Quantification and Isolation ofBacillus subtilisSpores using Cell Sorting and automated Gating

Abstract

The Gram-positive bacterium Bacillus subtilis is able to form endospores which have a variety of biotechnological applications. Due to this ability, B. subtilis is as well a model organism for cellular differentiation processes. Sporulating cultures of Bacillus subtilis form sub-populations which include vegetative cells, spore forming cells and spores. In order to readily and rapidly quantify spore formation we employed flow cytometric and fluorescence activated cell sorting techniques in combination with nucleic acid fluorescent staining in order to investigate the distribution of sporulating cultures on a single cell level. Moreover we tested different fluorescent dyes as well as different conditions in order to develop a method for optimal separation of distinct populations during sporulation. Automated gating procedures using k-means clustering and thresholding by gaussian mixture modeling were employed to avoid subjective gating and allow for the simultaneous measurement of controls. We utilized the presented method for monitoring sporulation over time in strains harboring different genome modifications. We identified the different subpopulations formed during sporulation by employing sorting and microscopy. Finally, we employed the technique to show that a double knock-out mutant of the phosphatase gene encoding Spo0E and of the spore killing factor SkfA results in faster spore formation.