![]() ![]() However, even under optimal sporulating conditions, only a part of the population undergoes sporulation. The levels of the master regulator of this process, Spo0A in its phosphorylated form define whether a cell will enter the sporulation pathway. The starving cells undergo an asymmetric division, governed by a complex regulatory network which results in the formation of metabolically inactive spores. subtilis is one of the most thoroughly investigated cellular differentiation programs, triggered by a combination of signals including nutrient exhaustion and cell density. Superfolder green fluorescent protein SYBR1,īacillus subtilis is a model organism extensively studied for its ability to differentiate depending on the growth conditions. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist.įluorescence channel 1, signal area (525/50 bandpass) for SYBR1 and SYBR2 FSC-A,įourier-transform infrared spectroscopy GMM, JK is supported by a LOEWE CompuGene grant. ![]() įunding: MK is supported by the BMBF grant Cascade Kit (FKZ: 031B0579A) and FB is supported by the FNR grant (FKZ: 22007413). Additionally data is available in a public gitlab. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: All relevant data are within the manuscript and its Supporting Information files. Received: FebruAccepted: JPublished: July 29, 2019Ĭopyright: © 2019 Karava et al. PLoS ONE 14(7):Įditor: Cinzia Calvio, Università degli Studi di Pavia, ITALY We confirmed the identity of the different subpopulations formed during sporulation by employing sorting and microscopy.Ĭitation: Karava M, Bracharz F, Kabisch J (2019) Quantification and isolation of Bacillus subtilis spores using cell sorting and automated gating. 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. A decrease in the sporulation efficiency of strain Bs02018, utilized for the display of sfGFP on the spores surface was observed. We utilized the presented method for monitoring sporulation over time in germination deficient strains harboring different genome modifications. Automated gating procedures using Gaussian mixture modeling (GMM) were employed to avoid subjective gating and allow for the simultaneous measurement of controls. 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. subtilis form sub-populations which include vegetative cells, sporulating cells and spores. subtilis is as well a model organism for cellular differentiation processes. The Gram-positive bacterium Bacillus subtilis is able to form endospores which have a variety of biotechnological applications. ![]()
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