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 B. subtilis form sub-populations which include vegetative cells, sporulating 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. Automated gating procedures using Gaussian mixture modeling (GMM) 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 germination deficient strains harboring different genome modifications. A decrease in the sporulation efficiency of strain Bs02018, utilized for the display of sfGFP on the spores surface was observed. On the contrary, a double knock-out mutant of the phosphatase gene encoding Spo0E and of the spore killing factor SkfA (Bs02025) exhibited the highest sporulation efficiency, as within 24 h of cultivation in sporulation medium, cultures of BS02025 already consisted of 80% spores as opposed to 18% for the control strain. We confirmed the identity of the different subpopulations formed during sporulation by employing sorting and microscopy.
Highlights
Bacillus subtilis is a model organism extensively studied for its ability to differentiate depending on the growth conditions [1]
Samples were respectively stained with SYBR Green 1 (SYBR1) and SYBR Green 2 (SYBR2) as described in materials and methods and subsequently analyzed by flow cytometry
To assess the capacity of separating spores from cells coming from the same sample, a 1:1 mix of vegetative cells and purified spores was stained with different concentrations of dye
Summary
Bacillus subtilis is a model organism extensively studied for its ability to differentiate depending on the growth conditions [1]. Sporulation in B. subtilis is one of the most thoroughly investigated cellular differentiation programs, triggered by a combination of signals including nutrient exhaustion and cell density [2,3]. The starving cells undergo an asymmetric division, governed by a complex regulatory network which results in the formation of metabolically inactive spores [4,5]. The levels of the master regulator of this process, Spo0A in its phosphorylated form define whether a cell will enter the sporulation pathway [6]. Even under optimal sporulating conditions, only a part of the population undergoes sporulation [7,8].
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