Slow Leakage of Ca-Dipicolinic Acid from Individual Bacillus Spores during Initiation of Spore Germination

When exposed to nutrient or nonnutrient germinants, individual Bacillus spores can return to life through germination followed by outgrowth. Laser tweezers, Raman spectroscopy, and either differential interference contrast or phase-contrast microscopy were used to analyze the slow dipicolinic acid (DPA) leakage (normally ∼20% of spore DPA) from individual spores that takes place prior to the lag time, T ₗₐg, when spores begin rapid release of remaining DPA. Major conclusions from this work with Bacillus subtilis spores were as follows: (i) slow DPA leakage from wild-type spores germinating with nutrients did not begin immediately after nutrient exposure but only at a later heterogeneous time T ₁; (ii) the period of slow DPA leakage (Δ T ₗₑₐₖₐgₑ = T ₗₐg − T ₁) was heterogeneous among individual spores, although the amount of DPA released in this period was relatively constant; (iii) increases in germination temperature significantly decreased T ₁ times but increased values of Δ T ₗₑₐₖₐgₑ; (iv) upon germination with l -valine for 10 min followed by addition of d -alanine to block further germination, all germinated spores had T ₁ times of less than 10 min, suggesting that T ₁ is the time when spores become committed to germinate; (v) elevated levels of SpoVA proteins involved in DPA movement in spore germination decreased T ₁ and T ₗₐg times but not the amount of DPA released in Δ T ₗₑₐₖₐgₑ; (vi) lack of the cortex-lytic enzyme CwlJ increased DPA leakage during germination due to longer Δ T ₗₑₐₖₐgₑ times in which more DPA was released; and (vii) there was slow DPA leakage early in germination of B. subtilis spores by the nonnutrients CaDPA and dodecylamine and in nutrient germination of Bacillus cereus and Bacillus megaterium spores. Overall, these findings have identified and characterized a new early event in Bacillus spore germination.