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

Spores of Bacillus and Clostridium species are metabolically dormant and very resistant to many stress agents including heat, radiation, and toxic chemicals (13, 24). As a consequence and because these organisms are commonly found in soils, spores of a number of these species are significant agents of food spoilage and food-borne disease (25). However, since spores are metabolically dormant, they must return to active growth to cause their deleterious effects. Spores return to active growth through the process of germination that is followed by outgrowth (16, 23). Spore germination is normally triggered by any of a variety of specific nutrients such as l-alanine and d-glucose. Metabolism of these nutrients does not trigger spore germination. Rather, the nutrients bind to specific receptors located in the spore's inner membrane, and this binding triggers the release of a variety of compounds from the spore's central region or core, including monovalent cations (H+, Na+, and K+), divalent cations (Ca2+, Mg2+, and Mn2+), and pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]). Release of the latter compound is especially important, since DPA makes up ∼10% of the total spore dry weight and ∼20% of the dry weight of the spore core where it is present in a 1:1 ratio of chelate with divalent cations, predominantly Ca2+. Indeed, during germination with nutrients, most DPA is likely released as the Ca2+ chelate (Ca-DPA). This Ca-DPA release is crucial to spore progression from germination into outgrowth, as water replaces the Ca-DPA, thus elevating the spore core water content significantly. The Ca-DPA release also triggers further events in spore germination, most importantly, the hydrolysis of the spore's peptidoglycan cortex that otherwise prevents the expansion of the spore's core. Following cortex hydrolysis, the spore core expands and takes up more water such that the core water content rises to that in the protoplast of a growing cell. This allows protein movement and enzyme action in the spore core and leads to early events in spore outgrowth including the resumption of energy metabolism and macromolecular synthesis (7, 16, 23). Since Ca-DPA release is such a pivotal event in spore germination, knowledge of the mechanisms and control of Ca-DPA release is crucial to the complete understanding of the overall process of germination. It was suggested a number of years ago that the proteins encoded by the spoVA operon are involved in DPA uptake into the developing spore during sporulation (9). The spoVA operon encodes six proteins, the majority of which are most likely membrane proteins. Since spoVA is transcribed exclusively in the developing forespore, the SpoVA proteins are likely to reside in the spore's inner membrane, and this is the case for at least the SpoVAD protein (32). The fact that SpoVA proteins are involved in DPA uptake during sporulation has been shown using strains with either deletion or temperature-sensitive (TS) mutations in spoVA (30, 31). The spores of TS strains formed at a permissive temperature are also TS for DPA release during nutrient-induced germination (31), consistent with a role for at least some SpoVA proteins in Ca-DPA release during spore germination. In addition to nutrients, spores can be germinated by a number of other agents including surfactants such as dodecylamine, exogenous Ca-DPA, and lysozyme treatment if spores are first decoated to allow the lysozyme to penetrate to the spore's cortex (16, 20, 23). These latter agents do not act through the spore's nutrient germinant receptors but in some cases (dodecylamine) appear to directly cause Ca-DPA release, with this release then triggering subsequent germination events. An obvious question is whether the Ca-DPA release triggered by germination agents other than nutrients also involves SpoVA proteins, as it is possible that these other agents directly stimulate the opening of a Ca-DPA channel. In this paper, we report the results of studies that indicate that Ca-DPA release triggered by both dodecylamine and lysozyme treatment of decoated spores requires SpoVA proteins, consistent with these proteins being components of a Ca-DPA channel.