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 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 Localization of the Cortex Lytic Enzyme CwlJ in Spores of Bacillus subtilis

2002 ◽  
Vol 184 (4) ◽  
pp. 1219-1224 ◽  
Author(s):  
Irina Bagyan ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
High Salt ◽  
Lytic Enzyme ◽  
Spore Coat ◽  
Coat Proteins ◽  
His Tag ◽  
Spore Coat Proteins ◽  
Triton X

ABSTRACT The enzyme CwlJ is involved in the depolymerization of cortex peptidoglycan during germination of spores of Bacillus subtilis. CwlJ with a C-terminal His tag was functional and was extracted from spores by procedures that remove spore coat proteins. However, this CwlJ was not extracted from disrupted spores by dilute buffer, high salt concentrations, Triton X-100, Ca2+-dipicolinic acid, dithiothreitol, or peptidoglycan digestion, disappeared during spore germination, and was not present in cotE spores in which the spore coat is aberrant. These findings indicate the following: (i) the reason decoated and cotE spores germinate poorly with dipicolinic acid is the absence of CwlJ from these spores; and (ii) CwlJ is located in the spore coat, presumably tightly associated with one or more other coat proteins.

scholarly journals Role of Ger Proteins in Nutrient and Nonnutrient Triggering of Spore Germination in Bacillus subtilis

2000 ◽  
Vol 182 (9) ◽  
pp. 2513-2519 ◽  
Author(s):  
Madan Paidhungat ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Mutant Strain ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Family Members ◽  
Wild Type ◽  
Receptor Proteins ◽  
Rich Media

ABSTRACT Dormant Bacillus subtilis spores germinate in the presence of particular nutrients called germinants. The spores are thought to recognize germinants through receptor proteins encoded by the gerA family of operons, which includesgerA, gerB, and gerK. We sought to substantiate this putative function of the GerA family proteins by characterizing spore germination in a mutant strain that contained deletions at all known gerA-like loci. As expected, the mutant spores germinated very poorly in a variety of rich media. In contrast, they germinated like wild-type spores in a chemical germinant, a 1-1 chelate of Ca2+ and dipicolinic acid (DPA). These observations showed that proteins encoded bygerA family members are required for nutrient-induced germination but not for chemical-triggered germination, supporting the hypothesis that the GerA family encodes receptors for nutrient germinants. Further characterization of Ca2+–DPA-induced germination showed that the effect of Ca2+–DPA on spore germination was saturated at 60 mM and had a Km of 30 mM. We also found that decoating spores abolished their ability to germinate in Ca2+–DPA but not in nutrient germinants, indicating that Ca2+–DPA and nutrient germinants probably act through parallel arms of the germination pathway.

scholarly journals Identification of a New Gene Essential for Germinationof Bacillus subtilis Spores withCa2+-Dipicolinate

2003 ◽  
Vol 185 (7) ◽  
pp. 2315-2329 ◽  
Author(s):  
Katerina Ragkousi ◽  
Patrick Eichenberger ◽  
Christiaan van Ooij ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Spore Germination ◽  
Function Analysis ◽  
Dipicolinic Acid ◽  
Functional Characterization ◽  
Lytic Enzyme ◽  
Spore Coat ◽  
Gene Encoding ◽  
Reading Frame ◽  
Genes Encoding

ABSTRACT Bacillus subtilis spores can germinate with a 1:1 chelate of Ca2+ and dipicolinic acid (DPA), a compound present at high levels in the spore core. Using a genetic screen to identify genes encoding proteins that are specifically involved in spore germination by Ca2+-DPA, three mutations were identified. One was in the gene encoding the cortex lytic enzyme, CwlJ, that was previously shown to be essential for spore germination by Ca2+-DPA. The other two were mapped to an open reading frame, ywdL, encoding a protein of unknown function. Analysis of ywdL expression showed that the gene is expressed during sporulation in the mother cell compartment of the sporulating cell and that its transcription is σE dependent. Functional characterization of YwdL demonstrated that it is a new spore coat protein that is essential for the presence of CwlJ in the spore coat. Assembly of YwdL itself into the spore coat is dependent on the coat morphogenetic proteins CotE and SpoIVA. However, other than lacking CwlJ, ywdL spores have no obvious defect in their spore coat. Because of the role for YwdL in a part of the spore germination process, we propose renaming ywdL as a spore germination gene, gerQ.

scholarly journals Role of GerD in Germination of Bacillus subtilis Spores

2006 ◽  
Vol 189 (3) ◽  
pp. 1090-1098 ◽  
Author(s):  
Patricia L. Pelczar ◽  
Takao Igarashi ◽  
Barbara Setlow ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Ectopic Expression ◽  
Direct Interaction ◽  
Cysteine Residue ◽  
Wild Type ◽  
Hypertonic Medium ◽  
Putative Signal Peptide

ABSTRACT Spores of a Bacillus subtilis strain with a gerD deletion mutation (ΔgerD) responded much slower than wild-type spores to nutrient germinants, although they did ultimately germinate, outgrow, and form colonies. Spores lacking GerD and nutrient germinant receptors also germinated slowly with nutrients, as did ΔgerD spores in which nutrient receptors were overexpressed. The germination defect of ΔgerD spores was not suppressed by many changes in the sporulation or germination conditions. Germination of ΔgerD spores was also slower than that of wild-type spores with a pressure of 150 MPa, which triggers spore germination through nutrient receptors. Ectopic expression of gerD suppressed the slow germination of ΔgerD spores with nutrients, but overexpression of GerD did not increase rates of spore germination. Loss of GerD had no effect on spore germination induced by agents that do not act through nutrient receptors, including a 1:1 chelate of Ca2+ and dipicolinic acid, dodecylamine, lysozyme in hypertonic medium, a pressure of 500 MPa, and spontaneous germination of spores that lack all nutrient receptors. Deletion of GerD's putative signal peptide or change of its likely diacylglycerylated cysteine residue to alanine reduced GerD function. The latter findings suggest that GerD is located in a spore membrane, most likely the inner membrane, where the nutrient receptors are located. All these data suggest that, while GerD is not essential for nutrient germination, this protein has an important role in spores' rapid response to nutrient germinants, by either direct interaction with nutrient receptors or some signal transduction essential for germination.

scholarly journals Genetic Requirements for Induction of Germination of Spores of Bacillus subtilis by Ca2+-Dipicolinate

2001 ◽  
Vol 183 (16) ◽  
pp. 4886-4893 ◽  
Author(s):  
Madan Paidhungat ◽  
Katerina Ragkousi ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Signaling Pathway ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Lytic Enzymes ◽  
Definitive Evidence ◽  
The Core ◽  
Early Germination ◽  
Hydrolysis Of

ABSTRACT Dormant Bacillus subtilis spores can be induced to germinate by nutrients, as well as by nonmetabolizable chemicals, such as a 1:1 chelate of Ca2+ and dipicolinic acid (DPA). Nutrients bind receptors in the spore, and this binding triggers events in the spore core, including DPA excretion and rehydration, and also activates hydrolysis of the surrounding cortex through mechanisms that are largely unknown. As Ca2+-DPA does not require receptors to induce spore germination, we asked if this process utilizes other proteins, such as the putative cortex-lytic enzymes SleB and CwlJ, that are involved in nutrient-induced germination. We found that Ca2+-DPA triggers germination by first activating CwlJ-dependent cortex hydrolysis; this mechanism is different from nutrient-induced germination where cortex hydrolysis is not required for the early germination events in the spore core. Nevertheless, since nutrients can induce release of the spore's DPA before cortex hydrolysis, we examined if the DPA excreted from the core acts as a signal to activate CwlJ in the cortex. Indeed, endogenous DPA is required for nutrient-induced CwlJ activation and this requirement was partially remedied by exogenous Ca2+-DPA. Our findings thus define a mechanism for Ca2+-DPA-induced germination and also provide the first definitive evidence for a signaling pathway that activates cortex hydrolysis in response to nutrients.

scholarly journals Analysis of Nucleoid Morphology during Germination and Outgrowth of Spores of Bacillus Species

2000 ◽  
Vol 182 (19) ◽  
pp. 5556-5562 ◽  
Author(s):  
Katerina Ragkousi ◽  
Ann E. Cowan ◽  
Margery A. Ross ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Dna Binding ◽  
Bacillus Megaterium ◽  
Spore Germination ◽  
Great Majority ◽  
Dna Binding Proteins ◽  
Soluble Proteins ◽  
Bacillus Species ◽  
Chromosomal Protein ◽  
Spore Outgrowth

ABSTRACT After a few minutes of germination, nucleoids in the great majority of spores of Bacillus subtilis and Bacillus megaterium were ring shaped. The major spore DNA binding proteins, the α/β-type small, acid-soluble proteins (SASP), colocalized to these nucleoid rings early in spore germination, as did the B. megaterium homolog of the major B. subtilis chromosomal protein HBsu. The percentage of ring-shaped nucleoids was decreased in germinated spores with lower levels of α/β-type SASP. As spore outgrowth proceeded, the ring-shaped nucleoids disappeared and the nucleoid became more compact. This change took place after degradation of most of the spores' pool of major α/β-type SASP and was delayed when α/β-type SASP degradation was delayed. Later in spore outgrowth, the shape of the nucleoid reverted to the diffuse lobular shape seen in growing cells.

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

2015 ◽  
Vol 197 (14) ◽  
pp. 2276-2283 ◽  
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.

scholarly journals Kinetics of Germination of Wet-Heat-Treated Individual Spores of Bacillus Species, Monitored by Raman Spectroscopy and Differential Interference Contrast Microscopy

2011 ◽  
Vol 77 (10) ◽  
pp. 3368-3379 ◽  
Author(s):  
Guiwen Wang ◽  
Pengfei Zhang ◽  
Peter Setlow ◽  
Yong-qing Li
Keyword(s):  
Heat Treatment ◽  
Raman Spectroscopy ◽  
Spore Germination ◽  
Great Majority ◽  
Lytic Enzyme ◽  
Differential Interference Contrast ◽  
Content Type ◽  
Heat Treated ◽  
Wet Heat ◽  
Kinetics Of

ABSTRACTRaman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the kinetics of nutrient and nonnutrient germination of multiple individual untreated and wet-heat-treated spores ofBacillus cereusandBacillus megaterium, as well as of several isogenicBacillus subtilisstrains. Major conclusions from this work were as follows. (i) More than 90% of these spores were nonculturable but retained their 1:1 chelate of Ca2+and dipicolinic acid (CaDPA) when incubated in water at 80 to 95°C for 5 to 30 min. (ii) Wet-heat treatment significantly increased the time,Tlag, at which spores began release of the great majority of their CaDPA during the germination ofB. subtilisspores with different nutrient germinants and also increased the variability ofTlagvalues. (iii) The time period, ΔTrelease, betweenTlagand the time,Trelease, at which a spore germinating with nutrients completed the release of the great majority of its CaDPA, was also increased in wet-heat-treated spores. (iv) Wet-heat-treated spores germinating with nutrients had higher values ofIrelease, the intensity of a spore's DIC image atTrelease, than did untreated spores and had much longer time periods, ΔTlys, for the reduction inIreleaseintensities to the basal value due to hydrolysis of the spore's peptidoglycan cortex, probably due at least in part to damage to the cortex-lytic enzyme CwlJ. (v) Increases inTlagand ΔTreleasewere also observed when wet-heat-treatedB. subtilisspores were germinated with the nonnutrient dodecylamine, while the change inIreleasewas less significant. (vi) The effects of wet-heat treatment on nutrient germination ofB. cereusandB. megateriumspores were generally similar to those onB. subtilisspores. These results indicate that (i) some proteins important in spore germination are damaged by wet-heat treatment, (ii) the cortex-lytic enzyme CwlJ is one germination protein damaged by wet heat, and (iii) the CaDPA release process itself seems likely to be the target of wet-heat damage which has the greatest effect on spore germination.

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