Characterization of Clostridium difficile Spores Lacking Either SpoVAC or Dipicolinic Acid Synthetase

ABSTRACTThe spore-forming obligate anaerobeClostridium difficileis a leading cause of antibiotic-associated diarrhea around the world. In order forC. difficileto 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. InC. difficile, cortex hydrolysis is necessary for DPA release, whereas inBacillus subtilis, DPA release is necessary for cortex hydrolysis. Given this difference, we tested whether DPA synthesis and/or release was required forC. difficilespore germination by constructing mutations in eitherspoVACordpaAB, which encode an ion channel predicted to transport DPA into the forespore and the enzyme complex predicted to synthesize DPA, respectively.C. difficilespoVACanddpaABmutant spores lacked DPA but could be stably purified and were more hydrated than wild-type spores; in contrast,B. subtilisspoVACanddpaABmutant spores were unstable. AlthoughC. difficilespoVACanddpaABmutant 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. difficilespoVACmutant spores were significantly more sensitive to heat treatment thandpaABmutant spores, indicating that SpoVAC plays additional roles in conferring heat resistance. Taken together, our results demonstrate that SpoVAC and DPA synthetase controlC. difficilespore resistance and reveal differential requirements for these proteins among theFirmicutes.IMPORTANCEClostridium difficileis a spore-forming obligate anaerobe that causes ∼500,000 infections per year in the United States. Although spore germination is essential forC. difficileto cause disease, the factors required for this process have been only partially characterized. This study describes the roles of two factors, DpaAB and SpoVAC, which control the synthesis and release of dipicolinic acid (DPA), respectively, from bacterial spores. Previous studies of these proteins in other spore-forming organisms indicated that they are differentially required for spore formation, germination, and resistance. We now show that the proteins are dispensable forC. difficilespore formation and germination but are necessary for heat resistance. Thus, our study further highlights the diverse functions of DpaAB and SpoVAC in spore-forming organisms.

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The Conserved Spore Coat Protein SpoVM Is Largely Dispensable in Clostridium difficile Spore Formation

mSphere ◽

10.1128/msphere.00315-17 ◽

2017 ◽

Vol 2 (5) ◽

Cited By ~ 23

Author(s):

John W. Ribis ◽

Priyanka Ravichandran ◽

Emily E. Putnam ◽

Keyan Pishdadian ◽

Aimee Shen

Keyword(s):

United States ◽

Bacillus Subtilis ◽

Clostridium Difficile ◽

Diarrheal Disease ◽

The United States ◽

Spore Formation ◽

Minor Role ◽

Obligate Anaerobe ◽

Content Type ◽

A Minor

ABSTRACT The spore-forming obligate anaerobe Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. When C. difficile spores are ingested by susceptible individuals, they germinate within the gut and transform into vegetative, toxin-secreting cells. During infection, C. difficile must also induce spore formation to survive exit from the host. Since spore formation is essential for transmission, understanding the basic mechanisms underlying sporulation in C. difficile could inform the development of therapeutic strategies targeting spores. In this study, we determine the requirement of the C. difficile homolog of SpoVM, a protein that is essential for spore formation in Bacillus subtilis due to its regulation of coat and cortex formation. We observed that SpoVM plays a minor role in C. difficile spore formation, in contrast with B. subtilis, indicating that this protein would not be a good target for inhibiting spore formation. The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health care-associated infections in the United States. In order for this obligate anaerobe to transmit infection, it must form metabolically dormant spores prior to exiting the host. A key step during this process is the assembly of a protective, multilayered proteinaceous coat around the spore. Coat assembly depends on coat morphogenetic proteins recruiting distinct subsets of coat proteins to the developing spore. While 10 coat morphogenetic proteins have been identified in Bacillus subtilis, only two of these morphogenetic proteins have homologs in the Clostridia: SpoIVA and SpoVM. C. difficile SpoIVA is critical for proper coat assembly and functional spore formation, but the requirement for SpoVM during this process was unknown. Here, we show that SpoVM is largely dispensable for C. difficile spore formation, in contrast with B. subtilis. Loss of C. difficile SpoVM resulted in modest decreases (~3-fold) in heat- and chloroform-resistant spore formation, while morphological defects such as coat detachment from the forespore and abnormal cortex thickness were observed in ~30% of spoVM mutant cells. Biochemical analyses revealed that C. difficile SpoIVA and SpoVM directly interact, similarly to their B. subtilis counterparts. However, in contrast with B. subtilis, C. difficile SpoVM was not essential for SpoIVA to encase the forespore. Since C. difficile coat morphogenesis requires SpoIVA-interacting protein L (SipL), which is conserved exclusively in the Clostridia, but not the more broadly conserved SpoVM, our results reveal another key difference between C. difficile and B. subtilis spore assembly pathways. IMPORTANCE The spore-forming obligate anaerobe Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. When C. difficile spores are ingested by susceptible individuals, they germinate within the gut and transform into vegetative, toxin-secreting cells. During infection, C. difficile must also induce spore formation to survive exit from the host. Since spore formation is essential for transmission, understanding the basic mechanisms underlying sporulation in C. difficile could inform the development of therapeutic strategies targeting spores. In this study, we determine the requirement of the C. difficile homolog of SpoVM, a protein that is essential for spore formation in Bacillus subtilis due to its regulation of coat and cortex formation. We observed that SpoVM plays a minor role in C. difficile spore formation, in contrast with B. subtilis, indicating that this protein would not be a good target for inhibiting spore formation.

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Spore Cortex Hydrolysis Precedes Dipicolinic Acid Release during Clostridium difficile Spore Germination

Journal of Bacteriology ◽

10.1128/jb.02575-14 ◽

2015 ◽

Vol 197 (14) ◽

pp. 2276-2283 ◽

Cited By ~ 47

Author(s):

Michael B. Francis ◽

Charlotte A. Allen ◽

Joseph A. Sorg

Keyword(s):

Bacillus Subtilis ◽

Bile Acid ◽

Clostridium Difficile ◽

Spore Germination ◽

Dipicolinic Acid ◽

Model Organism ◽

Stage I ◽

Content Type ◽

The Core ◽

Dormant Spore

ABSTRACTBacterial spore germination is a process whereby a dormant spore returns to active, vegetative growth, and this process has largely been studied in the model organismBacillus subtilis. InB. subtilis, the initiation of germinant receptor-mediated spore germination is divided into two genetically separable stages. Stage I is characterized by the release of dipicolinic acid (DPA) from the spore core. Stage II is characterized by cortex degradation, and stage II is activated by the DPA released during stage I. Thus, DPA release precedes cortex hydrolysis duringB. subtilisspore germination. Here, we investigated the timing of DPA release and cortex hydrolysis duringClostridium difficilespore germination and found that cortex hydrolysis precedes DPA release. Inactivation of either the bile acid germinant receptor,cspC, or the cortex hydrolase,sleC, prevented both cortex hydrolysis and DPA release. Because both cortex hydrolysis and DPA release duringC. difficilespore germination are dependent on the presence of the germinant receptor and the cortex hydrolase, the release of DPA from the core may rely on the osmotic swelling of the core upon cortex hydrolysis. These results have implications for the hypothesized glycine receptor and suggest that the initiation of germinant receptor-mediatedC. difficilespore germination proceeds through a novel germination pathway.IMPORTANCEClostridium difficileinfects antibiotic-treated hosts and spreads between hosts as a dormant spore. In a host, spores germinate to the vegetative form that produces the toxins necessary for disease.C. difficilespore germination is stimulated by certain bile acids and glycine. We recently identified the bile acid germinant receptor as the germination-specific, protease-like CspC. CspC is likely cortex localized, where it can transmit the bile acid signal to the cortex hydrolase, SleC. Due to the differences in location of CspC compared to theBacillus subtilisgerminant receptors, we hypothesized that there are fundamental differences in the germination processes between the model organism andC. difficile. We found thatC. difficilespore germination proceeds through a novel pathway.

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Involvement of Coat Proteins in Bacillus subtilis Spore Germination in High-Salinity Environments

Applied and Environmental Microbiology ◽

10.1128/aem.01817-15 ◽

2015 ◽

Vol 81 (19) ◽

pp. 6725-6735 ◽

Cited By ~ 8

Author(s):

Katja Nagler ◽

Peter Setlow ◽

Kai Reineke ◽

Adam Driks ◽

Ralf Moeller

Keyword(s):

Bacillus Subtilis ◽

Spore Germination ◽

High Salinity ◽

Dipicolinic Acid ◽

Food Microbiology ◽

Coat Proteins ◽

Wild Type ◽

Protective Function ◽

Content Type ◽

Bacillus Subtilis Spore

ABSTRACTThe germination of spore-forming bacteria in high-salinity environments is of applied interest for food microbiology and soil ecology. It has previously been shown that high salt concentrations detrimentally affectBacillus subtilisspore germination, rendering this process slower and less efficient. The mechanistic details of these salt effects, however, remained obscure. Since initiation of nutrient germination first requires germinant passage through the spores' protective integuments, the aim of this study was to elucidate the role of the proteinaceous spore coat in germination in high-salinity environments. Spores lacking major layers of the coat due to chemical decoating or mutation germinated much worse in the presence of NaCl than untreated wild-type spores at comparable salinities. However, the absence of the crust, the absence of some individual nonmorphogenetic proteins, and the absence of either CwlJ or SleB had no or little effect on germination in high-salinity environments. Although the germination of spores lacking GerP (which is assumed to facilitate germinant flow through the coat) was generally less efficient than the germination of wild-type spores, the presence of up to 2.4 M NaCl enhanced the germination of these mutant spores. Interestingly, nutrient-independent germination by high pressure was also inhibited by NaCl. Taken together, these results suggest that (i) the coat has a protective function during germination in high-salinity environments; (ii) germination inhibition by NaCl is probably not exerted at the level of cortex hydrolysis, germinant accessibility, or germinant-receptor binding; and (iii) the most likely germination processes to be inhibited by NaCl are ion, Ca2+-dipicolinic acid, and water fluxes.

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The Products of the spoVA Operon Are Involved in Dipicolinic Acid Uptake into Developing Spores of Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.184.2.584-587.2002 ◽

2002 ◽

Vol 184 (2) ◽

pp. 584-587 ◽

Cited By ~ 75

Author(s):

Federico Tovar-Rojo ◽

Monica Chander ◽

Barbara Setlow ◽

Peter Setlow

Keyword(s):

Bacillus Subtilis ◽

Heat Resistance ◽

Dipicolinic Acid ◽

Lytic Enzyme ◽

Acid Uptake ◽

Wild Type ◽

Wet Heat

ABSTRACT Bacillus subtilis cells with mutations in the spoVA operon do not complete sporulation. However, a spoVA strain with mutations that remove all three of the spore’s functional nutrient germinant receptors (termed the ger3 mutations) or the cortex lytic enzyme SleB (but not CwlJ) did complete sporulation. ger3 spoVA and sleB spoVA spores lack dipicolinic acid (DPA) and have lower core wet densities and levels of wet heat resistance than wild-type or ger3 spores. These properties of ger3 spoVA and sleB spoVA spores are identical to those of ger3 spoVF and sleB spoVF spores that lack DPA due to deletion of the spoVF operon coding for DPA synthetase. Sporulation in the presence of exogenous DPA restored DPA levels in ger3 spoVF spores to 53% of the wild-type spore levels, but there was no incorporation of exogenous DPA into ger3 spoVA spores. These data indicate that one or more products of the spoVA operon are involved in DPA transport into the developing forespore during sporulation.

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Dipicolinic Acid Release by Germinating Clostridium difficile Spores Occurs through a Mechanosensing Mechanism

mSphere ◽

10.1128/msphere.00306-16 ◽

2016 ◽

Vol 1 (6) ◽

Cited By ~ 22

Author(s):

Michael B. Francis ◽

Joseph A. Sorg

Keyword(s):

Clostridium Difficile ◽

Bile Acids ◽

Spore Germination ◽

Dipicolinic Acid ◽

Model Organism ◽

Lytic Enzyme ◽

Prior Work ◽

Spore Form ◽

Content Type ◽

Dormant Spore

ABSTRACT Clostridium difficile is transmitted between hosts in the form of a dormant spore, and germination by C. difficile spores is required to initiate infection, because the toxins that are necessary for disease are not deposited on the spore form. Importantly, the C. difficile spore germination pathway represents a novel pathway for bacterial spore germination. Prior work has shown that the order of events during C. difficile spore germination (cortex degradation and DPA release) is flipped compared to the events during B. subtilis spore germination, a model organism. Here, we further characterize the C. difficile spore germination pathway and summarize our findings indicating that DPA release by germinating C. difficile spores occurs through a mechanosensing mechanism in response to the degradation of the spore cortex. Classically, dormant endospores are defined by their resistance properties, particularly their resistance to heat. Much of the heat resistance is due to the large amount of dipicolinic acid (DPA) stored within the spore core. During spore germination, DPA is released and allows for rehydration of the otherwise-dehydrated core. In Bacillus subtilis, 7 proteins are encoded by the spoVA operon and are important for DPA release. These proteins receive a signal from the activated germinant receptor and release DPA. This DPA activates the cortex lytic enzyme CwlJ, and cortex degradation begins. In Clostridium difficile, spore germination is initiated in response to certain bile acids and amino acids. These bile acids interact with the CspC germinant receptor, which then transfers the signal to the CspB protease. Activated CspB cleaves the cortex lytic enzyme, pro-SleC, to its active form. Subsequently, DPA is released from the core. C. difficile encodes orthologues of spoVAC, spoVAD, and spoVAE. Of these, the B. subtilis SpoVAC protein was shown to be capable of mechanosensing. Because cortex degradation precedes DPA release during C. difficile spore germination (opposite of what occurs in B. subtilis), we hypothesized that cortex degradation would relieve the osmotic constraints placed on the inner spore membrane and permit DPA release. Here, we assayed germination in the presence of osmolytes, and we found that they can delay DPA release from germinating C. difficile spores while still permitting cortex degradation. Together, our results suggest that DPA release during C. difficile spore germination occurs though a mechanosensing mechanism. IMPORTANCE Clostridium difficile is transmitted between hosts in the form of a dormant spore, and germination by C. difficile spores is required to initiate infection, because the toxins that are necessary for disease are not deposited on the spore form. Importantly, the C. difficile spore germination pathway represents a novel pathway for bacterial spore germination. Prior work has shown that the order of events during C. difficile spore germination (cortex degradation and DPA release) is flipped compared to the events during B. subtilis spore germination, a model organism. Here, we further characterize the C. difficile spore germination pathway and summarize our findings indicating that DPA release by germinating C. difficile spores occurs through a mechanosensing mechanism in response to the degradation of the spore cortex.

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Maturation of Released Spores Is Necessary for Acquisition of Full Spore Heat Resistance during Bacillus subtilis Sporulation

Applied and Environmental Microbiology ◽

10.1128/aem.05031-11 ◽

2011 ◽

Vol 77 (19) ◽

pp. 6746-6754 ◽

Cited By ~ 39

Author(s):

Jose-Luis Sanchez-Salas ◽

Barbara Setlow ◽

Pengfei Zhang ◽

Yong-qing Li ◽

Peter Setlow

Keyword(s):

Bacillus Subtilis ◽

Heat Resistance ◽

Uv Radiation ◽

Dipicolinic Acid ◽

Sporulation Medium ◽

Content Type ◽

Dry Heat ◽

Lower Resistance ◽

Wet Heat ◽

Spore Maturation

ABSTRACTThe first ∼10% of spores released from sporangia (early spores) duringBacillus subtilissporulation were isolated, and their properties were compared to those of the total spores produced from the same culture. The early spores had significantly lower resistance to wet heat and hypochlorite than the total spores but identical resistance to dry heat and UV radiation. Early and total spores also had the same levels of core water, dipicolinic acid, and Ca and germinated similarly with several nutrient germinants. The wet heat resistance of the early spores could be increased to that of total spores if early spores were incubated in conditioned sporulation medium for ∼24 h at 37°C (maturation), and some hypochlorite resistance was also restored. The maturation of early spores took place in pH 8 buffer with Ca2+but was blocked by EDTA; maturation was also seen with early spores of strains lacking the CotE protein or the coat-associated transglutaminase, both of which are needed for normal coat structure. Nonetheless, it appears to be most likely that it is changes in coat structure that are responsible for the increased resistance to wet heat and hypochlorite upon early spore maturation.

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Properties of Aged Spores ofBacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.00231-19 ◽

2019 ◽

Vol 201 (14) ◽

Cited By ~ 4

Author(s):

Emily Camilleri ◽

George Korza ◽

Joshua Green ◽

Jianhui Yuan ◽

Yong-qing Li ◽

...

Keyword(s):

Protein Synthesis ◽

Spore Germination ◽

Dipicolinic Acid ◽

23S Rrna ◽

Published Data ◽

Sporulation Medium ◽

Content Type ◽

Phosphoglyceric Acid ◽

Wet Heat ◽

Almost All

ABSTRACTBacillusspores incubated on plates for 2 to 98 days at 37°C had identical Ca-dipicolinic acid contents, exhibited identical viability on rich- or poor-medium plates, germinated identically in liquid with all germinants tested, identically returned to vegetative growth in rich or minimal medium, and exhibited essentially identical resistance to dry heat and similar resistance to UV radiation. However, the oldest spores had a lower core water content and significantly higher wet heat and NaOCl resistance. In addition, 47- and 98-day spores had lost >98% of intact 16S and 23S rRNA and 97 to 99% of almost all mRNAs, although minimal amounts of mononucleotides were generated in 91 days. Levels of 3-phosphoglyceric acid (3PGA) also fell 30 to 60% in the oldest spores, but how the 3PGA was lost is not clear. These results indicate that (i) translation of dormant spore mRNA is not essential for completion of spore germination, nor is protein synthesis from any mRNA; (ii) in sporulation for up to 91 days at 37°C, the RNA broken down generates minimal levels of mononucleotides; and (iii) the lengths of time that spores are incubated in sporulation medium should be considered when determining conditions for spore inactivation by wet heat, in particular, in using spores to test for the efficacy of sterilization regimens.IMPORTANCEWe show that spores incubated at 37°C on sporulation plates for up to 98 days have lost almost all mRNAs and rRNAs, yet the aged spores germinated and outgrew as well as 2-day spores, and all these spores had identical viability. Thus, it is unlikely that spore mRNA, rRNA, or protein synthesis is important in spore germination. Spores incubated for 47 to 98 days also had much higher wet heat resistance than 2-day spores, suggesting that spore “age” should be considered in generating spores for tests of sterilization assurance. These data are the first to show complete survival of hydrated spores for ∼100 days, complementing published data showing dry-spore survival for years.

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Effects of High-Pressure Treatment on Spores of Clostridium Species

Applied and Environmental Microbiology ◽

10.1128/aem.01363-16 ◽

2016 ◽

Vol 82 (17) ◽

pp. 5287-5297 ◽

Cited By ~ 16

Author(s):

Christopher J. Doona ◽

Florence E. Feeherry ◽

Barbara Setlow ◽

Shiwei Wang ◽

William Li ◽

...

Keyword(s):

High Pressure ◽

Clostridium Difficile ◽

Clostridium Perfringens ◽

Dipicolinic Acid ◽

Food Contamination ◽

Opportunistic Pathogen ◽

Content Type ◽

Wet Heat ◽

Food Sterilization ◽

Temperature Levels

ABSTRACTThis work analyzes the high-pressure (HP) germination of spores of the food-borne pathogenClostridium perfringens(with inner membrane [IM] germinant receptors [GRs]) and the opportunistic pathogenClostridium difficile(with no IM GRs), which has growing implications as an emerging food safety threat. In contrast to those of spores ofBacillusspecies, mechanisms of HP germination of clostridial spores have not been well studied. HP treatments triggerBacillusspore germination through spores' IM GRs at ∼150 MPa or through SpoVA channels for release of spores' dipicolinic acid (DPA) at ≥400 MPa, and DPA-less spores have lower wet heat resistance than dormant spores. We found thatC. difficilespores exhibited no germination events upon 150-MPa treatment and were not heat sensitized. In contrast, 150-MPa-treated unactivatedC. perfringensspores released DPA and became heat sensitive, although most spores did not complete germination by fully rehydrating the spore core, but this treatment of heat-activated spores led to almost complete germination and greater heat sensitization. Spores of both clostridial organisms released DPA during 550-MPa treatment, butC. difficilespores did not complete germination and remained heat resistant. Heat-activated 550-MPa-HP-treatedC. perfringensspores germinated almost completely and became heat sensitive. However, unactivated 550-MPa-treatedC. perfringensspores did not germinate completely and were less heat sensitive than spores that completed germination. SinceC. difficileandC. perfringensspores use different mechanisms for sensing germinants, our results may allow refinement of HP methods for their inactivation in foods and other applications and may guide the development of commercially sterile low-acid foods.IMPORTANCESpores of various clostridial organisms cause human disease, sometimes due to food contamination by spores. Because of these spores' resistance to normal decontamination regimens, there is continued interest in ways to kill spores without compromising food quality. High hydrostatic pressure (HP) under appropriate conditions can inactivate bacterial spores. With growing use of HP for food pasteurization, advancement of HP for commercial production of sterile low-acid foods requires understanding of mechanisms of spores' interactions with HP. While much is known about HP germination and inactivation of spores ofBacillusspecies, how HP germinates and inactivates clostridial spores is less well understood. In this work we have tried to remedy this information deficit by examining germination of spores ofClostridium difficileandClostridium perfringensby several HP and temperature levels. The results may give insight that could facilitate more efficient methods for spore eradication in food sterilization or pasteurization, biodecontamination, and health care.

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Slow Leakage of Ca-Dipicolinic Acid from Individual Bacillus Spores during Initiation of Spore Germination

Journal of Bacteriology ◽

10.1128/jb.02490-14 ◽

2015 ◽

Vol 197 (6) ◽

pp. 1095-1103 ◽

Cited By ~ 19

Author(s):

Shiwei Wang ◽

Peter Setlow ◽

Yong-qing Li

Keyword(s):

Spore Germination ◽

Dipicolinic Acid ◽

Lytic Enzyme ◽

Early Event ◽

Laser Tweezers ◽

Wild Type ◽

Content Type ◽

Rapid Release ◽

Contrast Microscopy ◽

Bacillus Spores

When exposed to nutrient or nonnutrient germinants, individualBacillusspores 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 withBacillus subtilisspores 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 timeT1; (ii) the period of slow DPA leakage (ΔTleakage=Tlag−T1) was heterogeneous among individual spores, although the amount of DPA released in this period was relatively constant; (iii) increases in germination temperature significantly decreasedT1times but increased values of ΔTleakage; (iv) upon germination withl-valine for 10 min followed by addition ofd-alanine to block further germination, all germinated spores hadT1times of less than 10 min, suggesting thatT1is the time when spores become committed to germinate; (v) elevated levels of SpoVA proteins involved in DPA movement in spore germination decreasedT1andTlagtimes 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 ΔTleakagetimes in which more DPA was released; and (vii) there was slow DPA leakage early in germination ofB. subtilisspores by the nonnutrients CaDPA and dodecylamine and in nutrient germination ofBacillus cereusandBacillus megateriumspores. Overall, these findings have identified and characterized a new early event inBacillusspore germination.

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Properties of spores of Bacillus subtilis with or without a transposon that decreases spore germination and increases spore wet heat resistance

Journal of Applied Microbiology ◽

10.1111/jam.15163 ◽

2021 ◽

Author(s):

Yannong Luo ◽

George Korza ◽

Angela M. DeMarco ◽

Oscar P. Kuipers ◽

Yong‐qing Li ◽

...

Keyword(s):

Bacillus Subtilis ◽

Heat Resistance ◽

Spore Germination ◽

Wet Heat

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