Spores of Bacillus subtilis with a mutation in spoVF cannot synthesize dipicolinic acid (DPA) and are too unstable to be purified and studied in detail. However, the spores of a strain lacking the three major germinant receptors (termed ⌬ger3), as well as spoVF, can be isolated, although they spontaneously germinate much more readily than ⌬ger3 spores. The ⌬ger3 spoVF spores lack DPA and have higher levels of core water than ⌬ger3 spores, although sporulation with DPA restores close to normal levels of DPA and core water to ⌬ger3 spoVF spores. The DPA-less spores have normal cortical and coat layers, as observed with an electron microscope, but their core region appears to be more hydrated than that of spores with DPA. The ⌬ger3 spoVF spores also contain minimal levels of the processed active form (termed P 41 ) of the germination protease, GPR, a finding consistent with the known requirement for DPA and dehydration for GPR autoprocessing. However, any P 41 formed in ⌬ger3 spoVF spores may be at least transiently active on one of this protease's small acid-soluble spore protein (SASP) substrates, SASP-␥. Analysis of the resistance of wild-type, ⌬ger3, and ⌬ger3 spoVF spores to various agents led to the following conclusions: (i) DPA and core water content play no role in spore resistance to dry heat, dessication, or glutaraldehyde; (ii) an elevated core water content is associated with decreased spore resistance to wet heat, hydrogen peroxide, formaldehyde, and the iodine-based disinfectant Betadine; (iii) the absence of DPA increases spore resistance to UV radiation; and (iv) wild-type spores are more resistant than ⌬ger3 spores to Betadine and glutaraldehyde. These results are discussed in view of current models of spore resistance and spore germination.Spores of Bacillus and Clostridium species normally contain Ն10% of their dry weight as pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) (21,22,39). This compound is synthesized late in sporulation in the mother cell compartment of the sporulating cell but accumulates only in the developing forespore (6, 36). The great majority of the spore's DPA is in the spore core, where it is most likely chelated with divalent cations, predominantly Ca 2ϩ , although there are also significant amounts of Mg 2ϩ and Mn 2ϩ , with smaller amounts of other divalent cations (21,22,37,39). In the first minutes of spore germination the DPA is excreted, along with the associated divalent cations (36, 37).Since DPA is found only in dormant spores of Bacillus and Clostridium species and since these spores differ in a number of properties from vegetative cells, in particular in their dormancy and heat resistance, it is not surprising that DPA and divalent cations have been suggested to be involved in some of the spore's unique properties. There is some evidence in support of this suggestion, since mutants whose spores do not accumulate DPA have been isolated in several Bacillus species, and often these DPA-less spores are heat sensitive (1,4,25,42,43). Unfortunately, for some of ...

DormantBacillus subtilis spores can be induced to germinate by nutrients, as well as by nonmetabolizable chemicals, such as a 1:1 chelate of Ca 2؉ 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 Ca 2؉ -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 Ca 2؉ -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 Ca 2؉ -DPA. Our findings thus define a mechanism for Ca 2؉ -DPA-induced germination and also provide the first definitive evidence for a signaling pathway that activates cortex hydrolysis in response to nutrients.Germination is the process by which dormant bacterial spores resume metabolism and growth, and it is generally triggered by the presence of nutrients, including amino acids, sugars, and nucleosides (8, 25). The germination process triggered by nutrients consists of a number of events whose precise temporal order has not been unequivocally determined. Some of those events occur in the spore core and include rehydration of the spore's somewhat dehydrated cytoplasm and excretion of its large (ϳ10% of the spore's dry weight) depot of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) and divalent cations, predominantly Ca 2ϩ , which are likely present as a 1:1 chelate (25). A third major event early in spore germination is the breakdown of the spore's cortex, which is a special peptidoglycan (PG) layer that surrounds the spore core and inner membrane and is responsible in some fashion for the core's relative dehydration and enzymatic dormancy (6,25). It is currently thought that interaction of nutrients with their receptors in the spore's inner membrane (10, 24) induces some permeability change in that membrane leading to the release of DPA and cations from the spore core along with attendant water uptake (8, 29). It is also known that cortex hydrolysis is not needed for those events in the spore core but is absolutely necessary for subsequent steps in germination, including initiation of spore metabolism and growth of the germinated spore, that culminate in the formation of a viable cell (12,26,29). On that basis, nutrient-induced spore germination has been divided into stage I (29), which consists of events that occ...

DormantBacillus 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.Bacillus subtilis cells form metabolically dormant spores when starved for one or more nutrients (7). In the presence of particular nutrients, called germinants, the spores break dormancy through the process of germination and, after going through outgrowth, eventually resume vegetative growth (15). It is currently believed that spores recognize nutrient germinants through receptor proteins encoded by three loci (gerA, gerB, and gerK). This hypothesis is based on genetic studies which showed that spores containing mutations at any one of these loci fail to germinate in response to specific germinants (14,15,19). Further support for the receptor hypothesis came from the finding that dominant mutations in gerB allow spores to germinate in novel germinants (17). The gerA, gerB, and gerK loci are each tricistronic operons encoding proteins which share significant homology across the three operons (4,12,25). Two of the three proteins encoded by each operon are predicted to be membrane proteins, which is again consistent with the idea that they encode germinant receptors (4, 25). The proteins, however, have been refractory to in vitro biochemical manipulation, and thus the receptor hypothesis remains untested.Recent sequencing and genetic studies have identified members of the gerA family of operons in other endospore-forming bacteria including Bacillus cereus (3), Bacillus anthracis (9), Bacillus halodurans (23), Clostridium acetobutylicum (Genome Therapeutics Corporation), Clostridium difficile (Sanger Centre), and Clostridium pasteurianum (GenBank). However, thus far no gerA homologs have been identified in other bacterial groups. Furthermore, the B. cereus gerA operon homolog, gerI, has been implicated in inosine-induced spore germination in that ...

Previous studies attributed the yeast (Saccharomyces cerevisiae) cdc1(Ts) growth defect to loss of an Mn 2؉ -dependent function. In this report we show that cdc1(Ts) temperature-sensitive growth is also associated with an increase in cytosolic Ca 2؉ . We identified two recessive suppressors of the cdc1(Ts) temperature-sensitive growth which block Ca 2؉ uptake and accumulation, suggesting that cytosolic Ca 2؉ exacerbates or is responsible for the cdc1(Ts) growth defect. One of the cdc1(Ts) suppressors is identical to a gene, MID1, recently implicated in mating pheromone-stimulated Ca 2؉ uptake. The gene (CCH1) corresponding to the second suppressor encodes a protein that bears significant sequence similarity to the pore-forming subunit (␣1) of plasma membrane, voltage-gated Ca 2؉ channels from higher eukaryotes. Strains lacking Mid1 or Cch1 protein exhibit a defect in pheromone-induced Ca 2؉ uptake and consequently lose viability upon mating arrest. The mid1⌬ and cch1⌬ mutants also display reduced tolerance to monovalent cations such as Li ؉ , suggesting a role for Ca 2؉ uptake in the calcineurin-dependent ion stress response. Finally, mid1⌬ cch1⌬ double mutants are, by both physiological and genetic criteria, identical to single mutants. These and other results suggest Mid1 and Cch1 are components of a yeast Ca 2؉ channel that may mediate Ca 2؉ uptake in response to mating pheromone, salt stress, and Mn 2؉ depletion.In eukaryotic cells, cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ) fluctuates transiently to regulate such diverse processes as neurotransmitter release, muscle contraction, and T-cell activation (9, 31). Intracellular Ca 2ϩ levels are tightly regulated by numerous channels, pumps, and antiporters, such that [Ca 2ϩ ] i is normally maintained at an extremely low concentration (ϳ 100 nM) despite a 10,000-fold concentration difference across the plasma membrane (9). Eukaryotic cells utilize this gradient to generate Ca 2ϩ spikes by stimulating the opening of Ca 2ϩ channels and allowing Ca 2ϩ influx down the concentration gradient (9, 31). Among the best characterized of these entry pathways are the voltage-dependent Ca 2ϩ channels of the plasma membrane which open in response to membrane depolarization (3,9). Ca 2ϩ has been implicated in numerous processes of the budding yeast Saccharomyces cerevisiae (8,16,20,37), including stress-induced expression of ion transporter genes, bud formation, and viability upon pheromone-induced arrest. However, there is compelling evidence for a role for Ca 2ϩ in only the last of these processes (7,15,16,22). Haploid yeast cells exist as two mating types, a and ␣, that secrete mating pheromones a-and ␣-factor, respectively. In the presence of ␣-factor, a cells undergo a complex developmental process in preparation for mating. For example, pheromone-treated cells accumulate high levels of intracellular Ca 2ϩ , arrest in G 1 , and form a mating projection or shmoo (16). Cells arrested in medium lacking Ca 2ϩ lose viability within 5 h after pheromone treatment, and th...