The Effects of Heat Activation on Bacillus Spore Germination, with Nutrients or under High Pressure, with or without Various Germination Proteins

Luu, Stephanie; Cruz-Mora, Jose; Setlow, Barbara; Feeherry, Florence E.; Doona, Christopher J.; Setlow, Peter

doi:10.1128/aem.00193-15

Cited by 93 publications

(83 citation statements)

References 58 publications

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“…There is also evidence that a second CLE in C. perfringens spores, SleM, can play a small role in cortex hydrolysis in spore germination, although SleM alone is not sufficient for the germination of Ͼ99% of C. perfringens spores (22). The more rapid and complete germination of heat-activated C. perfringens spores exposed to 150 MPa of HP is consistent with the 150-MPa germination of spores of at least two Bacillus species being stimulated markedly by heat activation, a treatment that is thought to exert its effects primarily by increasing the responsiveness of IM GRs (16).…”

Section: Discussionsupporting

confidence: 52%

“…Treatment of B. subtilis spores at 150 MPa can also result in spores that are activated for germination, although these activated spores can decay to the unactivated dormant spore state depending on the incubation conditions (15). Interestingly, the 150-MPa HP germination of spores of some Bacillus species can be greatly potentiated by a heat activation step prior to HP treatment, most likely by activating at least some IM GRs (16). In contrast, HPs of 400 to 1,000 MPa (550 MPa and 50°C for B. subtilis spores) trigger the opening of a SpoVA protein channel in spores' IM, allowing the release of the spore core's huge depot (ϳ25% of dry weight) of dipicolinic acid (DPA) in a 1:1 chelate primarily with Ca 2ϩ (14).…”

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confidence: 99%

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Effects of High-Pressure Treatment on Spores of Clostridium Species

Doona

Feeherry

Setlow

et al. 2016

Appl Environ Microbiol

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This work analyzes the high-pressure (HP) germination of spores of the food-borne pathogen Clostridium perfringens (with inner membrane [IM] germinant receptors [GRs]) and the opportunistic pathogen Clostridium difficile (with no IM GRs), which has growing implications as an emerging food safety threat. In contrast to those of spores of Bacillus species, mechanisms of HP germination of clostridial spores have not been well studied. HP treatments trigger Bacillus spore germination through spores' IM GRs at ϳ150 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 that C. difficile spores exhibited no germination events upon 150-MPa treatment and were not heat sensitized. In contrast, 150-MPa-treated unactivated C. perfringens spores 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, but C. difficile spores did not complete germination and remained heat resistant. Heat-activated 550-MPa-HP-treated C. perfringens spores germinated almost completely and became heat sensitive. However, unactivated 550-MPa-treated C. perfringens spores did not germinate completely and were less heat sensitive than spores that completed germination. Since C. difficile and C. perfringens spores 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 of Bacillus species, 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 of Clostridium difficile and Clostridium perfringens by 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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Section: Discussionsupporting

confidence: 52%

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confidence: 99%

Effects of High-Pressure Treatment on Spores of Clostridium Species

Doona

Feeherry

Setlow

et al. 2016

Appl Environ Microbiol

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“…6). The similar or faster germination of the heat-treated spores was seen even though the untreated spores were given the heat activation treatments prior to germination shown to give maximal increases in spore germination (23,30).…”

Section: Resultsmentioning

confidence: 95%

“…The GR-dependent germinants used were as follows: B. subtilis spores, 10 mM L-valine or a mixture of 10 mM L-asparagine, 10 mM D-glucose, 10 mM D-fructose, and 10 mM KCl (AGFK); B. megaterium spores, 10 mM D-glucose or 50 mM KBr. Tb-DPA fluorescence was read every 5 min for 30 min to 2 h. Untreated spores were heat activated prior to germination as follows: 30 min or 2 h at 75°C for B. subtilis spores germinating with L-valine or AGFK, respectively, and 30 min at 60°C for B. megaterium spores (18,23). After heat activation, spores were cooled on ice for at least 15 min prior to germination experiments.…”

Section: Methodsmentioning

confidence: 99%

“…Spores of B. subtilis PS533 (wild type) with and without incubation at 80°C for 18 h were purified, purified untreated and heat-treated spores were germinated with either L-valine (A) or AGFK (B), and spore germination was monitored by measuring DPA release, all as described in Materials and Methods. Untreated spores were heat activated for 30 min or 2 h at 75°C prior to L-valine or AGFK germination, respectively, and then cooled; these heat activation conditions have been shown to be optimal for eliciting maximum spore germination with L-valine or AGFK (23). Symbols: OE, untreated spores; were degraded to mononucleotides, this would increase the levels of AMP in B. megaterium and B. subtilis spores 20-or 30-fold, respectively, and increase the levels of CMP, GMP, and UMP even more (11,28).…”

Section: Fig 6 Germination Of Spores Of B Subtilis With and Without mentioning

confidence: 99%

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Changes in Bacillus Spore Small Molecules, rRNA, Germination, and Outgrowth after Extended Sublethal Exposure to Various Temperatures: Evidence that Protein Synthesis Is Not Essential for Spore Germination

et al. 2016

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ABSTRACTrRNAs of dormant spores of Bacillus subtilis were >95% degraded during extended incubation at 50°C, as reported previously (E. Segev, Y. Smith, and S. Ben-Yehuda, Cell 148:139 -114, 2012, doi:http://dx.doi.org/10.1016/j.cell.2011.11.059), and this was also true of spores of Bacillus megaterium. Incubation of spores of these two species for ϳ20 h at 75 to 80°C also resulted in the degradation of all or the great majority of the 23S and 16S rRNAs, although this rRNA degradation was slower than nonenzymatic hydrolysis of purified rRNAs at these temperatures. This rRNA degradation at high temperature generated almost exclusively oligonucleotides with minimal levels of mononucleotides. RNase Y, suggested to be involved in rRNA hydrolysis during B. subtilis spore incubation at 50°C, did not play a role in B. subtilis spore rRNA breakdown at 80°C. Twenty hours of incubation of Bacillus spores at 70°C also decreased the already minimal levels of ATP in dormant spores 10-to 30-fold, to <0.01% of the total free adenine nucleotide levels. Spores depleted of rRNA were viable and germinated relatively normally, often even faster than starting spores. Their return to vegetative growth was also similar to that of untreated spores for B. megaterium spores and slower for heat-treated B. subtilis spores; accumulation of rRNA took place only after completion of spore germination. These findings thus strongly suggest that protein synthesis is not essential for Bacillus spore germination. Spores of Bacillus species formed in sporulation are metabolically dormant and extremely resistant to many severe treatments, including heat, radiation, and toxic chemicals (1-3). Although these spores can survive in this dormant resistant state for many years, if given the appropriate stimulus, generally small molecules that are found in environments that are appropriate for the growth of these bacteria, the spores can rapidly break dormancy in the process of spore germination and then outgrowth (1, 4, 5). Notably, spores' extreme resistance properties are lost in the process of germination and early outgrowth, and thus, germinated spores are much easier to kill than dormant spores. This property has significant applied implications, as dormant spores are the vectors for much food spoilage, as well as some foodborne and other human diseases. Consequently, there is significant interest in mechanisms that determine rates of spore germination and how sporulation conditions, as well as storage conditions, modify spores' resistance properties and their rates of germination.In addition to the applied interest in spore resistance and germination, there has also long been much basic interest in the mechanism of spore germination, in particular, the signal transduction pathways operating during the process (4, 5). The major proteins involved in Bacillus spore germination are as follows. (i) Germinant receptors (GRs) in spores' inner membrane (IM) recognize and respond to the physiological germinants specific for spores of each species/strain. (ii)...

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Bacterial Endospores

Flores¹,

Popham²

2020

Encyclopedia of Life Sciences

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Species of the genera Bacillus and Clostridium form dormant, resistant cells are known as endospores in response to stressful conditions. Endospores form inside of a mother cell, or sporangia, via a modified, asymmetrical cell division pathway followed by a variety of spore‐specific modifications to cell structure and content. Once the spore is formed, it is metabolically dormant and highly resistant to various insults such as heat, noxious chemicals, desiccation and UV irradiation. Several significant pathogens are spore formers, which makes disease prevention via food and environment sterilisation challenging. The resistance properties of spores are derived from chemical and physical alterations such as dehydration and mineralisation of the cytoplasm, protective alteration of DNA structure by specialised proteins and formation of impermeable proteinaceous spore coats. Dormancy is ended when the spore enters an environment where spore germination is favourable. Key Concepts Bacterial endospores can survive for decades and are highly resistant to numerous environmental insults. The resistance properties of bacterial endospores contribute to their roles in food spoilage and in human and animal pathogenesis. Bacterial endospores are cells with specialised modifications to their structure and contents. Bacterial endospores are produced via a simple developmental process involving cooperative and regulated gene expression of two cells. Heat resistance of bacterial endospores is determined by several factors, the most important being the relative dehydration of the spore core. Bacterial endospores possess a unique mode of resistance to UV irradiation involving specialised DNA‐binding proteins.

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The Effects of Heat Activation on Bacillus Spore Germination, with Nutrients or under High Pressure, with or without Various Germination Proteins

Cited by 93 publications

References 58 publications

Effects of High-Pressure Treatment on Spores of Clostridium Species

Effects of High-Pressure Treatment on Spores of Clostridium Species

Changes in Bacillus Spore Small Molecules, rRNA, Germination, and Outgrowth after Extended Sublethal Exposure to Various Temperatures: Evidence that Protein Synthesis Is Not Essential for Spore Germination

Bacterial Endospores

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