Calcium Release and Germination of Bacterial Spores

During germination and subsequent outgrowth of bacterial spores a number of well documented physical and biochemical changes occur (Gould & Dring, 1972). A wide variety of chemical and physical stimuli will initiate these changes, including potential metabolites such as amino acids, sugars and purine ribosides, non-nutrients such as EDTA and surfactants and physical treatments such as hydrostatic pressure and abrasion. The extreme diversity of these germination initiators renders it difficult to imagine how they might act through a common mechanism. We have been examining the possibility that many of these germinants might act by displacing calcium from spore structures. Dormant bacterial spores are characterized by an extremely high calcium content, probably in excess of 200m, which is thought to occur as a chelate with dipicolinic acid (pyridine-2,6-dicarboxylic acid). The bulk of this calcium is rapidly lost from the spore in the first minutes of germination. Rode & Foster (1961) observed that several n-alkyl primary amines would germinate Bacillus spores. Further, with respect to a large number of the changes typically observed during germination, these non-nutrient germinants produced results virtually identical with those produced by nutrient initiators. Provided the amines were removed rapidly after germination had been initiated, the germinated spores remained viable and capable of outgrowth into normal vegetative cells. The potency of these alkylamines as germinants increased with carbon chain length above seven carbon atoms. In an unrelated study of calcium displacement from phospholipid monolayers by pharmacologically active and other organic bases, Hauser & Dawson (1968) observed that the ability of a series of straight-chain aliphatic amines to displace calcium from phosphatidylinositol monolayers increased with the number of carbon atoms up to C12. Since a large number of other nutrient and non-nutrient spore germinants clearly possess a potential protonated amino group or alternatively have the ability to chelate calcium, we decided to investigate the effect of alkyl amines of various chain lengths and other germinants on calcium release from spores and spore lipid monolayers. Hauser & Dawson (1968) showed that when the logarithm of the concentration of amine required to displace 50 % of calcium bound to the phospholipid monolayers was plotted against the number of carbon atoms in the amine, a constant slope was obtained. Using the decrease in absorbance of spore suspensions as an index of germination, we repeated this experiment, plotting the logarithm of amine concentration required to give a fixed rate of absorbance decrease against carbon chain length. This gave a line of constant slope from Cs to Ci3. In a second series of experiments we examined the ability of the amines to displace 50% of the total calcium from spores produced in medium containing 'TaZ+. Once again a linear relationship was found between the logarithm of amine concentration required to release 50% of spore calcium and carbon chain length from C, to CIS. Spore lipids were then extracted and purified (Ellar & Posgate, 1974) and used to prepare ''Ca2+-containing monolayers. Measurements of the ability of the amines to displace calcium from these monolayers again revealed a linear relationship between release of 50% bound calcium and carbon chain length. As noted by Hauser & Dawson (1968) such linear relationships are also often found in any homologous series bctween the logarithm of the distribution coefficients between two immiscible phases and the number of carbon atoms in the molecule. At the present time the distribution of the high concentrations of endogenous calcium within spore structures is not known. Calcium is required for maintenance of the integrity of spore membranes (Fitz-James, 1971) and the relatively high concentrations of cardiolipin in spore membranes (Ellar & Posgate, 1974) might result in a greater affiity for this cation. Both our results and those of Hauser & Dawson (1968) demQnstrating the ability of