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Quantification and isolation of Bacillus subtilis spores using cell sorting and automated gating

Bracharz, Felix and Karava, Marianna and Kabisch, Johannes (2019):
Quantification and isolation of Bacillus subtilis spores using cell sorting and automated gating.
14, In: PLOS ONE, (7), PLOS, ISSN 1932-6203,
DOI: 10.1371/journal.pone.0219892,
Secondary publishing via sponsored Golden Open Access, [Article]

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Item Type: Article
Origin: Secondary publishing via sponsored Golden Open Access
Title: Quantification and isolation of Bacillus subtilis spores using cell sorting and automated gating
Language: English
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.

Journal or Publication Title: PLOS ONE
Volume: 14
Number: 7
Publisher: PLOS
Classification DDC: 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
Divisions: 10 Department of Biology > Yeast Membrane Biology
Date Deposited: 29 Oct 2019 15:00
Last Modified: 21 Jan 2020 15:22
DOI: 10.1371/journal.pone.0219892
URN: urn:nbn:de:tuda-tuprints-92240
URI: https://tuprints.ulb.tu-darmstadt.de/id/eprint/9224
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