Analysis of the Raman spectra of Ca2+-dipicolinic acid alone and in the bacterial spore core in both aqueous and dehydrated environments

Abstract

The core of dormant bacterial spores suspended in water contains a large depot of dipicolinic acid (DPA) chelated with divalent cations, predominantly Ca²⁺ (CaDPA), and surrounded by water molecules. Since the intensities of the vibration bands of CaDPA molecules depend significantly on the water content in the CaDPA's environment, the Raman spectra of CaDPA in spores may allow the determination of the spore core's hydration state. We have measured Raman spectra of single spores of three Bacillus species in different hydration states including the spores suspended in water, air-dried and vacuum-dried. As a comparison, we also measured the Raman spectra of CaDPA and DPA in different forms including in aqueous solution, and as amorphous powder and crystalline form. We also monitored changes in Raman spectra of an individual spore during dehydration under vacuum. The results indicated that (1) the state of CaDPA in the core of a spore suspended in water is close to an amorphous solid or a glassy state, but still mixed with water molecules; (2) the ratio of intensities of Raman bands at 1575 and 1017 cm⁻¹ (I₁₅₇₅/I₁₀₁₇) is sensitive to the water content in the CaDPA's environment; (3) variations in I₁₅₇₅/I₁₀₁₇ are small (∼4%) in a population of dormant Bacillus spores suspended in water; and (4) the I₁₅₇₅/I₁₀₁₇ ratio increases significantly during dehydration under vacuum. Consequently, measurement of the I₁₅₇₅/I₁₀₁₇ ratio of CaDPA in spores may allow a qualitative estimation of the degree of hydration of the bacterial spore's core.