Superdormant Spores of Bacillus Species Germinate Normally with High Pressure,Peptidoglycan Fragments,and Bryostatin

Superdormant spores of Bacillus cereus and Bacillus subtilis germinated just as well as dormant spores with pressures of 150 or 500 MPa and with or without heat activation. Superdormant B. subtilis spores also germinated as well as dormant spores with peptidoglycan fragments or bryostatin, a Ser/Thr protein kinase activator.Spores of Bacillus species are formed in sporulation, a process that is generally triggered by starvation for one or more nutrients (13, 19). These spores are metabolically dormant and extremely resistant to a large variety of environmental stresses, including heat, radiation, and toxic chemicals, and as a consequence of these properties, these spores can remain viable in their dormant state for many years (13, 18, 19). However, spores are constantly sensing their environment, and if nutrients return, the spores can rapidly return to growth through the process of spore germination (17). Spore germination is generally triggered by specific nutrients that bind to nutrient germinant receptors, with this binding alone somehow triggering germination. However, spore germination can also be triggered by many non-nutrient agents, including cationic surfactants such as dodecylamine, a 1:1 complex of Ca²⁺ with pyridine-2,6-dicarboxylic acid (dipicolinic acid DPA], a major spore small molecule), very high pressures, specific peptidoglycan fragments, and bryostatin, an activator of Ser/Thr protein kinases (17, 19, 20). For nutrient germinants in particular, spore germination is also potentiated by a prior sublethal heat treatment termed heat activation (17).While normally the great majority of spores in populations germinate relatively rapidly in response to nutrient germinants, a small percentage of spores germinate extremely slowly. These spores that are refractory to nutrient germination have been termed superdormant spores and are a major concern for the food industry (8). Recently superdormant spores of three Bacillus species have been isolated by repeated germination of spore populations with specific nutrient germinants and isolation of remaining dormant spores (5, 6). These superdormant spores germinate extremely poorly with the nutrient germinants used in superdormant spore isolation, as well as with other nutrient germinants. All of the specific defects leading to spore superdormancy are not known, although an increased level of receptors for specific nutrient germinants decreases levels of superdormant spores obtained with the nutrients that are ligands for these receptors (5). Superdormant spores also have significantly higher temperature optima for heat activation of nutrient germination than the spore population as a whole (7).In contrast to the poor germination of superdormant spores with nutrient germinants, superdormant spores germinate normally with dodecylamine and Ca-DPA (5, 6). This is consistent with possible roles of nutrient germinant receptor levels and/or heat activation temperature optima in affecting spore superdormancy, since neither dodecylamine nor Ca-DPA triggers Bacillus spore germination through nutrient germinant receptors, and germination with these agents is also not stimulated by heat activation (11, 15, 17). However, the effects of high pressures, peptidoglycan fragments, and bryostatin, all of which almost certainly trigger spore germination by mechanisms at least somewhat different than triggering of germination by nutrients, dodecylamine, and Ca-DPA (2, 3, 11, 15, 20, 22, 23), have not been tested for their effects on superdormant spores. Consequently, we have compared the germination of dormant and superdormant spores of two Bacillus species by high-pressures, peptidoglycan fragments, and bryostatin.The spores used in this work were from Bacillus subtilis PS533 (16), a derivative of strain 168 that also carries plasmid pUB110, providing resistance to kanamycin (10 μg/ml), and Bacillus cereus T (originally obtained from H. O. Halvorson). Spores of these strains were prepared and purified as described previously (6, 10, 12). Superdormant spores of B. subtilis were prepared by germination following heat activation at 75°C for 30 min by two germination treatments at 37°C with 10 mM l-valine for 2 h, followed by isolation of remaining dormant spores, all as described previously (5, 10, 12). These superdormant spores germinated extremely poorly with 10 mM valine at 37°C, giving ≤10% germination in 2 h at 37°C, while the initial spore population exhibited >95% germination under the same conditions (data not shown). Superdormant B. cereus spores were isolated similarly, although heat activation was at 65°C for 30 min and the germinant was 5 mM inosine as described previously (6). These superdormant B. cereus spores exhibited <5% germination with inosine in 2 h at 37°C compared to the >95% germination of the initial dormant spores under the same conditions (data not shown).