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

mSphere ◽  
2016 ◽  
Vol 1 (6) ◽  
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.

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 Reexamining the Germination Phenotypes of Several Clostridium difficile Strains Suggests Another Role for the CspC Germinant Receptor

2015 ◽  
Vol 198 (5) ◽  
pp. 777-786 ◽  
Author(s):  
Disha Bhattacharjee ◽  
Michael B. Francis ◽  
Xicheng Ding ◽  
Kathleen N. McAllister ◽  
Ritu Shrestha ◽  
...  
Keyword(s):  
Bile Acid ◽  
Clostridium Difficile ◽  
Bile Acids ◽  
Spore Germination ◽  
Germination Rate ◽  
Taurocholic Acid ◽  
Receptor Complex ◽  
Health Care Environment ◽  
Content Type ◽  
Downstream Processes

ABSTRACTClostridium difficilespore germination is essential for colonization and disease. The signals that initiateC. difficilespore germination are a combination of taurocholic acid (a bile acid) and glycine. Interestingly, the chenodeoxycholic acid class (CDCA) bile acids competitively inhibit taurocholic acid-mediated germination, suggesting that compounds that inhibit spore germination could be developed into drugs that prophylactically preventC. difficileinfection or reduce recurring disease. However, a recent report called into question the utility of such a strategy to prevent infection by describingC. difficilestrains that germinated in the apparent absence of bile acids or germinated in the presence of the CDCA inhibitor. Because the mechanisms ofC. difficilespore germination are beginning to be elucidated, the mechanism of germination in these particular strains could yield important information on howC. difficilespores initiate germination. Therefore, we quantified the interaction of these strains with taurocholic acid and CDCA, the rates of spore germination, the release of DPA from the spore core, and the abundance of the germinant receptor complex (CspC, CspB, and SleC). We found that strains previously observed to germinate in the absence of taurocholic acid correspond to more potent 50% effective concentrations (EC50values; the concentrations that achieve a half-maximum germination rate) of the germinant and are still inhibited by CDCA, possibly explaining the previous observations. By comparing the germination kinetics and the abundance of proteins in the germinant receptor complex, we revised our original model for CspC-mediated activation of spore germination and propose that CspC may activate spore germination and then inhibit downstream processes.IMPORTANCEClostridium difficileforms metabolically dormant spores that persist in the health care environment. In susceptible hosts,C. difficilespores germinate in response to certain bile acids and glycine. Blocking germination byC. difficilespores is an attractive strategy to prevent the initiation of disease or to block recurring infection. However, certainC. difficilestrains have been identified whose spores germinate in the absence of bile acids or are not blocked by known inhibitors ofC. difficilespore germination (calling into question the utility of such strategies). Here, we further investigate these strains and reestablish that bile acid activators and inhibitors of germination affect these strains and use these data to suggest another role for theC. difficilebile acid germinant receptor.

scholarly journals Analysis of the Dynamics of a Bacillus subtilis Spore Germination Protein Complex during Spore Germination and Outgrowth

2014 ◽  
Vol 197 (2) ◽  
pp. 252-261 ◽  
Author(s):  
Anthony J. Troiano ◽  
Jingqiao Zhang ◽  
Ann E. Cowan ◽  
Ji Yu ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Western Blot ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Interference Microscopy ◽  
Epifluorescence Microscopy ◽  
Focus Formation ◽  
Content Type ◽  
Protein Levels ◽  
Bacillus Subtilis Spore

Germination ofBacillus subtilisspores is normally initiated when nutrients from the environment interact with germinant receptors (GRs) in the spores' inner membrane (IM), in which most of the lipids are immobile. GRs and another germination protein, GerD, colocalize in the IM of dormant spores in a small focus termed the “germinosome,” and this colocalization or focus formation is dependent upon GerD, which is also essential for rapid GR-dependent spore germination. To determine the fate of the germinosome and germination proteins during spore germination and outgrowth, we employed differential interference microscopy and epifluorescence microscopy to track germinating spores with fluorescent fusions to germination proteins and used Western blot analyses to measure germination protein levels. We found that after initiation of spore germination, the germinosome foci ultimately changed into larger disperse patterns, with ≥75% of spore populations displaying this pattern in spores germinated for 1 h, although >80% of spores germinated for 30 min retained the germinosome foci. Western blot analysis revealed that levels of GR proteins and the SpoVA proteins essential for dipicolinic acid release changed minimally during this period, although GerD levels decreased ∼50% within 15 min in germinated spores. Since the dispersion of the germinosome during germination was slower than the decrease in GerD levels, either germinosome stability is not compromised by ∼2-fold decreases in GerD levels or other factors, such as restoration of rapid IM lipid mobility, are also significant in germinosome dispersion as spore germination proceeds.

scholarly journals Antibiotic-Induced Alterations of the Gut Microbiota Alter Secondary Bile Acid Production and Allow for Clostridium difficile Spore Germination and Outgrowth in the Large Intestine

mSphere ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Casey M. Theriot ◽  
Alison A. Bowman ◽  
Vincent B. Young
Keyword(s):  
Bile Acid ◽  
Clostridium Difficile ◽  
Gut Microbiota ◽  
Bile Acids ◽  
Large Intestine ◽  
Spore Germination ◽  
Secondary Bile Acid ◽  
Content Type ◽  
Secondary Bile Acids

ABSTRACT Antibiotics alter the gastrointestinal microbiota, allowing for Clostridium difficile infection, which is a significant public health problem. Changes in the structure of the gut microbiota alter the metabolome, specifically the production of secondary bile acids. Specific bile acids are able to initiate C. difficile spore germination and also inhibit C. difficile growth in vitro, although no study to date has defined physiologically relevant bile acids in the gastrointestinal tract. In this study, we define the bile acids C. difficile spores encounter in the small and large intestines before and after various antibiotic treatments. Antibiotics that alter the gut microbiota and deplete secondary bile acid production allow C. difficile colonization, representing a mechanism of colonization resistance. Multiple secondary bile acids in the large intestine were able to inhibit C. difficile spore germination and growth at physiological concentrations and represent new targets to combat C. difficile in the large intestine. It is hypothesized that the depletion of microbial members responsible for converting primary bile acids into secondary bile acids reduces resistance to Clostridium difficile colonization. To date, inhibition of C. difficile growth by secondary bile acids has only been shown in vitro. Using targeted bile acid metabolomics, we sought to define the physiologically relevant concentrations of primary and secondary bile acids present in the murine small and large intestinal tracts and how these impact C. difficile dynamics. We treated mice with a variety of antibiotics to create distinct microbial and metabolic (bile acid) environments and directly tested their ability to support or inhibit C. difficile spore germination and outgrowth ex vivo. Susceptibility to C. difficile in the large intestine was observed only after specific broad-spectrum antibiotic treatment (cefoperazone, clindamycin, and vancomycin) and was accompanied by a significant loss of secondary bile acids (deoxycholate, lithocholate, ursodeoxycholate, hyodeoxycholate, and ω-muricholate). These changes were correlated to the loss of specific microbiota community members, the Lachnospiraceae and Ruminococcaceae families. Additionally, physiological concentrations of secondary bile acids present during C. difficile resistance were able to inhibit spore germination and outgrowth in vitro. Interestingly, we observed that C. difficile spore germination and outgrowth were supported constantly in murine small intestinal content regardless of antibiotic perturbation, suggesting that targeting growth of C. difficile will prove most important for future therapeutics and that antibiotic-related changes are organ specific. Understanding how the gut microbiota regulates bile acids throughout the intestine will aid the development of future therapies for C. difficile infection and other metabolically relevant disorders such as obesity and diabetes. IMPORTANCE Antibiotics alter the gastrointestinal microbiota, allowing for Clostridium difficile infection, which is a significant public health problem. Changes in the structure of the gut microbiota alter the metabolome, specifically the production of secondary bile acids. Specific bile acids are able to initiate C. difficile spore germination and also inhibit C. difficile growth in vitro, although no study to date has defined physiologically relevant bile acids in the gastrointestinal tract. In this study, we define the bile acids C. difficile spores encounter in the small and large intestines before and after various antibiotic treatments. Antibiotics that alter the gut microbiota and deplete secondary bile acid production allow C. difficile colonization, representing a mechanism of colonization resistance. Multiple secondary bile acids in the large intestine were able to inhibit C. difficile spore germination and growth at physiological concentrations and represent new targets to combat C. difficile in the large intestine.

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

2015 ◽  
Vol 81 (19) ◽  
pp. 6725-6735 ◽  
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.

scholarly journals Effects of Sporulation Conditions on the Germination and Germination Protein Levels of Bacillus subtilis Spores

2012 ◽  
Vol 78 (8) ◽  
pp. 2689-2697 ◽  
Author(s):  
Arturo Ramirez-Peralta ◽  
Pengfei Zhang ◽  
Yong-qing Li ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Rich Medium ◽  
Microscope Slide ◽  
Content Type ◽  
Protein Levels ◽  
Germination Characteristics ◽  
The Difference ◽  
Lag Times

ABSTRACTBacillus subtilisspores prepared in rich medium germinated faster with nutrient germinants than poor-medium spores as populations in liquid and multiple individual spores on a microscope slide. Poor-medium spores had longer average lag times between mixing of spores with nutrient germinants and initiation of Ca-dipicolinic acid (CaDPA) release. Rich-medium spores made at 37°C germinated slightly faster with nutrient germinants than 23°C spores in liquid, but not when spores germinated on a slide. The difference in germination characteristics of these spore populations in liquid was paralleled by changes in expression levels of a transcriptionallacZfusion to thegerAoperon, encoding a germinant receptor (GR). Levels of GR subunits were 3- to 8-fold lower in poor-medium spores than rich-medium spores and 1.6- to 2-fold lower in 23°C spores than 37°C spores, and levels of the auxiliary germination protein GerD were 3.5- to 4-fold lower in poor medium and 23°C spores. In contrast, levels of another likely germination protein, SpoVAD, were similar in all these spores. These different spores germinated similarly with CaDPA, and poor-medium and 23°C spores germinated faster than rich-medium and 37°C spores, respectively, with dodecylamine. Since spore germination with CaDPA and dodecylamine does not require GerD or GRs, these results indicate that determinants of rates of nutrient germination of spores prepared differently are primarily the levels of the GRs that bind nutrient germinants and trigger germination and secondarily the levels of GerD.

scholarly journals Properties of Spores of Bacillus subtilis Blocked at an Intermediate Stage in Spore Germination

2001 ◽  
Vol 183 (16) ◽  
pp. 4894-4899 ◽  
Author(s):  
Barbara Setlow ◽  
Elizabeth Melly ◽  
Peter Setlow
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Uv Irradiation ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Intermediate Stage ◽  
Stage I ◽  
Wild Type ◽  
Resistance To Heat

ABSTRACT Germination of mutant spores of Bacillus subtilisunable to degrade their cortex is accompanied by excretion of dipicolinic acid and uptake of some core water. However, compared to wild-type germinated spores in which the cortex has been degraded, the germinated mutant spores accumulated less core water, exhibited greatly reduced enzyme activity in the spore core, synthesized neither ATP nor reduced pyridine or flavin nucleotides, and had significantly higher resistance to heat and UV irradiation. We propose that the germinated spores in which the cortex has not been degraded represent an intermediate stage in spore germination, which we term stage I.

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.

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.

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