Pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) in a 1:1 chelate with calcium ion (Ca-DPA) comprises 5 to 15% of the dry weight of spores of Bacillus species. Ca-DPA is important in spore resistance to many environmental stresses and in spore stability, and Ca-DPA levels in spore populations can vary with spore species/strains, as well as with sporulation conditions. We have measured levels of Ca-DPA in large numbers of individual spores in populations of a variety of Bacillus species and strains by using microfluidic Raman tweezers, in which a single spore is trapped in a focused laser beam and its Ca-DPA is quantitated from the intensity of the Ca-DPA-specific band at 1,017 cm ؊1 in Raman spectroscopy. Conclusions from these measurements include the following: (i) Ca-DPA concentrations in the spore core are >800 mM, well above Ca-DPA solubility; (ii) SpoVA proteins may be involved in Ca-DPA uptake in sporulation; and (iii) Ca-DPA levels differ significantly among individual spores in a population, but much of this variation could be due to variations in the sizes of individual spores.

The rates of germination of Bacillus subtilis spores with L-alanine were increased markedly, in particular at low L-alanine concentrations, by overexpression of the tricistronic gerA operon that encodes the spore's germinant receptor for L-alanine but not by overexpression of gerA operon homologs encoding receptors for other germinants. However, spores with elevated levels of the GerA proteins did not germinate more rapidly in a mixture of asparagine, glucose, fructose, and K ؉ (AGFK), a germinant combination that requires the participation of at least the germinant receptors encoded by the tricistronic gerB and gerK operons. Overexpression of the gerB or gerK operon or both the gerB and gerK operons also did not stimulate spore germination in AGFK. Overexpression of a mutant gerB operon, termed gerB*, that encodes a receptor allowing spore germination in response to either D-alanine or L-asparagine also caused faster spore germination with these germinants, again with the largest enhancement of spore germination rates at lower germinant concentrations. However, the magnitudes of the increases in the germination rates with D-alanine or L-asparagine in spores overexpressing gerB* were well below the increases in the spore's levels of the GerBA protein. Germination of gerB* spores with D-alanine or L-asparagine did not require participation of the products of the gerK operon, but germination with these agents was decreased markedly in spores also overexpressing gerA. These findings suggest that (i) increases in the levels of germinant receptors that respond to single germinants can increase spore germination rates significantly; (ii) there is some maximum rate of spore germination above which stimulation of GerA operon receptors alone will not further increase the rate of spore germination, as action of some protein other than the germinant receptors can become rate limiting; (iii) while previous work has shown that the wild-type GerB and GerK receptors interact in some fashion to cause spore germination in AGFK, there also appears to be an additional component required for AGFK-triggered spore germination; (iv) activation of the GerB receptor with D-alanine or L-asparagine can trigger spore germination independently of the GerK receptor; and (v) it is likely that the different germinant receptors interact directly and/or compete with each other for some additional component needed for initiation of spore germination. We also found that very high levels of overexpression of the gerA or gerK operon (but not the gerB or gerB* operon) in the forespore blocked sporulation shortly after the engulfment stage, although sporulation appeared normal with the lower levels of gerA or gerK overexpression that were used to generate spores for analysis of rates of germination.Spores of various Bacillus species can remain dormant for long periods of time but can rapidly "return to life" through the process of spore germination (18,19,20,28). Spore germination is normally triggered by the stereospecific binding of specific low-mol...

The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of ϳ9 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 HgCl 2 . 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 Ca 2؉ almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca 2؉ and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca 2؉ -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 Ca 2؉ -DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.

The products of the hexacistronic spoVA operon of Bacillus subtilis may be involved in the transport of dipicolinic acid into the forespore during sporulation and its release during spore germination. The major hydrophilic coding region of B. subtilis spoVAD was cloned, the protein was expressed in Escherichia coli as a His tag fusion protein, and a rabbit antiserum was raised against the purified protein. Western blot analyses of fractions from B. subtilis spores showed that SpoVAD is an integral inner membrane protein present at levels >50-fold higher than those of the spore's nutrient germinant receptors that are also present in the inner membrane. SpoVAD also persisted in outgrowing spores.Spore formation and spore germination are two crucial processes in the life cycle of spore-forming bacteria. Sporulation is induced by nutrient deprivation and generates a dormant spore that can survive long periods under unfavorable growth conditions. The process of spore germination and then outgrowth returns the spore to life in response to better conditions, in particular, the presence of nutrients. In addition, for spores of pathogenic species, spore germination can lead to rapid production of toxins or enzymes that cause disease or food spoilage.The mechanisms of spore formation and germination in Bacillus species, in particular, Bacillus subtilis, have been extensively studied (10,31,35). A characteristic feature of the spores of Bacillus and Clostridium species is high levels of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]), which usually comprises ϳ10% of the spore's dry weight (9). DPA is synthesized in the mother cell compartment of a sporulating cell, enters the developing spore by moving across the two membranes that surround the spore core, and likely exists in the core as a 1:1 chelate with divalent cations, predominantly Ca 2ϩ . The high DPA level in the spore core is important for spore resistance to wet heat and spore stability, as DPA-less spores lyse rapidly during sporulation and stabilized DPA-less spores are much more susceptible to wet heat than are wildtype spores (15,27). DPA is released, most likely as a 1:1 chelate with divalent cations, in the first minutes of spore germination triggered by nutrients. This DPA exit facilitates the rehydration of the spore core and also triggers subsequent steps in the germination process (30,35). Clearly, the entry of DPA into, its presence in, and its exit from the spore core, respectively, are essential processes for these spore-forming bacteria.Unfortunately, very little is known of the mechanisms of DPA entry into the developing forespore and its exit during spore germination, although it has been suggested that proteins encoded by the spoVA operon are involved in DPA entry (13). The B. subtilis spoVA operon encodes six proteins that are likely to be membrane proteins, and the operon is transcribed in the forespore by RNA polymerase containing G at or about the time of DPA synthesis in the mother cell (12,13,14,25,33,34,36). Evidence for the invol...

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.