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

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.

Download Full-text

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

Applied and Environmental Microbiology ◽

10.1128/aem.71.10.5879-5887.2005 ◽

2005 ◽

Vol 71 (10) ◽

pp. 5879-5887 ◽

Cited By ~ 90

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.

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.01613-06 ◽

2006 ◽

Vol 189 (5) ◽

pp. 1565-1572 ◽

Cited By ~ 82

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.

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.183.16.4886-4893.2001 ◽

2001 ◽

Vol 183 (16) ◽

pp. 4886-4893 ◽

Cited By ~ 207

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.

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.182.9.2513-2519.2000 ◽

2000 ◽

Vol 182 (9) ◽

pp. 2513-2519 ◽

Cited By ~ 198

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.

Download Full-text

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

Applied and Environmental Microbiology ◽

10.1128/aem.01817-15 ◽

2015 ◽

Vol 81 (19) ◽

pp. 6725-6735 ◽

Cited By ~ 8

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.

Download Full-text

Role of GerD in Germination of Bacillus subtilis Spores

Journal of Bacteriology ◽

10.1128/jb.01606-06 ◽

2006 ◽

Vol 189 (3) ◽

pp. 1090-1098 ◽

Cited By ~ 59

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.

Download Full-text

SleC Is Essential for Cortex Peptidoglycan Hydrolysis during Germination of Spores of the Pathogenic Bacterium Clostridium perfringens

Journal of Bacteriology ◽

10.1128/jb.01832-08 ◽

2009 ◽

Vol 191 (8) ◽

pp. 2711-2720 ◽

Cited By ~ 65

Author(s):

Daniel Paredes-Sabja ◽

Peter Setlow ◽

Mahfuzur R. Sarker

Keyword(s):

Clostridium Perfringens ◽

Spore Germination ◽

Dipicolinic Acid ◽

Pathogenic Bacterium ◽

Wild Type ◽

Lytic Enzymes ◽

Colony Forming Efficiency ◽

Forming Efficiency ◽

Peptidoglycan Hydrolysis

ABSTRACT Clostridial spore germination requires degradation of the spore's peptidoglycan (PG) cortex by cortex-lytic enzymes (CLEs), and two Clostridium perfringens CLEs, SleC and SleM, degrade cortex PG in vitro. We now find that only SleC is essential for cortex hydrolysis and viability of C. perfringens spores. C. perfringens sleC spores did not germinate completely with nutrients, KCl, or a 1:1 chelate of Ca2+ and dipicolinic acid (Ca-DPA), and the colony-forming efficiency of sleC spores was 103-fold lower than that of wild-type spores. However, sleC spores incubated with various germinants released most of their DPA, although slower than wild-type or sleM spores, and DPA release from sleC sleM spores was very slow. In contrast, germination and viability of sleM spores were similar to that of wild-type spores, although sleC sleM spores had 105-fold-lower viability. These results allow the following conclusions about C. perfringens spore germination: (i) SleC is essential for cortex hydrolysis; (ii) although SleM can degrade cortex PG in vitro, this enzyme is not essential; (iii) action of SleC alone or with SleM can accelerate DPA release; and (iv) Ca-DPA does not trigger spore germination by activation of CLEs.

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.183.16.4894-4899.2001 ◽

2001 ◽

Vol 183 (16) ◽

pp. 4894-4899 ◽

Cited By ~ 107

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.

Download Full-text

Effect of the cortex-lytic enzyme SleC from non-food-borne Clostridium perfringens on the germination properties of SleC-lacking spores of a food poisoning isolate

Canadian Journal of Microbiology ◽

10.1139/w10-083 ◽

2010 ◽

Vol 56 (11) ◽

pp. 952-958 ◽

Cited By ~ 5

Author(s):

Daniel Paredes-Sabja ◽

Mahfuzur R. Sarker

Keyword(s):

Clostridium Perfringens ◽

Spore Germination ◽

Type Strain ◽

Wild Type Strain ◽

Dipicolinic Acid ◽

Food Poisoning ◽

Lytic Enzyme ◽

Wild Type ◽

Lytic Enzymes ◽

Food Borne

The hallmark of bacterial spore germination is peptidoglycan cortex hydrolysis by cortex-lytic enzymes. In spores of Clostridium perfringens wild-type strain SM101, which causes food poisoning, the sole essential cortex-lytic enzyme SleC is activated by a unique serine protease CspB. Interestingly, the non-food-borne wild-type strain F4969 encodes a significantly divergent SleC variant (SleCF4969) and 3 serine proteases (CspA, CspB, and CspC). Consequently, in this study we evaluated the functional compatibility of SleCF4969and SleCSM101by complementing the germination phenotypes of SM101ΔsleC spores with sleCF4969. Our results show that although pro-SleCF4969was processed into mature SleCF4969in the SM101ΔsleC spores, it partially restored spore germination with nutrient medium, with a mixture of l-asparagine and KCl, or with a 1:1 chelate of Ca2+and dipicolinic acid. While the amount of dipicolinic acid released was lower, the amount of hexosamine-containing material released during germination of SM101ΔsleC(sleCF4969) spores was similar to the amount released during germination of SM101 wild-type spores. The viability of SM101ΔsleC(sleCF4969) spores was 8- and 3-fold lower than that of SM101 and F4969 spores, respectively. Together, these data indicate that the peptidoglycan cortex hydrolysis machinery in the food poisoning isolate SM101 is functionally divergent than that in the non-food-borne isolate F4969.

Download Full-text