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 Ca(2+) (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) (I(1575)/I(1017)) is sensitive to the water content in the CaDPA's environment; (3) variations in I(1575)/I(1017) are small (∼4%) in a population of dormant Bacillus spores suspended in water; and (4) the I(1575)/I(1017) ratio increases significantly during dehydration under vacuum. Consequently, measurement of the I(1575)/I(1017) ratio of CaDPA in spores may allow a qualitative estimation of the degree of hydration of the bacterial spore's core.

Proton dynamics in bacterial spores, a neutron scattering investigation

“…This would confirm the suggested idea of immobilized network of proteins in the core [3,7]. It could also reflect in part the amorphous solid state of Ca-DPA [5]. This finding should be related to the proposition of Sunde et al [3] that core proteins are surrounded by at most one water layer.…”

“…However, it would rather be attributed to the way out of confinement of the core after being heat-killed. An increase of macromolecules' dynamics associated with the swelling of the core and the rehydration of macromolecules [5] after the lethal heat treatment could indeed explain the larger MSD observed at 320 K.…”

“…It is characterized by low water content (0.6 h, where h = g water /g dry matter ) 3 , an elevated concentration of ions and a complex of dipicolinic acid (DPA) and (Ca 2+ ) 2 . DPA is accumulated in high quantity in the core up to 10-15% of spore dry weight [5], largely beyond its solubility and it is involved in the dehydration of the core. The multi-resistance of the spores is the consequence of various protective strategies involving, but not limited to, the detoxication through external layer of the spore (i.e.…”

Water Behavior in Bacterial Spores by Deuterium NMR Spectroscopy

“…Amorphous calcium dipicolinate was also seen using Raman spectroscopy of B. cereus spores. 24,25 The other theory is that the core water is in a gel-like state. This hypothesis was put forward initially by Black and Gerhardt 26 as a means of explaining their results on water uptake in spores using tritium-labeled H 2 O; they proposed that the spore core is an insoluble gel whose components are cross-linked but water-permeable.…”

Analysis of the Loss in Heat and Acid Resistance during Germination of Spores of Bacillus Species

“…The occurrence of all these events well before CaDPA release during spore germination suggests that they are due to one fundamental change that commits a spore to germinate. For example, if there is an increase in the permeability of the spores' IM associated with or the cause of commitment, this would explain the release of monovalent cations, which would almost certainly be more soluble in spores than is the spores' huge CaDPA depot, the great majority of which is most likely insoluble (20,21). This putative IM permeability change might also explain the increase in spores' Hg 2ϩ sensitivity well before CaDPA release.…”