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

2002 ◽  
Vol 184 (2) ◽  
pp. 584-587 ◽  
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.

scholarly journals Molecular Physiological Characterization of a High Heat Resistant Spore Forming Bacillus subtilis Food Isolate

2021 ◽  
Vol 9 (3) ◽  
pp. 667
Author(s):  
Zhiwei Tu ◽  
Peter Setlow ◽  
Stanley Brul ◽  
Gertjan Kramer
Keyword(s):  
Bacillus Subtilis ◽  
Heat Resistance ◽  
Dipicolinic Acid ◽  
Laboratory Strain ◽  
High Heat ◽  
High Heat Resistance ◽  
Vegetative Cells ◽  
Heat Resistant ◽  
Food Isolate ◽  
Wet Heat

Bacterial endospores (spores) are among the most resistant living forms on earth. Spores of Bacillus subtilis A163 show extremely high resistance to wet heat compared to spores of laboratory strains. In this study, we found that spores of B. subtilis A163 were indeed very wet heat resistant and released dipicolinic acid (DPA) very slowly during heat treatment. We also determined the proteome of vegetative cells and spores of B. subtilis A163 and the differences in these proteomes from those of the laboratory strain PY79, spores of which are much less heat resistant. This proteomic characterization identified 2011 proteins in spores and 1901 proteins in vegetative cells of B. subtilis A163. Surprisingly, spore morphogenic protein SpoVM had no homologs in B. subtilis A163. Comparing protein expression between these two strains uncovered 108 proteins that were differentially present in spores and 93 proteins differentially present in cells. In addition, five of the seven proteins on an operon in strain A163, which is thought to be primarily responsible for this strain’s spores high heat resistance, were also identified. These findings reveal proteomic differences of the two strains exhibiting different resistance to heat and form a basis for further mechanistic analysis of the high heat resistance of B. subtilis A163 spores.

scholarly journals Characterization of Spores of Bacillus subtilis Which Lack Dipicolinic Acid

2000 ◽  
Vol 182 (19) ◽  
pp. 5505-5512 ◽  
Author(s):  
Madan Paidhungat ◽  
Barbara Setlow ◽  
Adam Driks ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Water Content ◽  
Dipicolinic Acid ◽  
Active Form ◽  
Core Region ◽  
Wild Type ◽  
Dry Heat ◽  
Spore Resistance ◽  
Wet Heat

ABSTRACT Spores of Bacillus subtilis with a mutation inspoVF cannot synthesize dipicolinic acid (DPA) and are too unstable to be purified and studied in detail. However, the spores of a strain lacking the three major germinant receptors (termed Δger3), as well as spoVF, can be isolated, although they spontaneously germinate much more readily than Δger3 spores. The Δger3 spoVF spores lack DPA and have higher levels of core water than Δger3spores, although sporulation with DPA restores close to normal levels of DPA and core water to Δger3 spoVF spores. The DPA-less spores have normal cortical and coat layers, as observed with an electron microscope, but their core region appears to be more hydrated than that of spores with DPA. The Δger3 spoVF spores also contain minimal levels of the processed active form (termed P41) of the germination protease, GPR, a finding consistent with the known requirement for DPA and dehydration for GPR autoprocessing. However, any P41 formed in Δger3 spoVF spores may be at least transiently active on one of this protease's small acid-soluble spore protein (SASP) substrates, SASP-γ. Analysis of the resistance of wild-type, Δger3, and Δger3 spoVF spores to various agents led to the following conclusions: (i) DPA and core water content play no role in spore resistance to dry heat, dessication, or glutaraldehyde; (ii) an elevated core water content is associated with decreased spore resistance to wet heat, hydrogen peroxide, formaldehyde, and the iodine-based disinfectant Betadine; (iii) the absence of DPA increases spore resistance to UV radiation; and (iv) wild-type spores are more resistant than Δger3 spores to Betadine and glutaraldehyde. These results are discussed in view of current models of spore resistance and spore germination.

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

2011 ◽  
Vol 77 (19) ◽  
pp. 6746-6754 ◽  
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.

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

2016 ◽  
Vol 198 (11) ◽  
pp. 1694-1707 ◽  
Author(s):  
M. Lauren Donnelly ◽  
Kelly A. Fimlaid ◽  
Aimee Shen
Keyword(s):  
Clostridium Difficile ◽  
Heat Resistance ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
The United States ◽  
Spore Formation ◽  
Obligate Anaerobe ◽  
Wild Type ◽  
Content Type ◽  
Wet Heat

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.

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

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

scholarly journals Role of SpoVA Proteins in Release of Dipicolinic Acid during Germination of Bacillus subtilis Spores Triggered by Dodecylamine or Lysozyme

2006 ◽  
Vol 189 (5) ◽  
pp. 1565-1572 ◽  
Author(s):  
Venkata Ramana Vepachedu ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Small Molecules ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Ph Optimum ◽  
Inner Membrane ◽  
Temperature Sensitive Mutants

ABSTRACT The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of ∼9 and a temperature optimum of 60°C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl2. Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca2+ almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca2+ and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca2+-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca2+-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.

scholarly journals Commitment to sporulation in Bacillus subtilis and its relationship to development of actinomycin resistance

1969 ◽  
Vol 113 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Janet M. Sterlini ◽  
J. Mandelstam
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Subtilis ◽  
Heat Resistance ◽  
Messenger Rna ◽  
Actinomycin D ◽  
Dipicolinic Acid ◽  
Vegetative State ◽  
Single Point

1. Experiments to determine the point of commitment to sporulation were carried out by restoring nutrients at different times to suspensions of sporulating Bacillus subtilis. 2. No single point of commitment to the process as a whole was found. Instead, the cells became committed in turn to the following successive events connected with sporulation: formation of alkaline phosphatase, development of refractility, synthesis of dipicolinic acid and development of heat-resistance. 3. Each point of commitment was followed within about 30min. by a period in which the event concerned ceased to be inhibited by actinomycin D. 4. The implication of these results is that each point of commitment is probably due to the formation of a species of long-lived messenger RNA and that, in any case, sporulation is regulated at the level of both transcription and translation. 5. It is also shown that sporulation and growth are perhaps not mutually exclusive functions and that histidase, an enzyme typical of the vegetative state, can be induced in sporulating suspensions.

scholarly journals Heat Shock Proteins Do Not Influence Wet Heat Resistance of Bacillus subtilis Spores

2001 ◽  
Vol 183 (2) ◽  
pp. 779-784 ◽  
Author(s):  
Elizabeth Melly ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Heat Shock ◽  
Heat Shock Proteins ◽  
Heat Resistance ◽  
Significant Role ◽  
Vegetative Cell ◽  
Heat Shock Genes ◽  
Wet Heat ◽  
Shock Proteins

ABSTRACT Spores of Bacillus subtilis are significantly more resistant to wet heat than are their vegetative cell counterparts. Analysis of the effects of mutations in and the expression of fusions of a coding gene for a thermostable β-galactosidase to a number of heat shock genes has shown that heat shock proteins play no significant role in the wet heat resistance of B. subtilis spores.

scholarly journals Role of Dipicolinic Acid in the Germination, Stability, and Viability of Spores of Bacillus subtilis

2008 ◽  
Vol 190 (14) ◽  
pp. 4798-4807 ◽  
Author(s):  
Anil Magge ◽  
Amanda C. Granger ◽  
Paul G. Wahome ◽  
Barbara Setlow ◽  
Venkata R. Vepachedu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Small Molecules ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Great Majority ◽  
Lytic Enzyme ◽  
Spore Proteins ◽  
Low Levels ◽  
Made In

ABSTRACT Spores of Bacillus subtilis spoVF strains that cannot synthesize dipicolinic acid (DPA) but take it up during sporulation were prepared in medium with various DPA concentrations, and the germination and viability of these spores as well as the DPA content in individual spores were measured. Levels of some other small molecules in DPA-less spores were also measured. These studies have allowed the following conclusions. (i) Spores with no DPA or low DPA levels that lack either the cortex-lytic enzyme (CLE) SleB or the receptors that respond to nutrient germinants could be isolated but were unstable and spontaneously initiated early steps in spore germination. (ii) Spores that lacked SleB and nutrient germinant receptors and also had low DPA levels were more stable. (iii) Spontaneous germination of spores with no DPA or low DPA levels was at least in part via activation of SleB. (iv) The other redundant CLE, CwlJ, was activated only by the release of high levels of DPA from spores. (v) Low levels of DPA were sufficient for the viability of spores that lacked most α/β-type small, acid-soluble spore proteins. (vi) DPA levels accumulated in spores prepared in low-DPA-containing media varied greatly between individual spores, in contrast to the presence of more homogeneous DPA levels in individual spores made in media with high DPA concentrations. (vii) At least the great majority of spores of several spoVF strains that contained no DPA also lacked other major spore small molecules and had gone through some of the early reactions in spore germination.

scholarly journals Effects of Mn and Fe Levels onBacillus subtilisSpore Resistance and Effects of Mn2+, Other Divalent Cations, Orthophosphate, and Dipicolinic Acid on Protein Resistance to Ionizing Radiation

2010 ◽  
Vol 77 (1) ◽  
pp. 32-40 ◽  
Author(s):  
Amanda C. Granger ◽  
Elena K. Gaidamakova ◽  
Vera Y. Matrosova ◽  
Michael J. Daly ◽  
Peter Setlow
Keyword(s):  
Ionizing Radiation ◽  
Deinococcus Radiodurans ◽  
Dipicolinic Acid ◽  
Divalent Cations ◽  
Wild Type ◽  
Divalent Metal Ions ◽  
Protein Resistance ◽  
Wet Heat ◽  
Resistance To Ionizing Radiation

ABSTRACTSpores ofBacillus subtilisstrains with (wild type) or without (α−β−) most DNA-binding α/β-type small, acid-soluble proteins (SASP) were prepared in medium with additional MnCl2concentrations of 0.3 μM to 1 mM. These haploid spores had Mn levels that varied up to 180-fold and Mn/Fe ratios that varied up to 300-fold. However, the resistance of these spores to desiccation, wet heat, dry heat, and in particular ionizing radiation was unaffected by their level of Mn or their Mn/Fe ratio; this was also the case for wild-type spore resistance to hydrogen peroxide (H2O2). However, α−β−spores were more sensitive to H2O2when they had high Mn levels and a high Mn/Fe ratio. These results suggest that Mn levels alone are not essential for wild-type bacterial spores' extreme resistance properties, in particular ionizing radiation, although high Mn levels sensitize α−β−spores to H2O2, probably by repressing expression of the auxiliary DNA-protective protein MrgA. Notably, Mn2+complexed with the abundant spore molecule dipicolinic acid (DPA) with or without inorganic phosphate was very effective at protecting a restriction enzyme against ionizing radiationin vitro, and Ca2+complexed with DPA and phosphate was also very effective in this regard. These latter data suggest that protein protection in spores against treatments such as ionizing radiation that generate reactive oxygen species may be due in part to the spores' high levels of DPA conjugated to divalent metal ions, predominantly Ca2+, much like high levels of Mn2+complexed with small molecules protect the bacteriumDeinococcus radioduransagainst ionizing radiation.

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