Intensities of Calcium Dipicolinate and Bacillus Subtilis Spore Raman Spectra Excited with 244 nm Light

Ultraviolet (UV) resonance Raman spectra of Bacillus subtilis endospores have been excited at 244 nm. Spectra can be interpreted in terms of contributions from calcium dipicolinate and nucleic acid components. Differences between spectra of spores and vegetative cells are very large and are due to the dominance of the dipicolinate features in the spore spectra. Because the DNA and RNA composition of B. subtilis spores is known and because the cross-sections of Raman bands belonging to DNA and RNA bases are known, it is possible to calculate resonance Raman spectral cross-sections for the spore Raman peaks associated with the nucleic acids. The cross-sections of peaks associated with calcium dipicolinate have been measured from aqueous solutions. Cross-section values of the dominant 1017 cm−1 calcium dipicolinate peak measured from the Bacillus spores have been shown to be consistent with a calcium dipicolinate composition of ten percent or less by weight in the spores. It is suggested that spectral cross-sections of endospores excited at 244 nm can be estimated to be the sum of the cross-sections of the calcium dipicolinate, DNA, and RNA components of the spore. It appears that the peaks due to DNA and RNA can be used as an internal standard in the calculation of spore Raman peak cross-sections, and potentially the amount of calcium dipicolinate in spores. It is estimated on the basis of known nucleic acid base cross-sections that the most intense Raman band of the Bacillus subtilis spore spectra has a cross-section of no more than 4 × 10−18 cm2/mol-sr.

[1]  Ramasamy Manoharan,et al.  UV Resonance Raman Studies of Bacteria , 1992 .

[2]  W. Nelson,et al.  UV resonance Raman spectra of bacteria, bacterial spores, protoplasts and calcium dipicolinate , 1990 .

[3]  S. P. Fodor,et al.  Deep-ultraviolet Raman excitation profiles and vibronic scattering mechanisms of phenylalanine, tyrosine, and tryptophan , 1989 .

[4]  Y. Hathout,et al.  Identification of Bacillus Spores by Matrix-Assisted Laser Desorption Ionization–Mass Spectrometry , 1999, Applied and Environmental Microbiology.

[5]  D B Kell,et al.  Detection of the dipicolinic acid biomarker in Bacillus spores using Curie-point pyrolysis mass spectrometry and Fourier transform infrared spectroscopy. , 2000, Analytical chemistry.

[6]  T. Spiro,et al.  Raman spectroscopy in vivo: evidence on the structure of dipicolinate in intact spores of Bacillus megaterium. , 1974, Biochemical and biophysical research communications.

[7]  Q Wu,et al.  Intensities of E. coli nucleic acid Raman spectra excited selectively from whole cells with 251-nm light. , 2000, Analytical chemistry.

[8]  Sanford A. Asher,et al.  Wavelength dependence of the preresonance Raman cross sections of CH3CN, SO42−, ClO4−, and NO3− , 1985 .

[9]  Bettina Warscheid,et al.  Characterization of Bacillus spore species and their mixtures using postsource decay with a curved-field reflectron. , 2003, Analytical chemistry.

[10]  Ramasamy Manoharan,et al.  UV Resonance Raman Spectra of Bacillus Spores , 1992 .

[11]  Pedro Carmona,et al.  Vibrational spectra and structure of crystalline dipicolinic acid and calcium dipicolinate trihydrate , 1980 .

[12]  J. Ramírez,et al.  Tandem mass spectrometry of intact proteins for characterization of biomarkers from Bacillus cereus T spores. , 2001, Analytical chemistry.

[13]  Q Wu,et al.  UV Raman spectral intensities of E. coli and other bacteria excited at 228.9, 244.0, and 248.2 nm. , 2001, Analytical chemistry.

[14]  G. Thomas,et al.  Structure and interactions of the single-stranded DNA genome of filamentous virus fd: investigation by ultraviolet resonance raman spectroscopy. , 1997, Biochemistry.

[15]  Discrimination between bacterial spore types using time-of-flight mass spectrometry and matrix-free infrared laser desorption and ionization. , 2001, Analytical chemistry.

[16]  D. Britt,et al.  Ultraviolet Resonance Raman Spectra of Escherichia Coli with 222.5–251.0 nm Pulsed Laser Excitation , 1988 .

[17]  J. Powell.  Isolation of dipicolinic acid (pyridine-2:6-dicarboxylic acid) from spores of Bacillus megatherium. , 1953, The Biochemical journal.

[18]  Dimitra N. Stratis-Cullum,et al.  A miniature biochip system for detection of aerosolized Bacillus globigii spores. , 2003, Analytical chemistry.

[19]  Thomas Huser,et al.  Analysis of Single Bacterial Spores by Micro-Raman Spectroscopy , 2003, Applied spectroscopy.

[20]  Laser power dependence of mass spectral signatures from individual bacterial spores in bioaerosol mass spectrometry. , 2003, Analytical chemistry.

[21]  G. Gould,et al.  The Bacterial Spore , 1984 .

[22]  A. D. Warth.  Molecular structure of the bacterial spore. , 1978, Advances in microbial physiology.

[23]  Y. Hachisuka.  [The bacterial spore]. , 1966, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[24]  M. S. Zubairy,et al.  FAST CARS: Engineering a laser spectroscopic technique for rapid identification of bacterial spores , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[25]  T. Hadfield,et al.  Electron monochromator mass spectrometry for the analysis of whole bacteria and bacterial spores. , 2000, Analytical chemistry.

[26]  W. H. Nelson,et al.  Ultraviolet micro-Raman spectrograph for the detection of small numbers of bacterial cells , 1993 .

[27]  D. Britt,et al.  An Ultraviolet (242 nm Excitation) Resonance Raman Study of Live Bacteria and Bacterial Components , 1987 .

[28]  G. Thomas,et al.  Demonstration by ultraviolet resonance Raman spectroscopy of differences in DNA organization and interactions in filamentous viruses Pf1 and fd. , 1999, Biochemistry.

[29]  C. Fenselau,et al.  MALDI analysis of Bacilli in spore mixtures by applying a quadrupole ion trap time-of-flight tandem mass spectrometer. , 2003, Analytical chemistry.

[30]  G. Thomas,et al.  UV resonance Raman spectroscopy of DNA and protein constituents of viruses: assignments and cross sections for excitations at 257, 244, 238, and 229 nm. , 1998, Biopolymers.

[31]  Sandra E Thompson,et al.  Identification of Bacterial Spores Using Statistical Analysis of Fourier Transform Infrared Photoacoustic Spectroscopy Data , 2003, Applied spectroscopy.