UV Resonance Raman Spectra of Bacillus Spores

Abstract

UV resonance Raman spectra of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis endospores have been excited at 222.7,230.7,242.5, and 251.1 nm, and spectra have been compared with those of vegetative cells. The resonance Raman spectra of aqueous solutions of dipicolinic acid and calcium dipicolinate have been measured at the same wavelengths. Spectra of endospores and their corresponding germinated spores show only modest differences when excited at 222, 231, and 251 nm. However, very substantial differences appear when excitation occurs at 242 nm. Difference spectra obtained at 242 nm by subtracting spectra of germinated spores of Bacillus cereus from spectra of their corresponding endospores are attributed almost entirely to dipicolinate. Vegetative cells and endospores show large spectral dissimilarities at all exciting wavelengths. These spectral differences, which vary strongly with exciting wavelength, appear to be the result of large differences in the amounts and composition of proteins and nucleic acids, especially ribosomal RNA. The very substantial resonance enhancement of Raman spectra has been obtained from aqueous solutions of pure dipicolinic acid and of sodium and calcium dipicolinate salts, as well as spores at the various exciting wavelengths. The strong enhancement of dipicolinate spectra in spores, however, was noted only with 242-nm excitation. Consequently, only with 242-nm light was it possible to selectively and sensitively excite and study calcium dipicolinate in spores. Resonance enhancement of the dipicolinate spectra with 242-nm excitation appears due primarily to resonance interactions with n-π* electronic transitions associated with the pyridine ring and/or the carboxylate group.