scholarly journals Factors Influencing Germination of Bacillus subtilis Spores via Activation of Nutrient Receptors by High Pressure

2005 ◽  
Vol 71 (10) ◽  
pp. 5879-5887 ◽  
Author(s):  
Elaine P. Black ◽  
Kasia Koziol-Dube ◽  
Dongsheng Guan ◽  
Jie Wei ◽  
Barbara Setlow ◽  
...  
Keyword(s):  
High Pressure ◽  
Bacillus Subtilis ◽  
Fatty Acid ◽  
Unsaturated Fatty Acid ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Major Effect ◽  
Strong Inhibition ◽  
Wild Type ◽  
Factors Influencing

ABSTRACT Different nutrient receptors varied in triggering germination of Bacillus subtilis spores with a pressure of 150 MPa, the GerA receptor being more responsive than the GerB receptor and even more responsive than the GerK receptor. This hierarchy in receptor responsiveness to pressure was the same as receptor responsiveness to a mixture of nutrients. The levels of nutrient receptors influenced rates of pressure germination, since the GerA receptor is more abundant than the GerB receptor and elevated levels of individual receptors increased spore germination by 150 MPa of pressure. However, GerB receptor variants with relaxed specificity for nutrient germinants responded as well as the GerA receptor to this pressure. Spores lacking dipicolinic acid did not germinate with this pressure, and pressure activation of the GerA receptor required covalent addition of diacylglycerol. However, pressure activation of the GerB and GerK receptors displayed only a partial (GerB) or no (GerK) diacylglycerylation requirement. These effects of receptor diacylglycerylation on pressure germination are similar to those on nutrient germination. Wild-type spores prepared at higher temperatures germinated more rapidly with a pressure of 150 MPa than spores prepared at lower temperatures; this was also true for spores with only one receptor, but receptor levels did not increase in spores made at higher temperatures. Changes in inner membrane unsaturated fatty acid levels, lethal treatment with oxidizing agents, or exposure to chemicals that inhibit nutrient germination had no major effect on spore germination by 150 MPa of pressure, except for strong inhibition by HgCl2.

scholarly journals Mechanisms of Induction of Germination of Bacillus subtilis Spores by High Pressure

2002 ◽  
Vol 68 (6) ◽  
pp. 3172-3175 ◽  
Author(s):  
Madan Paidhungat ◽  
Barbara Setlow ◽  
William B. Daniels ◽  
Dallas Hoover ◽  
Efstathia Papafragkou ◽  
...  
Keyword(s):  
High Pressure ◽  
Bacillus Subtilis ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Wild Type ◽  
Lytic Enzymes

ABSTRACT Spores of Bacillus subtilis lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa. However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination. Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not attacked by these enzymes was not induced by a pressure of 550 MPa, but treatment of these mutant spores with this pressure caused the release of dipicolinic acid. These data suggest the following conclusions: (i) a pressure of 100 MPa induces spore germination by activating the germinant receptors; and (ii) a pressure of 550 MPa opens channels for release of dipicolinic acid from the spore core, which leads to the later steps 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 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 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 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 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 Analysis of factors influencing the rate of germination of spores of Bacillus subtilis by very high pressure

2007 ◽  
Vol 102 (1) ◽  
pp. 65-76 ◽  
Author(s):  
E.P. Black ◽  
J. Wei ◽  
S. Atluri ◽  
D.E. Cortezzo ◽  
K. Koziol-Dube ◽  
...  
Keyword(s):  
High Pressure ◽  
Bacillus Subtilis ◽  
Factors Influencing ◽  
Very High

scholarly journals The different pathways of spore germination and inactivation of Bacillus subtilis under high pressure and elevated temperatures

2011 ◽  
Vol 1 ◽  
pp. 792-799 ◽  
Author(s):  
Kai Reineke ◽  
Isabel Doehner ◽  
Daniel Baier ◽  
Alexander Mathys ◽  
Dietrich Knorr
Keyword(s):  
High Pressure ◽  
Bacillus Subtilis ◽  
Spore Germination ◽  
Elevated Temperatures

Effect of Small, Acid-Soluble Proteins on Spore Resistance and Germination under a Combination of Pressure and Heat Treatment

2007 ◽  
Vol 70 (9) ◽  
pp. 2168-2171
Author(s):  
JONG-KYUNG LEE ◽  
SARA MOVAHEDI ◽  
STEPHEN E. HARDING ◽  
BERNARD M. MACKEY ◽  
WILLIAM M. WAITES
Keyword(s):  
Heat Treatment ◽  
High Pressure ◽  
High Temperature ◽  
Spore Germination ◽  
Soluble Proteins ◽  
Wild Type ◽  
Spore Resistance ◽  
Pressure Resistance ◽  
The One

To find the range of pressure required for effective high-pressure inactivation of bacterial spores and to investigate the role of α/β-type small, acid-soluble proteins (SASP) in spores under pressure treatment, mild heat was combined with pressure (room temperature to 65°C and 100 to 500 MPa) and applied to wild-type and SASP-α−/β− Bacillus subtilis spores. On the one hand, more than 4 log units of wild-type spores were reduced after pressurization at 100 to 500 MPa and 65°C. On the other hand, the number of surviving mutant spores decreased by 2 log units at 100 MPa and by more than 5 log units at 500 MPa. At 500 MPa and 65°C, both wild-type and mutant spore survivor counts were reduced by 5 log units. Interestingly, pressures of 100, 200, and 300 MPa at 65°C inactivated wild-type SASP-α+/β+ spores more than mutant SASP-α−/β− spores, and this was attributed to less pressure-induced germination in SASP-α−/β− spores than in wild-type SASP-α+/β+ spores. However, there was no difference in the pressure resistance between SASP-α+/β+ and SASP-α−/β− spores at 100 MPa and ambient temperature (approximately 22°C) for 30 min. A combination of high pressure and high temperature is very effective for inducing spore germination, and then inactivation of the germinated spore occurs because of the heat treatment. This study showed that α/β-type SASP play a role in spore inactivation by increasing spore germination under 100 to 300 MPa at high temperature.

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.

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