Germination of spores of Bacillus subtilis with dodecylamine

AIMS: To determine the properties of Bacillus subtilis spores germinated with the alkylamine dodecylamine, and the mechanism of dodecylamine-induced spore germination. METHODS AND RESULTS: Spores of B. subtilis prepared in liquid medium were germinated efficiently by dodecylamine, while spores prepared on solid medium germinated more poorly with this agent. Dodecylamine germination of spores was accompanied by release of almost all spore dipicolinic acid (DPA), degradation of the spore's peptidoglycan cortex, release of the spore's pool of free adenine nucleotides and the killing of the spores. The dodecylamine-germinated spores did not initiate metabolism, did not degrade their pool of small, acid-soluble spore proteins efficiently and had a significantly lower level of core water than did spores germinated by nutrients. As measured by DPA release, dodecylamine readily induced germination of B. subtilis spores that: (a) were decoated, (b) lacked all the receptors for nutrient germinants, (c) lacked both the lytic enzymes either of which is essential for cortex degradation, or (d) had a cortex that could not be attacked by the spore's cortex-lytic enzymes. The DNA in dodecylamine-germinated wild-type spores was readily stained, while the DNA in dodecylamine-germinated spores of strains that were incapable of spore cortex degradation was not. These latter germinated spores also did not release their pool of free adenine nucleotides. CONCLUSIONS: These results indicate that: (a) the spore preparation method is very important in determining the rate of spore germination with dodecylamine, (b) wild-type spores germinated by dodecylamine progress only part way through the germination process, (c) dodecylamine may trigger spore germination by a novel mechanism involving the activation of neither the spore's nutrient germinant receptors nor the cortex-lytic enzymes, and (d) dodecylamine may trigger spore germination by directly or indirectly activating release of DPA from the spore core, through the opening of channels for DPA in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new insight into the mechanism of spore germination with the cationic surfactant dodecylamine, and also into the mechanism of spore germination in general. New knowledge of mechanisms to stimulate spore germination may have applied utility, as germinated spores are much more sensitive to processing treatments than are dormant spores.     

The formation of actin rods composed of actin tubules in Dictyostelium discoideum spores

A new type of actin rod formed in both the nucleus and the cytoplasm, as well as tyrosine phosphorylation of actin, is implicated in the maintenance of dormancy and viability of Dictyostelium discoideum spores. Here the ultrastructure of the rods and their relationship to the phosphorylation of actin were examined. The rods first appeared in premature spores at the midculmination stage as bundles composed of actin tubules hexagonally cross-linked. The 13-nm-diameter bundles were composed of three actin filaments. Formation of the actin rods begins during the late culmination stage and proceeds until 2 days after completion of fruiting bodies. The physical events occur in the following order; association of several modules of bundles, close packing and decrease in diameter of actin tubules, elongation of rods across the nucleus or the cytoplasm. Actin phosphorylation levels increased at the late culmination stage and reached a maximum level 12 h later. Immediately following activation of spore germination, actin was rapidly dephosphorylated, followed shortly thereafter by the disappearance of rods. Shortened actin tubules once again became arranged in a hexagonal pattern. This hexagonal arrangement of actin tubules is possibly involved in rod formation and disappearance and does not depend upon actin phosphorylation. In contrast, rod-maturation processes may correlate with actin phosphorylation.     

Effect of mechanical abrasion on the viability, disruption and germination of spores of Bacillus subtilis

AIMS: To elucidate the factors influencing the sensitivity of Bacillus subtilis spores in killing and disrupting by mechanical abrasion, and the mechanism of stimulation of spore germination by abrasion. METHODS AND RESULTS: Spores of B. subtilis strains were abraded by shaking with glass beads in liquid or the dry state, and spore killing, disruption and germination were determined. Dormant spores were more resistant to killing and disruption by abrasion than were growing cells or germinated spores. However, dormant spores of the wild-type strain with or without most coat proteins removed, spores of strains with mutations causing spore coat defects, spores lacking their large depot of dipicolinic acid (DPA) and spores with defects in the germination process exhibited essentially identical rates of killing and disruption by abrasion. When spores lacking all nutrient germinant receptors were enumerated by plating directly on nutrient medium, abrasion increased the plating efficiency of these spores before killing them. Spores lacking all nutrient receptors and either of the two redundant cortex-lytic enzymes behaved similarly in this regard, but the plating efficiency of spores lacking both cortex-lytic enzymes was not stimulated by abrasion. CONCLUSIONS: Dormant spores are more resistant to killing and disruption by abrasion than are growing cells or germinated spores, and neither the complete coats nor DPA are important in spore resistance to such treatments. Germination is not essential for spore killing by abrasion, although abrasion can trigger spore germination by activation of either of the spore's cortex-lytic enzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the mechanisms of the killing, disruption and germination of spores by abrasion and makes the surprising finding that at least much of the spore coat is not important in spore resistance to abrasion.     

Influence of cellular differentiation on ultraviolet induced DNA damage and its repair mechanisms in B. cereus

Susceptibility to UV irradiation of B. cereus BIS-59 spores undergoing germination at various stages-dormant spores to vegetative cell stage and their ability to recover from radiation damage were studied. For a given dose of radiation, the number of spore photoproducts (SPP) formed in the DNA of dormant spores was about 5-times greater than that of thymine dimers (TT) formed in the DNA of vegetative cells. At intermediate stages of the germination cycle, there was a rapid decline in the UV radiation-induced SPP formed in DNA with a concomitant increase in the UV radiation-induced TT formed in DNA. Bacterial spores undergoing germination (up to 3 hr) in the low nutrient medium (0.3% yeast extract) displayed much higher resistance to UV radiation than those germinating in the rich nutrient medium, even though there was no discernible difference under the two incubation conditions in respect of the extent of germination and the time at which the outgrowth stage appeared (3 hr). This was due to the formation TT in the DNA of spores germinating in the low nutrient as compared to that of spores germinating in the rich-nutrient medium. In UV-irradiated dormant spores, SPP formed in the spore DNA did not disappear even after prolonged incubation in the non-germinating medium. However, when the UV-irradiated dormant spores were germinated in low or rich nutrient medium, a significant proportion of SPP in DNA was eliminated. The dormant spores incubated in either of the germinating media for 15 min and then UV-irradiated were capable of eliminating SPP (presumably by monomerization) even by incubation in a non-germinating medium and in the complete absence of protein synthesis (buffer holding recovery), thereby implying that spore-repair enzymes were activated in response to initial's germination. The acquisition of photo-reactivation ability appeared in spores subjected to germination only in the rich-nutrient medium at the outgrowth stage and required de novo synthesis of the required enzymes.     

Spore pool glutamic acid as a metabolite in germination

Spore glutamic acid pools were examined in dormant and germinating spores using colorimetric and (14)C analytical procedures. Germination of spores of Bacillus megaterium (parent strain), initiated by d-glucose, was accompanied by a rapid drop in the level of spore pool glutamate, from 12.0 mug/mg of dry spores to 7.7 mug/mg of dry spores after 30 sec of germination. Similar decreases in extractable spore pool glutamate were observed with l-alanine-initiated germination of B. licheniformis spores. On the other hand, glutamate pools of mutant spores of B. megaterium, with a requirement of gamma-aminobutyric acid for spore germination, remained unchanged for 9 min of germination, at which time more than 50% of the spore population had germinated. Evidence for conversion of spore pool glutamate to gamma-aminobutyric acid during germination of spores of B. megaterium (parent strain) was obtained.     

Role of GerD in germination of Bacillus subtilis spores

Spores of a Bacillus subtilis strain with a gerD deletion mutation (Delta gerD) 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 Delta gerD spores in which nutrient receptors were overexpressed. The germination defect of Delta gerD spores was not suppressed by many changes in the sporulation or germination conditions. Germination of Delta gerD 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 Delta gerD 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.     

Steady-state fluorescence anisotropy changes of 1,6-diphenyl-1,3,5,-hexatriene in membranes from Bacillus megaterium spores

The steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene incorporated into isolated Bacillus megaterium spore membranes was measured. Compounds capable of triggering spore germination in vivo caused an increase in the anisotropy of diphenylhexatriene. These increases in anisotropy of diphenylhexatriene in spore membranes are likely to represent at least a portion of the trigger mechanism for spore germination based on the following observations. First, there was an exceptional positive correlation between compounds that both triggered germination in vivo and caused changes in anisotropy in vitro. Second. the capacity of membranes to respond to germinants by increases in anisotropy was unique to membranes from spores but disappeared after germination. Third, alteration of spores chemically or genetically to block the in vivo triggering of germination by l-proline also blocked the in vitro anisotropy change with l-proline but not d-glucose. Finally, there was no correlation between the transport activities of specific compounds and the ability of these compounds to either trigger germination or alter the anisotropy of diphenylhexatriene in the membranes. Although we do not known the nature of the molecular interactions giving rise to the anisotropy changes, we hypothesize that they are due to changes in protein conformation that alter protein-protein and/or protein-lipid interactions. Such modifications of membrane structures could account for the rapid release of small molecular weight compounds such as K⁺ and Ca²⁺ early in germination.     

Analysis of the action of compounds that inhibit the germination of spores of Bacillus species

AIMS: To determine the mechanism of action of inhibitors of the germination of spores of Bacillus species, and where these inhibitors act in the germination process. METHODS AND RESULTS: Spores of various Bacillus species are significant agents of food spoilage and food-borne disease, and inhibition of spore germination is a potential means of reducing such problems. Germination of the following spores was studied: (i) wild-type B. subtilis spores; (ii) B. subtilis spores with a nutrient receptor variant allowing recognition of a novel germinant; (iii) B. subtilis spores with elevated levels of either the variant nutrient receptor or its wild-type allele; (iv) B. subtilis spores lacking all nutrient receptors and (v) wild-type B. megaterium spores. Spores were germinated with a variety of nutrient germinants, Ca2+-dipicolinic acid (DPA) and dodecylamine for B. subtilis spores, and KBr for B. megaterium spores. Compounds tested as inhibitors of germination included alkyl alcohols, a phenol derivative, a fatty acid, ion channel blockers, enzyme inhibitors and several other compounds. Assays used to assess rates of spore germination monitored: (i) the fall in optical density at 600 nm of spore suspensions; (ii) the release of the dormant spore's large depot of DPA; (iii) hydrolysis of the dormant spore's peptidoglycan cortex and (iv) generation of CFU from spores that lacked all nutrient receptors. The results with B. subtilis spores allowed the assignment of inhibitory compounds into two general groups: (i) those that inhibited the action of, or response to, one nutrient receptor and (ii) those that blocked the action of, or response to, several or all of the nutrient receptors. Some of the compounds in groups 1 and 2 also blocked action of at least one cortex lytic enzyme, however, this does not appear to be the primary site of their action in inhibiting spore germination. The inhibitors had rather different effects on germination of B. subtilis spores with nutrients or non-nutrients, consistent with previous work indicating that germination of B. subtilis spores by non-nutrients does not involve the spore's nutrient receptors. In particular, none of the compounds tested inhibited spore germination with dodecylamine, and only three compounds inhibited Ca2+-DPA germination. In contrast, all compounds had very similar effects on the germination of B. megaterium spores with either glucose or KBr. The effects of the inhibitors tested on spores of both Bacillus species were largely reversible. CONCLUSIONS: This work indicates that inhibitors of B. subtilis spore germination fall into two classes: (i) compounds (most alkyl alcohols, N-ethylmaleimide, nifedipine, phenols, potassium sorbate) that inhibit the action of, or response to, primarily one nutrient receptor and (ii) compounds [amiloride, HgCl2, octanoic acid, octanol, phenylmethylsulphonylfluoride (PMSF), quinine, tetracaine, tosyl-l-arginine methyl ester, trifluoperazine] that inhibit the action of, or response to, several nutrient receptors. Action of these inhibitors, is reversible. The similar effects of inhibitors on B. megaterium spore germination by glucose or KBr indicate that inorganic salts likely trigger germination by activating one or more nutrient receptors. The lack of effect of all inhibitors on dodecylamine germination suggests that this compound stimulates germination by creating channels in the spore's inner membrane allowing DPA release. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the steps in spore germination that are inhibited by various chemicals, and the mechanism of action of these inhibitors. The work also provides new insights into the process of spore germination itself.     

Effects of a gerF (lgt) mutation on the germination of spores of Bacillus subtilis

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.     

Yeast spore germination: a requirement for Ras protein activity during re-entry into the cell cycle.

Saccharomyces cerevisiae spore germination is a process in which quiescent, non-dividing spores become competent for mitotic cell division. Using a novel assay for spore uncoating, we found that spore germination was a multi-step process whose nutritional requirements differed from those for mitotic division. Although both processes were controlled by nutrient availability, efficient spore germination occurred in conditions that did not support cell division. In addition, germination did not require many key regulators of cell cycle progression including the cyclin-dependent kinase, Cdc28p. However, two processes essential for cell growth, protein synthesis and signaling through the Ras protein pathway, were required for spore germination. Moreover, increasing Ras protein activity in spores resulted in an accelerated rate of germination and suggested that activation of the Ras pathway was rate-limiting for entry into the germination program. An early step in germination, commitment, was identified as the point at which spores became irreversibly destined to complete the uncoating process even if the original stimulus for germination was removed. Spore commitment to germination required protein synthesis and Ras protein activity; in contrast, post-commitment events did not require ongoing protein synthesis. Altogether, these data suggested a model for Ras function during transitions between periods of quiescence and cell cycle progression.     

Dynamics of spore germination and mycelial growth of streptomycetes under low humidity conditions

At extremely low values of moisture pressure (?96.4 MPa; a_w 0.50), the spores of xerotolerant streptomycetes (Streptomyces odorifer and S. rubiginosohelvolus) germinated, their germ lengths increased, and lateral branching of the mycelium was observed after 5 days of incubation in a thin layer of agarized nutrient medium. At ?22.6 MPa (a_w 0.86), the mycelium begins to branch after a 2-day incubation; over a 5-day incubation at ?2.8 MPa (a_w 0.98), it goes through a reproduction cycle, which culminates in spore formation. The mathematical model approach enabled us to elucidate the behavioral patterns of Streptomyces spores in a thin layer of agarized nutrient medium at low humidity levels. The dynamics of spore germination is governed by the exponential law, which allows calculation of the average duration of the period a before spore germination, as well as the time needed for 50% of viable spores to germinate.     

Bacillus thermoamylovorans Spores with Very-High-Level Heat Resistance Germinate Poorly in Rich Medium despite the Presence of ger Clusters but Efficiently upon Exposure to Calcium-Dipicolinic Acid

High-level heat resistance of spores of Bacillus thermoamylovorans poses challenges to the food industry, as industrial sterilization processes may not inactivate such spores, resulting in food spoilage upon germination and outgrowth. In this study, the germination and heat resistance properties of spores of four food-spoiling isolates were determined. Flow cytometry counts of spores were much higher than their counts on rich medium (maximum, 5%). Microscopic analysis revealed inefficient nutrient-induced germination of spores of all four isolates despite the presence of most known germination-related genes, including two operons encoding nutrient germinant receptors (GRs), in their genomes. In contrast, exposure to nonnutrient germinant calcium-dipicolinic acid (Ca-DPA) resulted in efficient (50 to 98%) spore germination. All four strains harbored cwlJ and gerQ genes, which are known to be essential for Ca-DPA-induced germination in Bacillus subtilis. When determining spore survival upon heating, low viable counts can be due to spore inactivation and an inability to germinate. To dissect these two phenomena, the recoveries of spores upon heat treatment were determined on plates with and without preexposure to Ca-DPA. The high-level heat resistance of spores as observed in this study (D_120°C, 1.9 ± 0.2 and 1.3 ± 0.1 min; z value, 12.2 ± 1.8°C) is in line with survival of sterilization processes in the food industry. The recovery of B. thermoamylovorans spores can be improved via nonnutrient germination, thereby avoiding gross underestimation of their levels in food ingredients.     

Cooperativity between different nutrient receptors in germination of spores of Bacillus subtilis and reduction of this cooperativity by alterations in the GerB receptor

The GerA nutrient receptor alone triggers germination of Bacillus subtilis spores with L-alanine or L-valine, and these germinations were stimulated by glucose and K+ plus the GerK nutrient receptor. The GerB nutrient receptor alone did not trigger spore germination with any nutrients but required glucose, fructose, and K+ (GFK) (termed cogerminants) plus GerK for triggering of germination with a number of L-amino acids. GerB and GerA also triggered spore germination cooperatively with l-asparagine, fructose, and K+ and either L-alanine or L-valine. Two GerB variants (termed GerB*s) that were previously isolated by their ability to trigger spore germination in response to D-alanine do not respond to D-alanine but respond to the same L-amino acids that stimulate germination via GerB plus GerK and GFK. GerB*s alone triggered spore germination with these L-amino acids, although GerK plus GFK stimulated the rates of these germinations. In contrast to l-alanine germination via GerA, spore germination via L-alanine and GerB or GerB* was not inhibited by D-alanine. These data support the following conclusions. (i) Interaction with GerK, glucose, and K+ somehow stimulates spore germination via GerA. (ii) GerB can bind and respond to L-amino acids, although normally either the binding site is inaccessible or its occupation is not sufficient to trigger spore germination. (iii) Interaction of GerB with GerK and GFK allows GerB to bind or respond to amino acids. (iv) In addition to spore germination due to the interaction between GerA and GerK, and GerB and GerK, GerB can interact with GerA to trigger spore germination in response to appropriate nutrients. (v) The amino acid sequence changes in GerB*s reduce these receptor variants' requirement for GerK and cogerminants in their response to L-amino acids. (vi) GerK binds glucose, GerB interacts with fructose in addition to L-amino acids, and GerA interacts only with L-valine, L-alanine, and its analogs. (vii) The amino acid binding sites in GerA and GerB are different, even though both respond to L-alanine. These new conclusions are integrated into models for the signal transduction pathways that initiate spore germination.     

Spore germination promoter of Dictyostelium discoideum excreted by Aerobacter aerogenes.

The nutrient medium in which Aerobacter aerogenes was grown, contains a spore germination promoter (SGP) for the cellular slime mould Dictyostelium discoideum. SGP can cuase synchronous spore germination in a short time, and triggers the germination process in just a few minutes. Germination-promoting capacity of SGP decreases as it comes in contact with increasing number of spores. When spores activated by SGP are stored at 4 degrees C, they gradually return to the dormant state. SGP is comparatively heat-stable, but is unstable at pH above 10 or under 3.     

Role of ger proteins in nutrient and nonnutrient triggering of spore germination in Bacillus subtilis

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 includes gerA, 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 Ca(2+) and dipicolinic acid (DPA). These observations showed that proteins encoded by gerA 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 Ca(2+)-DPA-induced germination showed that the effect of Ca(2+)-DPA on spore germination was saturated at 60 mM and had a K(m) of 30 mM. We also found that decoating spores abolished their ability to germinate in Ca(2+)-DPA but not in nutrient germinants, indicating that Ca(2+)-DPA and nutrient germinants probably act through parallel arms of the germination pathway.     

Role of dipicolinic acid in survival of Bacillus subtilis spores exposed to artificial and solar UV radiation

Pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) constitutes approximately 10% of Bacillus subtilis spore dry weight and has been shown to play a significant role in the survival of B. subtilis spores exposed to wet heat and to 254-nm UV radiation in the laboratory. However, to date, no work has addressed the importance of DPA in the survival of spores exposed to environmentally relevant solar UV radiation. Air-dried films of spores containing DPA or lacking DPA due to a null mutation in the DPA synthetase operon dpaAB were assayed for their resistance to UV-C (254 nm), UV-B (290 to 320 nm), full-spectrum sunlight (290 to 400 nm), and sunlight from which the UV-B portion was filtered (325 to 400 nm). In all cases, air-dried DPA-less spores were significantly more UV sensitive than their isogenic DPA-containing counterparts. However, the degree of difference in UV resistance between the two strains was wavelength dependent, being greatest in response to radiation in the UV-B portion of the spectrum. In addition, the inactivation responses of DPA-containing and DPA-less spores also depended strongly upon whether spores were exposed to UV as air-dried films or in aqueous suspension. Spores lacking the gerA, gerB, and gerK nutrient germination pathways, and which therefore rely on chemical triggering of germination by the calcium chelate of DPA (Ca-DPA), were also more UV sensitive than wild-type spores to all wavelengths tested, suggesting that the Ca-DPA-mediated spore germination pathway may consist of a UV-sensitive component or components.     

Analysis of factors influencing the rate of germination of spores of Bacillus subtilis by very high pressure

AIMS: To elucidate the factors that determine the rate of germination of Bacillus subtilis spores with very high pressure (VHP) and the mechanism of VHP germination. METHODS AND RESULTS: Spores of B. subtilis were germinated rapidly with a VHP of 500 MPa at 50 degrees C. This VHP germination did not require the spore's nutrient-germinant receptors, as found previously, and did not require diacylglycerylation of membrane proteins. However, the spore's pool of dipicolinic acid (DPA) was essential. Either of the two redundant enzymes that degrade the spore's peptidoglycan cortex, and thus allow completion of spore germination, was essential for completion of VHP germination. However, neither of these enzymes was needed for DPA release triggered by VHP treatment. Completion of spore germination as well as DPA release with VHP had an optimum temperature of approx. 60 degrees C, in contrast to an optimum temperature of 40 degrees C for germination with the moderately high pressure of 150 MPa. The rate of spore germination by VHP decreased approx. fourfold when the sporulation temperature increased from 23 degrees C to 44 degrees C, and decreased twofold when 1 mol l(-1) salt was present in sporulation. However, large variations in levels of unsaturated fatty acids in the spore's inner membranes did not affect rates of VHP germination. Complete germination of spores by VHP was not inhibited significantly by killing of spores with several oxidizing agents, and was not inhibited by ethanol, octanol or o-chlorophenol at concentrations that abolish nutrient germination. Completion of spore germination by VHP was also inhibited by Hg(2+), but this ion did not inhibit DPA release caused by VHP. In contrast, dodecylamine, a surfactant that can trigger spore germination, strongly inhibited DPA release caused by VHP treatment. CONCLUSIONS: VHP does not cause spore germination by acting upon the spore's nutrient-germinant receptors, but by directly causing DPA release. This DPA release then leads to subsequent completion of germination. VHP likely acts on the spore's inner membrane to cause DPA release, targeting either a membrane protein or the membrane itself. However, the precise identity of this target is not yet clear. SIGNIFICANCE AND IMPACT OF THE STUDY: There is significant interest in the use of VHP to eliminate or reduce levels of bacterial spores in foods. As at least partial spore germination by pressure is almost certainly essential for subsequent spore killing, knowledge of factors involved and the mechanism of VHP germination are crucial to the understanding of spore killing by VHP. This work provides new insight into factors that can affect the rate of B. subtilis spore germination by VHP, and into the mechanism of VHP germination itself.     

Role of dipicolinic acid in the germination, stability, and viability of spores of Bacillus subtilis

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 alpha/beta-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.