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 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 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 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 Integrative Analysis of Proteome and Transcriptome Dynamics during Bacillus subtilis Spore Revival

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Bhagyashree Swarge ◽  
Wishwas Abhyankar ◽  
Martijs Jonker ◽  
Huub Hoefsloot ◽  
Gertjan Kramer ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Spore Germination ◽  
Molecular Mechanisms ◽  
Transformation Process ◽  
Comprehensive Overview ◽  
Vegetative Cells ◽  
Content Type ◽  
Protein Levels ◽  
Bacillus Subtilis Spore ◽  
Proteome Changes

ABSTRACT Bacillus subtilis spores can reactivate their metabolism through germination upon contact with germinants and can develop into vegetative cells upon outgrowth. However, the mechanisms at the basis of the molecular machinery that triggers the spore germination and outgrowth processes are still largely unclear. To gain further insights into these processes, the transcriptome and proteome changes occurring during the conversion of spores to vegetative cells were analyzed in the present study. For each time point sampled, the changes in the spore proteome were quantitatively monitored relative to the proteome of metabolically 15N-labeled vegetative cells. Of the quantified proteins, 60% are shared by vegetative cells and spores, indicating that the spores have a minimal protein set, sufficient to resume metabolism upon completion of germination. These shared proteins thus represent the most basic “survival kit” for spore-based life. We observed no significant change in the proteome or the transcriptome until the spore’s completion of germination. Our analysis identified 34 abundant mRNA transcripts in the dormant spores, 31 of which are rapidly degraded after germination. In outgrowing spores, we identified 3,152 differentially expressed genes and have demonstrated the differential expression of 322 proteins with our mass spectrometry analyses. Our data also showed that 173 proteins from dormant spores, including both proteins unique to spores and proteins shared with vegetative cells, were lost after completion of germination. The observed diverse timings of synthesis of different protein sets in spore outgrowth revealed a putative core strategy underlying the revival of ‘life’ from the B. subtilis spore. IMPORTANCE This study demonstrated the progress of macromolecular synthesis during Bacillus subtilis spore germination and outgrowth. The transcriptome analysis has additionally allowed us to trace gene expression during this transformation process. For the first time, the basic survival kit for spore-based life has been identified. In addition, in this analysis based on monitoring of protein levels in germinating and outgrowing spores, the transition from (ribo)nucleotide and amino acid biosynthesis to the restoration of all metabolic pathways can be clearly seen. The integrative multi-omics approach applied in this study thus has helped us to achieve a comprehensive overview of the molecular mechanisms at the basis of spore germination and outgrowth as well as to identify important knowledge gaps in need of further study.

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 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 Spore Heat Activation Requirements and Germination Responses Correlate with Sequences of Germinant Receptors and with the Presence of a Specific spoVA2mob Operon in Foodborne Strains of Bacillus subtilis

2017 ◽  
Vol 83 (7) ◽  
Author(s):  
Antonina O. Krawczyk ◽  
Anne de Jong ◽  
Jimmy Omony ◽  
Siger Holsappel ◽  
Marjon H. J. Wells-Bennik ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Heat Resistance ◽  
Spore Germination ◽  
Food Industry ◽  
Heat Treatments ◽  
Phenotypic Traits ◽  
Content Type ◽  
Heat Resistant ◽  
Heat Activation ◽  
High Level

ABSTRACT 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. High-level 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+ (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.

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 Investigating Synthesis of the MalS Malic Enzyme during Bacillus subtilis Spore Germination and Outgrowth and the Influence of Spore Maturation and Sporulation Conditions

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Bhagyashree Swarge ◽  
Chahida Nafid ◽  
Norbert Vischer ◽  
Gertjan Kramer ◽  
Peter Setlow ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Bacillus Subtilis ◽  
Fluorescence Intensity ◽  
Spore Germination ◽  
Malic Enzyme ◽  
Present Report ◽  
Quiescent State ◽  
Content Type ◽  
Gfp Fluorescence ◽  
Spore Maturation

ABSTRACT Spore-forming bacteria of the orders Bacillales and Clostridiales play a major role in food spoilage and foodborne diseases. When environmental conditions become favorable, these spores can germinate as the germinant receptors located on the spore’s inner membrane are activated via germinant binding. This leads to the formation of vegetative cells via germination and subsequent outgrowth and potential deleterious effects on foods. The present report focuses on analysis of the synthesis of the MalS (malic enzyme) protein during Bacillus subtilis spore germination by investigating the dynamics of the presence and fluorescence level of a MalS-GFP (MalS-green fluorescent protein) fusion protein using time-lapse fluorescence microscopy. Our results show an initial increase in MalS-GFP fluorescence intensity within the first 15 min of germination, followed by a discernible drop and stabilization of the fluorescence throughout spore outgrowth as reported previously (L. Sinai, A. Rosenberg, Y. Smith, E. Segev, and S. Ben-Yehuda, Mol Cell 57:695–707, 2015, https://doi.org/10.1016/j.molcel.2014.12.019). However, in contrast to the earlier report, both Western blotting and SILAC (stable isotopic labeling of amino acids in cell culture) analysis showed there was no increase in MalS-GFP levels during the 15 min after the addition of germinants and that MalS synthesis did not begin until more than 90 min after germinant addition. Thus, the increase in MalS-GFP fluorescence early in germination is not due to new protein synthesis but is perhaps due to a change in the physical environment of the spore cores. Our findings also show that different sporulation conditions and spore maturation times affect expression of MalS-GFP and the germination behavior of the spores, albeit to a minor extent, but still result in no changes in MalS-GFP levels early in spore germination. IMPORTANCE The spores formed by Bacillus subtilis remain in a quiescent state for extended periods due to their dormancy and resistance features. Dormancy is linked to a very low level of core water content and a phase-bright state of spores. The present report, focusing on proteins MalS and PdhD (pyruvate dehydrogenase subunit D) and complementary to our companion report published in this issue, aims to shed light on a major dilemma in the field, i.e., whether protein synthesis, in particular that of MalS, takes place in phase-bright spores. Clustered MalS-GFP in dormant spores diffuses throughout the spore as germination proceeds. However, fluorescence intensity measurements, supported by Western blot analysis and SILAC proteomics, confirm that there is no new MalS protein synthesis in bright-phase dormant spores.

Sign in / Sign up

Export Citation Format

Share Document