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Updated: Jul 15, 2026
Single-cell Microfluidic Analysis of Bacillus subtilis
Published on: January 26, 2018
Levels of Ca2+-dipicolinic acid in individual bacillus spores determined using microfluidic Raman tweezers
Shu-shi Huang1, De Chen, Patricia L Pelczar
1Department of Physics, East Carolina University, Greenville, NC 27858-4353, USA.
Abstract:
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.
Insights
Calcium-dipicolinic acid (Ca-DPA) is crucial for Bacillus spore resistance and stability. This study quantifies Ca-DPA in individual spores, revealing high core concentrations and potential roles for SpoVA proteins in its uptake.
Area of Science:
- Microbiology
- Biochemistry
- Spectroscopy
Background:
- Calcium-dipicolinic acid (Ca-DPA) is a vital component of Bacillus spores, comprising 5-15% of their dry weight.
- Ca-DPA contributes significantly to spore resistance against environmental stressors and overall spore stability.
- Ca-DPA levels can fluctuate based on Bacillus species/strains and sporulation conditions.
Purpose of the Study:
- To accurately quantify Ca-DPA levels in individual Bacillus spores.
- To investigate the spatial distribution and concentration of Ca-DPA within the spore core.
- To explore potential mechanisms and proteins involved in Ca-DPA uptake during sporulation.
Main Methods:
- Utilized microfluidic Raman tweezers to isolate and analyze single Bacillus spores.
- Employed Raman spectroscopy to measure Ca-DPA concentration via the specific band at 1,017 cm(-1).
- Quantitated Ca-DPA levels across large populations of diverse Bacillus species and strains.
Main Results:
- Determined Ca-DPA concentrations within the spore core to be exceptionally high, exceeding 800 mM, surpassing its solubility limit.
- Observed significant variations in Ca-DPA levels among individual spores within populations.
- Identified potential involvement of SpoVA proteins in the process of Ca-DPA uptake during bacterial sporulation.
Conclusions:
- The high intracellular Ca-DPA concentration suggests a unique mechanism for its maintenance within Bacillus spores.
- SpoVA proteins are implicated as potential transporters or regulators of Ca-DPA accumulation during sporulation.
- Observed heterogeneity in Ca-DPA levels is likely influenced by variations in spore size, alongside biological factors.
