Inducement of a Heat-Shock Requirement for Germination and Production of Increased Heat Resistance in Bacillus fastidiosus Spores by Manganous Ions

Bacillus fastidiosus, which requires uric acid or allantoin, grows and sporulates on a simple medium containing 59.5 mM uric acid, 5.7 mM K₂HPO₄, and 2% agar in distilled water. Seventy to ninety percent sporulation was achieved in 96 h. Spores obtained on this medium do not need a heat shock prior to germination. The necessary germination conditions for this organism are 30 C, phosphate or this(hydroxymethyl)aminomethane buffer at pH 7.0, and 5.95 mM uric acid. Sporulation occurred earlier (48 h) and with higher frequency (greater than 99%) when Mn²⁺ was added to the growth medium. However, these spores germinated only after heat activation (70 C, 30 min). The effectiveness of heat activation was directly dependent upon the concentration of Mn²⁺ in the growth medium; 10⁻⁵ M Mn²⁺ was the minimal concentration for the effect. This phenomenon was not found upon addition of Ca²⁺, Mg²⁺, Fe²⁺, Zn²⁺, or Cu²⁺ to the medium. The Mn²⁺ content of the spores depended upon the concentration of Mn²⁺ in the sporulation medium. There was a significant difference in heat resistance between spores harvested from unsupplemented medium and those harvested from medium supplemented with 5 × 10⁻⁵ M Mn²⁺. A D_{85 C} value of 6.5 min was determined with the former, whereas the latter had a value of 17.0 min. Very little change in either Ca²⁺ or dipicolinic acid content was detected in spores harvested from various Mn²⁺-supplemented media. Thus Mn²⁺ may play a role in the inducement of the heat-shock requirement and the formation of spores with increased heat resistance.