Function of the SpoVAEa and SpoVAF Proteins of Bacillus subtilis Spores

Pérez-Valdespino, Abigail; Li, Yunfeng; Setlow, Barbara; Ghosh, Sumana; Pan, Di; Korza, George; Feeherry, Florence E.; Doona, Christopher J.; Li, Yongqing; Hao, Bing; Setlow, Peter

doi:10.1128/jb.01546-14

Cited by 35 publications

(57 citation statements)

References 25 publications

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“…SpoVA proteins are present in spores' IM and are involved in CaDPA release during germination with nutrients, most likely directly (3,(13)(14)(15). Previous work showed Table 3) exhibited I lag values similar to those of germinating wild-type spores, indicating that elevated SpoVA levels did not result in more CaDPA leakage prior to T lag .…”

Section: Slowmentioning

confidence: 81%

“…The B. subtilis strains used in this study were PS832 (wild type), a prototrophic 168 strain, and its isogenic derivatives, including (i) PS533 (also a wild-type strain), strain PS832 carrying plasmid pUB110 providing resistance to kanamycin (10 g/ml) (12); (ii) PS3411, termed 1SpoVA, which overexpresses SpoVA proteins ϳ4-fold in spores (13) (the SpoVA proteins are almost certainly components of the channel in spores' IM through which CaDPA is released in stage I of spore germination [3,[13][14][15]); and (iii) FB111, which lacks the CLE CwlJ (16). Bacillus cereus T was originally obtained from H. O. Halvorson, and Bacillus megaterium QM B1551 was originally obtained from H. S. Levinson.…”

Section: Methodsmentioning

confidence: 99%

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

Wang

Setlow

2015

J Bacteriol

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b 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 ϳ20% of spore DPA) from individual spores that takes place prior to the lag time, T lag , 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 T 1 ; (ii) the period of slow DPA leakage (⌬T leakage ‫؍‬ T lag ؊ T 1 ) was heterogeneous among individual spores, although the amount of DPA released in this period was relatively constant; (iii) increases in germination temperature significantly decreased T 1 times but increased values of ⌬T leakage ; (iv) upon germination with L-valine for 10 min followed by addition of D-alanine to block further germination, all germinated spores had T 1 times of less than 10 min, suggesting that T 1 is the time when spores become committed to germinate; (v) elevated levels of SpoVA proteins involved in DPA movement in spore germination decreased T 1 and T lag times but not the amount of DPA released in ⌬T leakage ; (vi) lack of the cortex-lytic enzyme CwlJ increased DPA leakage during germination due to longer ⌬T leakage 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. Spores of Bacillus species can remain dormant for long periods and are extremely resistant to a variety of environmental stresses. However, under appropriate conditions, normally upon the binding of specific nutrients to spores' nutrient germinant receptors (GRs), spores can rapidly return to life in the process of germination followed by outgrowth (1-3). Germination is important not only to spores but also to the food and medical product industries, since spores of a number of species are major agents of food spoilage and foodborne disease and must germinate to cause their deleterious effects (3). In contrast to the resistant dormant spores that are hard to kill, germinated spores have lost most dormant spore resistance properties and are killed relatively easily (2, 3). Consequently, there is much interest in the mechanism of spore germination, since preventing this process or promoting it efficiently could have significant applied microbiology applications.Germination of Bacillus subtilis spores can be triggered by L-alanine or L-valine or a combination of L-asparagine, D-glucose, D-fructose, and K ϩ (AGFK). These nutrient germinants trigger germination by binding to and in...

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Section: Slowmentioning

confidence: 81%

Section: Methodsmentioning

confidence: 99%

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

Wang

Setlow

2015

J Bacteriol

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“…The only gene that was missing was spoVAEa, but the absence of this gene is in fact not uncommon for spore-forming species of the Bacillales and Clostridiales orders (54). In B. subtilis, deletion of spoVAEa has been associated with a slower nutrientinduced germination phenotype (54), but this fairly moderate decrease does not fully explain the dramatic loss in germination efficiency in B. thermoamylovorans (Fig.…”

Section: Discussionmentioning

confidence: 99%

Bacillus thermoamylovorans Spores with Very-High-Level Heat Resistance Germinate Poorly in Rich Medium despite the Presence of ger Clusters but Efficiently upon Exposure to Calcium-Dipicolinic Acid

Berendsen

Krawczyk

Klaus

et al. 2015

Appl Environ Microbiol

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c High-level heat resistance of spores of Bacillus thermoamylovorans poses challenges to the food industry, as industrial sterilization processes may not inactivate such spores, resulting in food spoilage upon germination and outgrowth. In this study, the germination and heat resistance properties of spores of four food-spoiling isolates were determined. Flow cytometry counts of spores were much higher than their counts on rich medium (maximum, 5%). Microscopic analysis revealed inefficient nutrientinduced germination of spores of all four isolates despite the presence of most known germination-related genes, including two operons encoding nutrient germinant receptors (GRs), in their genomes. In contrast, exposure to nonnutrient germinant calcium-dipicolinic acid (Ca-DPA) resulted in efficient (50 to 98%) spore germination. All four strains harbored cwlJ and gerQ genes, which are known to be essential for Ca-DPA-induced germination in Bacillus subtilis. When determining spore survival upon heating, low viable counts can be due to spore inactivation and an inability to germinate. To dissect these two phenomena, the recoveries of spores upon heat treatment were determined on plates with and without preexposure to Ca-DPA. The high-level heat resistance of spores as observed in this study (D 120°C , 1.9 ؎ 0.2 and 1.3 ؎ 0.1 min; z value, 12.2 ؎ 1.8°C) is in line with survival of sterilization processes in the food industry. The recovery of B. thermoamylovorans spores can be improved via nonnutrient germination, thereby avoiding gross underestimation of their levels in food ingredients. Bacillus endospores (spores) are widely present in nature and may contaminate food ingredients and food products. Due to the intrinsic stability of spores, which allows them to withstand environmental insults, sufficient inactivation of spores in commercially sterile food products is a major challenge for the food industry (1-4).Bacillus thermoamylovorans produces spores with high-level heat resistance (3), and the spores are known to survive industrial food sterilization processes. The organism is facultatively anaerobic and has the ability to grow at temperatures between 40°C and 58°C (5, 6). In our experience, strains of B. thermoamylovorans are able to grow at 37°C but not at 30°C. The organism was first described as a nonsporogenous species (5, 7), but in an amended species description the formation of spores was reported (8). The occurrence of B. thermoamylovorans in a gelatin production plant and at dairy farms has been reported (3, 9). The genome sequence of one non-food-related B. thermoamylovorans strain from a biogas plant was published recently (6). Overall, the species has not been well characterized, and little is known about the spore properties that are important for control in foods, including spore resistance to various processing conditions and germination of spores that survive.When spores exit dormancy via germination, food spoilage can occur upon outgrowth. These processes have been well studied in Bacillus subtil...

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“…In fact, during the 2-h exposure to 10 mM AGFK, DPA was released less efficiently from B4417 spores (31.6% Ϯ 0.7% DPA released) than from B4417ΔspoVA 2mob (45.8% Ϯ 3.1%) and 168 (67.1% Ϯ 6.3%) spores, as monitored by fluorescence of released DPA with terbium (Tb 3ϩ -DPA) (data not shown) measured in a fluorescence plate reader, as similarly described before (65). In contrast to the regular conserved spoVA operon, spoVA 2mob does not encode homologs of the SpoVAEa and SpoVAF proteins, which are reportedly important for DPA release during GR-dependent spore germination but not for DPA uptake during spore formation (66). For this reason, the spoVA 2mob products may not be able to support DPA transport during germination.…”

Section: Discussionmentioning

confidence: 99%

Spore Heat Activation Requirements and Germination Responses Correlate with Sequences of Germinant Receptors and with the Presence of a Specific spoVA ^2mob Operon in Foodborne Strains of Bacillus subtilis

Krawczyk

Jong

Omony

et al. 2017

Appl Environ Microbiol

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Spore heat resistance, germination, and outgrowth are problematic bacterial properties compromising food safety and quality. Large interstrain variation in these properties makes prediction and control of spore behavior challenging. Highlevel heat resistance and slow germination of spores of some natural Bacillus subtilis isolates, encountered in foods, have been attributed to the occurrence of the spoVA 2mob operon carried on the Tn1546 transposon. In this study, we further investigate the correlation between the presence of this operon in high-level-heat-resistant spores and their germination efficiencies before and after exposure to various sublethal heat treatments (heat activation, or HA), which are known to significantly improve spore responses to nutrient germinants. We show that high-level-heat-resistant spores harboring spoVA 2mob required higher HA temperatures for efficient germination than spores lacking spoVA 2mob . The optimal spore HA requirements additionally depended on the nutrients used to trigger germination, L-alanine (L-Ala), or a mixture of L-asparagine, D-glucose, D-fructose, and K ϩ (AGFK). The distinct HA requirements of these two spore germination pathways are likely related to differences in properties of specific germinant receptors. Moreover, spores that germinated inefficiently in AGFK contained specific changes in sequences of the GerB and GerK germinant receptors, which are involved in this germination response. In contrast, no relation was found between transcription levels of main germination genes and spore germination phenotypes. The findings presented in this study have great implications for practices in the food industry, where heat treatments are commonly used to inactivate pathogenic and spoilage microbes, including bacterial spore formers.IMPORTANCE This study describes a strong variation in spore germination capacities and requirements for a heat activation treatment, i.e., an exposure to sublethal heat that increases spore responsiveness to nutrient germination triggers, among 17 strains of B. subtilis, including 9 isolates from spoiled food products. Spores of industrial foodborne isolates exhibited, on average, less efficient and slower germination responses and required more severe heat activation than spores from other sources. High heat activation requirements and inefficient, slow germination correlated with elevated resistance of spores to heat and with specific genetic features, indicating a common genetic basis of these three phenotypic traits. Clearly, interstrain variation and numerous factors that shape spore germination behavior challenge standardization of methods to recover highly heat-resistant spores from the environment and have an impact on the efficacy of preservation techniques used by the food industry to control spores.

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Function of the SpoVAEa and SpoVAF Proteins of Bacillus subtilis Spores

Cited by 35 publications

References 25 publications

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

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

Bacillus thermoamylovorans Spores with Very-High-Level Heat Resistance Germinate Poorly in Rich Medium despite the Presence of ger Clusters but Efficiently upon Exposure to Calcium-Dipicolinic Acid

Spore Heat Activation Requirements and Germination Responses Correlate with Sequences of Germinant Receptors and with the Presence of a Specific spoVA ^2mob Operon in Foodborne Strains of Bacillus subtilis

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Function of the SpoVAEa and SpoVAF Proteins of Bacillus subtilis Spores

Cited by 35 publications

References 25 publications

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

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

Bacillus thermoamylovorans Spores with Very-High-Level Heat Resistance Germinate Poorly in Rich Medium despite the Presence of ger Clusters but Efficiently upon Exposure to Calcium-Dipicolinic Acid

Spore Heat Activation Requirements and Germination Responses Correlate with Sequences of Germinant Receptors and with the Presence of a Specific spoVA 2mob Operon in Foodborne Strains of Bacillus subtilis

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Spore Heat Activation Requirements and Germination Responses Correlate with Sequences of Germinant Receptors and with the Presence of a Specific spoVA ^2mob Operon in Foodborne Strains of Bacillus subtilis