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Levels of Ca²⁺-Dipicolinic Acid in Individual Bacillus Spores Determined Using Microfluidic Raman Tweezers

Department of Physics, East Carolina University, Greenville, North Carolina 27858-4353,¹ Biophysics Laboratory, Guangxi Academy of Sciences, Nanning, Guangxi 530003, People's Republic of China,² Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut 06030-3305³

Received 21 February 2007/ Accepted 18 April 2007

Pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) in a 1:1 chelate with calcium ion (Ca-DPA) comprises 5 to 15% of the dry weight of spores of Bacillus species. Ca-DPA is important in spore resistance to many environmental stresses and in spore stability, and Ca-DPA levels in spore populations can vary with spore species/strains, as well as with sporulation conditions. We have measured levels of Ca-DPA in large numbers of individual spores in populations of a variety of Bacillus species and strains by using microfluidic Raman tweezers, in which a single spore is trapped in a focused laser beam and its Ca-DPA is quantitated from the intensity of the Ca-DPA-specific band at 1,017 cm^–1 in Raman spectroscopy. Conclusions from these measurements include the following: (i) Ca-DPA concentrations in the spore core are >800 mM, well above Ca-DPA solubility; (ii) SpoVA proteins may be involved in Ca-DPA uptake in sporulation; and (iii) Ca-DPA levels differ significantly among individual spores in a population, but much of this variation could be due to variations in the sizes of individual spores.

Published ahead of print on 27 April 2007.

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