other germinants, and iii) the irreversible commitment step was more sensitive to inhibition by lowering aw than were DPA release or cortex peptidoglycan hydrolysis. Presently, we use the three-step Quasi-chemical Germination Kinetics (QCGK) model, Transition State Theory, and Extrathermodynamics' Bunnett equation to determine the effects of humectants on Spore germination rates. The QCGK model is an ordinary differential equation system that postulates Spore germination occurs as a sequence of two productive steps with associated rate constants (k1 is irreversible and k2) and one nonproductive step (k3). The QCGK model fits the germination kinetics data well for changes in the observed dynamics with decreasing aw. Transition State Theory calculated negative activation volumes (ΔV∗) from the pressure-dependences of k1 and k2, and Bunnett equation calculations established linear relationships for the model's rate parameters, including rate constants and maximum germination rates, with variations in aw. Taken together, these results suggest that the irreversible step (k1) is rate-limiting and the inverse dependence of k1 and k2 on humectant concentration could occur through shielding of the activated complex by the solvated nonelectrolytes at low aw, which prevents the germination intermediate from linking to the aqueous environment. Practical applications High pressure processing is a food safety method that provides the least alteration of food appearance, texture and flavor. This research shows that food additives such as sugar can diminish the efficiency of high pressure food processing.
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A Quasi-chemical Model for Bacterial Spore Germination Kinetics by High Pressure
Food Engineering Reviews, 2017Co-Authors: Christopher J. Doona, X. Philip Ye, Kenneth Kustin, Florence E. Feeherry, Runze Huang, Haiqing Chen, Peter SetlowAbstract:High pressure processing (HPP) is an emerging non-thermal technology that is growing exponentially in use worldwide for the pasteurization of commercial foodstuffs. At combinations of elevated pressures and temperatures, HPP inactivates Bacterial Spores, but HPP has not yet been implemented commercially for food sterilization. Studies of the mechanisms of Bacterial Spore inactivation by HPP using primarily Spores of Bacillus species have shown that Spore germination precedes inactivation, with the release of dipicolinic acid from the Spore core as the rate-determining step. Investigations probing Spore resistance to and germination by HPP using Bacillus subtilis , a number of selected B. subtilis mutants, Bacillus amyloliquefaciens , and Clostridium difficile Spores have compiled a wealth of detailed mechanistic information, while also accumulating abundant germination kinetics data that has not previously been analyzed by predictive models. Presently, we devise a “quasi-chemical” model for Bacterial Spore germination dynamics by HPP. This quasi-chemical germination model (QCGM) hypothesizes a three-step mechanism and derives a set of ordinary differential equations to model the observed germination dynamics. The results with this model are viewed in the context of historical studies of Spore activation, germination, and inactivation, with an eye toward potentially integrating differential equation models for germination and inactivation into a single, comprehensive model for Spore dynamics by HPP. With the increasing use of high hydrostatic pressure to investigate mechanisms of Bacterial Spore resistance and physiology, the QCGM results help promote the efficient control of Bacterial Spores, whether for the inactivation of Clostridium botulinum Spores in low-acid foods or aerosolized Bacillus anthracis Spores on textiles used in protective clothing, tents, or shelters.
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Bacterial Spore structures and their protective role in biocide resistance
Journal of Applied Microbiology, 2012Co-Authors: Mark James Leggett, Peter Setlow, Gerald E Mcdonnell, Stephen Paul Denyer, Jeanyves MaillardAbstract:The structure and chemical composition of Bacterial Spores differ considerably from those of vegetative cells. These differences largely account for the unique resistance properties of the Spore to environmental stresses, including disinfectants and sterilants, resulting in the emergence of Spore-forming bacteria such as Clostridium difficile as major hospital pathogens. Although there has been considerable work investigating the mechanisms of action of many sporicidal biocides against Bacillus subtilis Spores, there is far less information available for other species and particularly for various Clostridia. This paucity of information represents a major gap in our knowledge given the importance of Clostridia as human pathogens. This review considers the main Spore structures, highlighting their relevance to Spore resistance properties and detailing their chemical composition, with a particular emphasis on the differences between various Spore formers. Such information will be vital for the rational design and development of novel sporicidal chemistries with enhanced activity in the future.
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Characterization of Bacterial Spore germination using phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers
Nature Protocols, 2011Co-Authors: Lingbo Kong, Pengfei Zhang, Guiwen Wang, Peter SetlowAbstract:This protocol describes a method combining phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers to characterize the germination of single Bacterial Spores. The characterization consists of the following steps: (i) loading heat-activated dormant Spores into a temperature-controlled microscope sample holder containing a germinant solution plus a nucleic acid stain; (ii) capturing a single Spore with optical tweezers; (iii) simultaneously measuring phase-contrast images, Raman spectra and fluorescence images of the optically captured Spore at 2- to 10-s intervals; and (iv) analyzing the acquired data for the loss of Spore refractility, changes in Spore-specific molecules (in particular, dipicolinic acid) and uptake of the nucleic acid stain. This information leads to precise correlations between various germination events, and takes 1–2 h to complete. The method can also be adapted to use multi-trap Raman spectroscopy or phase-contrast microscopy of Spores adhered on a cover slip to simultaneously obtain germination parameters for multiple individual Spores.
Kumaran S Ramamurthi - One of the best experts on this subject based on the ideXlab platform.
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a versatile nano display platform from Bacterial Spore coat proteins
Nature Communications, 2015Co-Authors: Kedar Narayan, Jeanphilippe Castaing, Fang Tian, Sriram Subramaniam, Kumaran S RamamurthiAbstract:The densely crosslinked protein coats of Bacterial Spores are among the most durable static structures in biology. Wu et al. reconstitute the basement layer of a Bacterial Spore coat on membrane-coated beads, and generate covalently-modified Spore-like particles with therapeutic potential.
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a versatile nano display platform from Bacterial Spore coat proteins
Nature Communications, 2015Co-Authors: Kedar Narayan, Jeanphilippe Castaing, Fang Tian, Sriram Subramaniam, Kumaran S RamamurthiAbstract:Dormant Bacterial Spores are encased in a thick protein shell, the 'coat', which contains ∼70 different proteins. The coat protects the Spore from environmental insults, and is among the most durable static structures in biology. Owing to extensive cross-linking among coat proteins, this structure has been recalcitrant to detailed biochemical analysis, so molecular details of how it assembles are largely unknown. Here, we reconstitute the basement layer of the coat atop spherical membranes supported by silica beads to create artificial Spore-like particles. We report that these synthetic Spore husk-encased lipid bilayers (SSHELs) assemble and polymerize into a static structure, mimicking in vivo basement layer assembly during sporulation in Bacillus subtilis. In addition, we demonstrate that SSHELs may be easily covalently modified with small molecules and proteins. We propose that SSHELs may be versatile display platforms for drugs and vaccines in clinical settings, or for enzymes that neutralize pollutants for environmental remediation.
Lingbo Kong - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Bacterial Spore germination using phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers
Nature Protocols, 2011Co-Authors: Lingbo Kong, Pengfei Zhang, Guiwen Wang, Peter SetlowAbstract:This protocol describes a method combining phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers to characterize the germination of single Bacterial Spores. The characterization consists of the following steps: (i) loading heat-activated dormant Spores into a temperature-controlled microscope sample holder containing a germinant solution plus a nucleic acid stain; (ii) capturing a single Spore with optical tweezers; (iii) simultaneously measuring phase-contrast images, Raman spectra and fluorescence images of the optically captured Spore at 2- to 10-s intervals; and (iv) analyzing the acquired data for the loss of Spore refractility, changes in Spore-specific molecules (in particular, dipicolinic acid) and uptake of the nucleic acid stain. This information leads to precise correlations between various germination events, and takes 1–2 h to complete. The method can also be adapted to use multi-trap Raman spectroscopy or phase-contrast microscopy of Spores adhered on a cover slip to simultaneously obtain germination parameters for multiple individual Spores.
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characterization of Bacterial Spore germination using phase contrast and fluorescence microscopy raman spectroscopy and optical tweezers
Nature Protocols, 2011Co-Authors: Lingbo Kong, Peter Setlow, Pengfei Zhang, Guiwen Wang, Jing Yu, Yongqing LiAbstract:Characterization of Bacterial Spore germination using phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers
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characterization of Bacterial Spore germination using integrated phase contrast microscopy raman spectroscopy and optical tweezers
Analytical Chemistry, 2010Co-Authors: Lingbo Kong, Pengfei Zhang, Peter SetlowAbstract:We present a methodology that combines external phase contrast microscopy, Raman spectroscopy, and optical tweezers to monitor a variety of changes during the germination of single Bacillus cereus Spores in both nutrient (l-alanine) and non-nutrient (Ca-dipicolinic acid (DPA)) germinants with a temporal resolution of ∼2 s. Phase contrast microscopy assesses changes in refractility of individual Spores during germination, while Raman spectroscopy gives information on changes in Spore-specific molecules. The results obtained include (1) the brightness of the phase contrast image of an individual dormant Spore is proportional to the level of CaDPA in that Spore; (2) the end of the first Stage of germination, revealed as the end of the rapid drop in Spore refractility by phase contrast microscopy, precisely corresponds to the completion of the release of CaDPA as revealed by Raman spectroscopy; and (3) the correspondence between the rapid drop in Spore refractility and complete CaDPA release was observed not ...
Kedar Narayan - One of the best experts on this subject based on the ideXlab platform.
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a versatile nano display platform from Bacterial Spore coat proteins
Nature Communications, 2015Co-Authors: Kedar Narayan, Jeanphilippe Castaing, Fang Tian, Sriram Subramaniam, Kumaran S RamamurthiAbstract:The densely crosslinked protein coats of Bacterial Spores are among the most durable static structures in biology. Wu et al. reconstitute the basement layer of a Bacterial Spore coat on membrane-coated beads, and generate covalently-modified Spore-like particles with therapeutic potential.
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a versatile nano display platform from Bacterial Spore coat proteins
Nature Communications, 2015Co-Authors: Kedar Narayan, Jeanphilippe Castaing, Fang Tian, Sriram Subramaniam, Kumaran S RamamurthiAbstract:Dormant Bacterial Spores are encased in a thick protein shell, the 'coat', which contains ∼70 different proteins. The coat protects the Spore from environmental insults, and is among the most durable static structures in biology. Owing to extensive cross-linking among coat proteins, this structure has been recalcitrant to detailed biochemical analysis, so molecular details of how it assembles are largely unknown. Here, we reconstitute the basement layer of the coat atop spherical membranes supported by silica beads to create artificial Spore-like particles. We report that these synthetic Spore husk-encased lipid bilayers (SSHELs) assemble and polymerize into a static structure, mimicking in vivo basement layer assembly during sporulation in Bacillus subtilis. In addition, we demonstrate that SSHELs may be easily covalently modified with small molecules and proteins. We propose that SSHELs may be versatile display platforms for drugs and vaccines in clinical settings, or for enzymes that neutralize pollutants for environmental remediation.
Robert C Andrews - One of the best experts on this subject based on the ideXlab platform.
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2001 Chlorine dioxide inactivation of Cryptosporidium parvum oocysts and Bacterial Spore indicators
2016Co-Authors: Christian Chauret, Chris Z Radziminski, Michael Lepuil, Robin Creason, Robert C AndrewsAbstract:Cryptosporidium parvum, which is resistant to chlorine concentrations typically used in water treatment, is recognized as a significant waterborne pathogen. Recent studies have demonstrated that chlorine dioxide is a more efficient disinfectant than free chlorine against Cryptosporidium oocysts. It is not known, however, if oocysts from different suppliers are equally sensitive to chlorine dioxide. This study used both a most-probable-number–cell culture infectivity assay and in vitro excystation to evaluate chlorine dioxide inactivation kinetics in laboratory water at pH 8 and 21°C. The two viability methods produced significantly different results (P < 0.05). Products of disinfectant concentration and contact time (Ct values) of 1,000 mg z min/liter were needed to inactivate approximately 0.5 log10 and 2.0 log10 units (99 % inactivation) of C. parvum as measured by in vitro excystation and cell infectivity, respectively, suggesting that excystation is not an adequate viability assay. Purified oocysts originating from three different suppliers were evaluated and showed marked differences with respect to their resistance to inactivation when using chlorine dioxide. Ct values of 75, 550, and 1,000 mg z min/liter were required to achieve approximately 2.0 log10 units of inactivation with oocysts from different sources. Finally, the study compared the relationship between easily measured indicators, including Bacillus subtilis (aerobic) Spores and Clostridium sporogenes (anaerobic) Spores, and C. parvum oocysts. The Bacterial Spores were found to be more sensitive to chlorine dioxide than C. parvum oocysts and therefore could not b
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chlorine dioxide inactivation of cryptosporidium parvum oocysts and Bacterial Spore indicators
Applied and Environmental Microbiology, 2001Co-Authors: Christian Chauret, Chris Z Radziminski, Michael Lepuil, Robin Creason, Robert C AndrewsAbstract:Cryptosporidium parvum, which is resistant to chlorine concentrations typically used in water treatment, is recognized as a significant waterborne pathogen. Recent studies have demonstrated that chlorine dioxide is a more efficient disinfectant than free chlorine against Cryptosporidium oocysts. It is not known, however, if oocysts from different suppliers are equally sensitive to chlorine dioxide. This study used both a most-probablenumber‐cell culture infectivity assay and in vitro excystation to evaluate chlorine dioxide inactivation kinetics in laboratory water at pH 8 and 21°C. The two viability methods produced significantly different results (P < 0.05). Products of disinfectant concentration and contact time (Ct values) of 1,000 mg z min/liter were needed to inactivate approximately 0.5 log10 and 2.0 log10 units (99% inactivation) of C. parvum as measured by in vitro excystation and cell infectivity, respectively, suggesting that excystation is not an adequate viability assay. Purified oocysts originating from three different suppliers were evaluated and showed marked differences with respect to their resistance to inactivation when using chlorine dioxide. Ct values of 75, 550, and 1,000 mg z min/liter were required to achieve approximately 2.0 log10 units of inactivation with oocysts from different sources. Finally, the study compared the relationship between easily measured indicators, including Bacillus subtilis (aerobic) Spores and Clostridium sporogenes (anaerobic) Spores, and C. parvum oocysts. The Bacterial Spores were found to be more sensitive to chlorine dioxide than C. parvum oocysts and therefore could not be used as direct indicators of C. parvum inactivation for this disinfectant. In conclusion, it is suggested that future studies address issues such as oocyst purification protocols and the genetic diversity of C. parvum, since these factors might affect oocyst disinfection sensitivity.