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 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 GerO, a Putative Na+/H+-K+ Antiporter, Is Essential for Normal Germination of Spores of the Pathogenic Bacterium Clostridium perfringens

2009 ◽  
Vol 191 (12) ◽  
pp. 3822-3831 ◽  
Author(s):  
Daniel Paredes-Sabja ◽  
Peter Setlow ◽  
Mahfuzur R. Sarker
Keyword(s):  
Clostridium Perfringens ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Ectopic Expression ◽  
Monovalent Cation ◽  
Wild Type ◽  
Cell Compartment ◽  
E Coli ◽  
Modeled Structure ◽  
Cation Transporters

ABSTRACT The genome of the pathogen Clostridium perfringens encodes two proteins, GerO and GerQ, homologous to monovalent cation transporters suggested to have roles in the germination of spores of some Bacillus species. GerO and GerQ were able to transport monovalent cations (K+ and/or Na+) in Escherichia coli, and gerO and gerQ were expressed only in the mother cell compartment during C. perfringens sporulation. C. perfringens spores lacking GerO were defective in germination with a rich medium, KCl, l-asparagine, and a 1:1 chelate of Ca2+ and dipicolinic acid (DPA), but not with dodecylamine, and the defect was prior to DPA release in germination. All defects in gerO spores were complemented by ectopic expression of wild-type gerO. Loss of GerQ had much smaller effects on spore germination, and these effects were most evident in spores also lacking GerO. A modeled structure of GerO was similar to that of the E. coli Na+/H+ antiporter NhaA, and GerO, but not GerQ contained two adjacent Asp residues thought to be important in the function of this group of cation transporters. Replacement of these adjacent Asp residues in GerO with Asn reduced the protein's ability to complement the germination defect in gerO spores but not the ability to restore cation transport to E. coli cells defective in K+ uptake. Together, these data suggest that monovalent cation transporters play some role in C. perfringens spore germination. However, it is not clear whether this role is directly in germination or perhaps in spore formation.

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 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 Protective Role of Spore Structural Components in Determining Bacillus subtilis Spore Resistance to Simulated Mars Surface Conditions

2012 ◽  
Vol 78 (24) ◽  
pp. 8849-8853 ◽  
Author(s):  
Ralf Moeller ◽  
Andrew C. Schuerger ◽  
Günther Reitz ◽  
Wayne L. Nicholson
Keyword(s):  
Bacillus Subtilis ◽  
Dipicolinic Acid ◽  
Protective Role ◽  
Structural Components ◽  
Wild Type ◽  
Content Type ◽  
Surface Conditions ◽  
Bacillus Subtilis Spore ◽  
Spore Resistance

ABSTRACTSpores of wild-type and mutantBacillus subtilisstrains lacking various structural components were exposed to simulated Martian atmospheric and UV irradiation conditions. Spore survival and mutagenesis were strongly dependent on the functionality of all of the structural components, with small acid-soluble spore proteins, coat layers, and dipicolinic acid as key protectants.

scholarly journals Unmasking Novel Sporulation Genes in Bacillus subtilis

2004 ◽  
Vol 186 (23) ◽  
pp. 8089-8095 ◽  
Author(s):  
Jessica M. Silvaggi ◽  
David L. Popham ◽  
Adam Driks ◽  
Patrick Eichenberger ◽  
Richard Losick
Keyword(s):  
Bacillus Subtilis ◽  
Double Mutant ◽  
Dipicolinic Acid ◽  
Muramic Acid ◽  
Wild Type ◽  
Triple Mutant ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Sporulation Genes

ABSTRACT The Bacillus subtilis transcription factor σE directs the expression of a regulon of 262 genes, but null mutations in only a small fraction of these genes severely impair sporulation. We have previously reported that mutations in seven σE-controlled genes cause a mild (2- to 10-fold) defect in sporulation. In this study, we found that pairwise combinations of some of these seven mutations led to strong synthetic sporulation phenotypes, especially those involving the ytrHI operon and ybaN. Double mutants of ybaN and ytrH and of ybaN and ytrI had >10,000-fold lower sporulation efficiencies than the wild type. Thin-section electron microscopy revealed a block in cortex formation for the ybaN ytrH double mutant and coat defects for the ybaN single and ybaN ytrI double mutants. Sporulating cells of a ybaN ytrI double mutant and of a ybaN ytrHI triple mutant exhibited a pronounced loss of dipicolinic acid (DPA) between hours 8 and 24 of sporulation, in contrast to the constant levels seen for the wild type. An analysis of the spore cortex peptidoglycans of the ybaN ytrI and ybaN ytrHI mutants showed striking decreases in the levels of total muramic acid by hour 24 of sporulation. These data, along with the loss of DPA in the mutants, suggest that the developing spores were unstable and that the cortex underwent degradation late in sporulation. The existence of otherwise hidden sporulation pathways indicates that functional redundancy may mask the role of hitherto unrecognized sporulation genes.

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 a gerF (lgt) Mutation on the Germination of Spores of Bacillus subtilis

2004 ◽  
Vol 186 (10) ◽  
pp. 2984-2991 ◽  
Author(s):  
Takao Igarashi ◽  
Barbara Setlow ◽  
Madan Paidhungat ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Cysteine Residue ◽  
Receptor Function ◽  
Recognition Sequence ◽  
N Terminus ◽  
Membrane Bound

ABSTRACT One of the proteins of the membrane-bound receptors that recognize individual nutrients that trigger germination of spores of Bacillus subtilis contains the recognition sequence for diacylglycerol addition to a cysteine residue near the protein's N terminus. B. subtilis spores lacking the gerF (lgt) gene that codes for prelipoprotein diacylglycerol transferase exhibited significantly slowed germination in response to nutrient germinants as found previously, but germination of gerF spores with a mixture of Ca2+ and dipicolinic acid or with dodecylamine was normal, as was the spontaneous germination of gerF spores lacking all nutrient germinant receptors. The deleterious effects of the gerF mutation on nutrient germination were highest on germination triggered by the GerA nutrient receptor and were less so (but still significant) on germination triggered by the GerB nutrient receptor. However, there was little, if any, effect on GerK nutrient receptor-mediated spore germination. As predicted from the latter results, replacement by alanine of the cysteine residue to which diacylglycerol is thought to be added to these nutrient receptors had a large effect on GerA receptor function, less effect on GerB receptor function, and little, if any, effect on GerK receptor function.

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.

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