Raman Spectral Predictors of Spore Germination Efficiency in Natural Isolates of Myxococcus xanthus

Due to the prevalence and importance of dormant microbial forms in regulating microbial ecosystems, the generation of dormant structures, like spores, has been extensively studied. However, several aspects of the exit of bacterial spores from dormancy, i.e., the germination of spores, remain relatively unclear. Since the biology of cells before transitioning into spores and the biology of the spores themselves could potentially influence the germination process, we used Myxococcus xanthus as a model organism to demonstrate that Raman spectroscopy can be used to characterize the factors that affect the ability of individual cells and spores to sporulate and germinate, respectively. M. xanthus is a Gram-negative soil bacterium that forms spore-filled multicellular fruiting bodies upon starvation. Single-cell Raman spectral profiling revealed lower Raman peak intensities of nucleic acids as a marker for the beginning of sporulation. Moreover, Raman profiles of sporulating cells of M. xanthus demonstrated that the lipid peaks increased during the initial sporulation phase before decreasing during the late sporulation phase. We also observed higher carotenoid peaks in spores than in cells, which might explain the reason for spores being more tolerant to oxidative stress than the cells. Significantly, the trends in Raman bands of nucleic acids and proteins observed in the lab strain were also observed in the natural isolates. Furthermore, partial least squares regression (PLSR) analysis of peak intensities of the most significantly affected chemical groups demonstrated a strong correlation between Raman spectra and germination efficiencies of spores, suggesting that such spectral markers are potential indicators of the germination efficiency of the spore population.