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Bacterial Endospores: Stress Resistance and Germination

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Microbiology".

Deadline for manuscript submissions: closed (31 July 2022) | Viewed by 24348

Special Issue Editors


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Guest Editor
Swammerdam Institute for Life Sciences, Amsterdam, The Netherlands
Interests: spore germination; spore proteomics; microbiota; antimicrobial resistance

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Guest Editor
Department of Molecular Biology and Biophysics, UConn Health, 263 Farmington Avenue, Farmington, CT 06030-3305, USA
Interests: spores; germination; spore inactivation
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Special Issue Information

Dear Colleagues,

Bacterial spores are the ultimate survival capsule occurring in nature. They have been around for eons and have evolved an unprecedented survival ability amongst all Life on Earth, even in the face of many adverse environmental conditions, such as: i) extreme acid and alkaline conditions; ii) the environmental niche of the gastrointestinal tract of hosts; iii) desiccation; iv) extreme temperatures, including those used in contemporary food manufacturing. The resistance conferred by the spores of both anaerobes, such as Clostridia and aerobic organisms, from the genus Bacillus has been long enigmatic, as the structures involved in spore resistance were, by nature, refractive to detailed biochemical analysis methods. In recent decades, genetic analyses and, more recently, genomic and proteomic analysis have contributed to unravelling some of the secrets of bacterial spore stress resistance, as well as spore germination mechanisms. Nevertheless, the mechanism(s) of the extreme high heat resistance of many spore formers remains unresolved, as do questions about spore germination triggering and the stress resistance of spores of many recently identified gut microbes.

Interestingly, imaging and molecular techniques applied to spore biology have shown tremendous phenotypical heterogeneity in genetically homogeneous populations, demonstrating this at the actual germination level by assessing the kinetics of spore water uptake and calcium dipicolinic acid secretion,, as well as at the level of individual germination proteins. It has been shown that these latter spore proteins are organized differently, at least in spores of the human pathogen Clostridiades diffcile, compared to the most extensively studied Bacillus subtilis. In the latter, germinant receptor (GR) proteins are organized in what has been termed germinosome complexes in the spore inner membrane (IM), and GR activation triggers germination, whereas in the former, such IM GRs are not observed. Instead, C. difficile spores have pseudoproteases and proteases in their cortex, which interact with (co-)germinants and activate a cortex lytic enzyme zymogen, thus starting germination. Finally, both mechanisms converge upon the need to open a SpoVA channel, allowing secretion of the spore core’s huge depot of calcium dipicolinc acid and, in a yet-to-be-defined manner, the uptake of water. This exemplifies the open major research questions in the field, such as: (1) where do the germinants actually bind on germinant receptor proteins or their functional equivalents; (2) what structural modification does such binding induce to allow for a functional interaction with the SpoVA channel proteins and what exactly is this “interaction”; (3) what drives the heterogeneity in the molecular composition of the germination apparatus of spores within genetically homogeneous populations; (4) which structures confer extreme heat resistance upon spores and how does wet heat kill spores; and (5) how widespread are the spore germination molecular physiological characteristics identified in Bacillus subtilis?

Prof. Dr. Stanley Brul
Prof. Dr. Peter Setlow
Guest Editors

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Keywords

  • spores
  • microbiome
  • microbiota
  • food chain
  • stress resistance
  • heat stress
  • germination heterogeneity
  • germinosome
  • SpoVA channel
  • calcium dipicolinc acid

Published Papers (6 papers)

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Research

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17 pages, 3588 KiB  
Article
Sporulation Strategies and Potential Role of the Exosporium in Survival and Persistence of Clostridium botulinum
by Inês M. Portinha, François P. Douillard, Hannu Korkeala and Miia Lindström
Int. J. Mol. Sci. 2022, 23(2), 754; https://doi.org/10.3390/ijms23020754 - 11 Jan 2022
Cited by 11 | Viewed by 4714
Abstract
Clostridium botulinum produces the botulinum neurotoxin that causes botulism, a rare but potentially lethal paralysis. Endospores play an important role in the survival, transmission, and pathogenesis of C. botulinum. C. botulinum strains are very diverse, both genetically and ecologically. Group I strains [...] Read more.
Clostridium botulinum produces the botulinum neurotoxin that causes botulism, a rare but potentially lethal paralysis. Endospores play an important role in the survival, transmission, and pathogenesis of C. botulinum. C. botulinum strains are very diverse, both genetically and ecologically. Group I strains are terrestrial, mesophilic, and produce highly heat-resistant spores, while Group II strains can be terrestrial (type B) or aquatic (type E) and are generally psychrotrophic and produce spores of moderate heat resistance. Group III strains are either terrestrial or aquatic, mesophilic or slightly thermophilic, and the heat resistance properties of their spores are poorly characterized. Here, we analyzed the sporulation dynamics in population, spore morphology, and other spore properties of 10 C. botulinum strains belonging to Groups I–III. We propose two distinct sporulation strategies used by C. botulinum Groups I–III strains, report their spore properties, and suggest a putative role for the exosporium in conferring high heat resistance. Strains within each physiological group produced spores with similar characteristics, likely reflecting adaptation to respective environmental habitats. Our work provides new information on the spores and on the population and single-cell level strategies in the sporulation of C. botulinum. Full article
(This article belongs to the Special Issue Bacterial Endospores: Stress Resistance and Germination)
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18 pages, 1868 KiB  
Article
Insights into the Structure and Protein Composition of Moorella thermoacetica Spores Formed at Different Temperatures
by Tiffany Malleck, Fatima Fekraoui, Isabelle Bornard, Céline Henry, Eloi Haudebourg, Stella Planchon and Véronique Broussolle
Int. J. Mol. Sci. 2022, 23(1), 550; https://doi.org/10.3390/ijms23010550 - 04 Jan 2022
Cited by 1 | Viewed by 2225
Abstract
The bacterium Moorella thermoacetica produces the most heat-resistant spores of any spoilage-causing microorganism known in the food industry. Previous work by our group revealed that the resistance of these spores to wet heat and biocides was lower when spores were produced at a [...] Read more.
The bacterium Moorella thermoacetica produces the most heat-resistant spores of any spoilage-causing microorganism known in the food industry. Previous work by our group revealed that the resistance of these spores to wet heat and biocides was lower when spores were produced at a lower temperature than the optimal temperature. Here, we used electron microcopy to characterize the ultrastructure of the coat of the spores formed at different sporulation temperatures; we found that spores produced at 55 °C mainly exhibited a lamellar inner coat tightly associated with a diffuse outer coat, while spores produced at 45 °C showed an inner and an outer coat separated by a less electron-dense zone. Moreover, misarranged coat structures were more frequently observed when spores were produced at the lower temperature. We then analyzed the proteome of the spores obtained at either 45 °C or 55 °C with respect to proteins putatively involved in the spore coat, exosporium, or in spore resistance. Some putative spore coat proteins, such as CotSA, were only identified in spores produced at 55 °C; other putative exosporium and coat proteins were significantly less abundant in spores produced at 45 °C. Altogether, our results suggest that sporulation temperature affects the structure and protein composition of M. thermoacetica spores. Full article
(This article belongs to the Special Issue Bacterial Endospores: Stress Resistance and Germination)
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16 pages, 3423 KiB  
Article
Dynamics of Germinosome Formation and FRET-Based Analysis of Interactions between GerD and Germinant Receptor Subunits in Bacillus cereus Spores
by Yan Wang, Ronald M. P. Breedijk, Mark A. Hink, Lars Bults, Norbert O. E. Vischer, Peter Setlow and Stanley Brul
Int. J. Mol. Sci. 2021, 22(20), 11230; https://doi.org/10.3390/ijms222011230 - 18 Oct 2021
Cited by 6 | Viewed by 2131
Abstract
Spores of the bacterium Bacillus cereus can cause disease in humans due to contamination of raw materials for food manufacturing. These dormant, resistant spores can survive for years in the environment, but can germinate and grow when their surroundings become suitable, and spore [...] Read more.
Spores of the bacterium Bacillus cereus can cause disease in humans due to contamination of raw materials for food manufacturing. These dormant, resistant spores can survive for years in the environment, but can germinate and grow when their surroundings become suitable, and spore germination proteins play an important role in the decision to germinate. Since germinated spores have lost dormant spores’ extreme resistance, knowledge about the formation and function of germination proteins could be useful in suggesting new preservation strategies to control B. cereus spores. In this study, we confirmed that the GerR germinant receptor’s (GR) A, B, and C subunits and GerD co-localize in B. cereus spore inner membrane (IM) foci termed germinosomes. The interaction between these proteins was examined by using fusions to the fluorescent reporter proteins SGFP2 and mScarlet-I and Förster Resonance Energy Transfer (FRET). This work found that the FRET efficiency was 6% between GerR(A-C-B)–SGFP2 and GerD–mScarlet-I, but there was no FRET between GerD–mScarlet-I and either GerRA–SGFP2 or GerRC–SGFP2. These results and that GerD does not interact with a GR C-subunit in vitro suggest that, in the germinosome, GerD interacts primarily with the GR B subunit. The dynamics of formation of germinosomes with GerR(A-C-B)–SGFP2 and GerD–mScarlet-I was also followed during sporulation. Our results showed heterogeneity in the formation of FRET positive foci of GerR(A-C-B)–SGFP2 and GerD–mScarlet-I; and while some foci formed at the same time, the formation of foci in the FRET channel could be significantly delayed. The latter finding suggests that either the GerR GR can at least transiently form IM foci in the absence of GerD, or that, while GerD is essential for GerR foci formation, the time to attain the final germinosome structure with close contacts between GerD and GerR can be heterogeneous. Full article
(This article belongs to the Special Issue Bacterial Endospores: Stress Resistance and Germination)
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14 pages, 2146 KiB  
Article
High Resolution Analysis of Proteome Dynamics during Bacillus subtilis Sporulation
by Zhiwei Tu, Henk L. Dekker, Winfried Roseboom, Bhagyashree N. Swarge, Peter Setlow, Stanley Brul and Gertjan Kramer
Int. J. Mol. Sci. 2021, 22(17), 9345; https://doi.org/10.3390/ijms22179345 - 28 Aug 2021
Cited by 5 | Viewed by 2308
Abstract
Bacillus subtilis vegetative cells switch to sporulation upon nutrient limitation. To investigate the proteome dynamics during sporulation, high-resolution time-lapse proteomics was performed in a cell population that was induced to sporulate synchronously. Here, we are the first to comprehensively investigate the changeover of [...] Read more.
Bacillus subtilis vegetative cells switch to sporulation upon nutrient limitation. To investigate the proteome dynamics during sporulation, high-resolution time-lapse proteomics was performed in a cell population that was induced to sporulate synchronously. Here, we are the first to comprehensively investigate the changeover of sporulation regulatory proteins, coat proteins, and other proteins involved in sporulation and spore biogenesis. Protein co-expression analysis revealed four co-expressed modules (termed blue, brown, green, and yellow). Modules brown and green are upregulated during sporulation and contain proteins associated with sporulation. Module blue is negatively correlated with modules brown and green, containing ribosomal and metabolic proteins. Finally, module yellow shows co-expression with the three other modules. Notably, several proteins not belonging to any of the known transcription regulons were identified as co-expressed with modules brown and green, and might also play roles during sporulation. Finally, levels of some coat proteins, for example morphogenetic coat proteins, decreased late in sporulation. Full article
(This article belongs to the Special Issue Bacterial Endospores: Stress Resistance and Germination)
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Review

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26 pages, 792 KiB  
Review
Mechanisms and Applications of Bacterial Sporulation and Germination in the Intestine
by Nienke Koopman, Lauren Remijas, Jurgen Seppen, Peter Setlow and Stanley Brul
Int. J. Mol. Sci. 2022, 23(6), 3405; https://doi.org/10.3390/ijms23063405 - 21 Mar 2022
Cited by 11 | Viewed by 8398
Abstract
Recent studies have suggested a major role for endospore forming bacteria within the gut microbiota, not only as pathogens but also as commensal and beneficial members contributing to gut homeostasis. In this review the sporulation processes, spore properties, and germination processes will be [...] Read more.
Recent studies have suggested a major role for endospore forming bacteria within the gut microbiota, not only as pathogens but also as commensal and beneficial members contributing to gut homeostasis. In this review the sporulation processes, spore properties, and germination processes will be explained within the scope of the human gut. Within the gut, spore-forming bacteria are known to interact with the host’s immune system, both in vegetative cell and spore form. Together with the resistant nature of the spore, these characteristics offer potential for spores’ use as delivery vehicles for therapeutics. In the last part of the review, the therapeutic potential of spores as probiotics, vaccine vehicles, and drug delivery systems will be discussed. Full article
(This article belongs to the Special Issue Bacterial Endospores: Stress Resistance and Germination)
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12 pages, 2665 KiB  
Review
Enigmatic Pilus-Like Endospore Appendages of Bacillus cereus Group Species
by Ephrem Debebe Zegeye, Brajabandhu Pradhan, Ann-Katrin Llarena and Marina Aspholm
Int. J. Mol. Sci. 2021, 22(22), 12367; https://doi.org/10.3390/ijms222212367 - 16 Nov 2021
Cited by 3 | Viewed by 2980
Abstract
The endospores (spores) of many Bacillus cereus sensu lato species are decorated with multiple hair/pilus-like appendages. Although they have been observed for more than 50 years, all efforts to characterize these fibers in detail have failed until now, largely due to their extraordinary [...] Read more.
The endospores (spores) of many Bacillus cereus sensu lato species are decorated with multiple hair/pilus-like appendages. Although they have been observed for more than 50 years, all efforts to characterize these fibers in detail have failed until now, largely due to their extraordinary resilience to proteolytic digestion and chemical solubilization. A recent structural analysis of B. cereus endospore appendages (Enas) using cryo-electron microscopy has revealed the structure of two distinct fiber morphologies: the longer and more abundant “Staggered-type” (S-Ena) and the shorter “Ladder-like” type (L-Ena), which further enabled the identification of the genes encoding the S-Ena. Ena homologs are widely and uniquely distributed among B. cereus sensu lato species, suggesting that appendages play important functional roles in these species. The discovery of ena genes is expected to facilitate functional studies involving Ena-depleted mutant spores to explore the role of Enas in the interaction between spores and their environment. Given the importance of B. cereus spores for the food industry and in medicine, there is a need for a better understanding of their biological functions and physicochemical properties. In this review, we discuss the current understanding of the Ena structure and the potential roles these remarkable fibers may play in the adhesion of spores to biotic and abiotic surfaces, aggregation, and biofilm formation. Full article
(This article belongs to the Special Issue Bacterial Endospores: Stress Resistance and Germination)
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