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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	separation protocol	First author	Year	EV-METRIC
	EV140046	1/1	Bacillus subtilis	Bacteria	DG (d)(U)C Filtration	Brown L	2014	57%
Study summary Full title Extracellular vesicles produced by the Gram-positive bacterium Bacillus subtilis are disrupted by the lipopeptide surfactin All authors Brown L, Kessler A, Cabezas-Sanchez P, Luque-Garcia JL, Casadevall A Journal Mol Microbiol Abstract Previously, extracellular vesicle production in Gram-positive bacteria was dismissed due to the abse (show more...)Previously, extracellular vesicle production in Gram-positive bacteria was dismissed due to the absence of an outer membrane, where Gram-negative vesicles originate, and the difficulty in envisioning how such a process could occur through the cell wall. However, recent work has shown that Gram-positive bacteria produce extracellular vesicles and that the vesicles are biologically active. In this study, we show that Bacillus subtilis produces extracellular vesicles similar in size and morphology to other bacteria, characterized vesicles using a variety of techniques, provide evidence that these vesicles are actively produced by cells, show differences in vesicle production between strains, and identified a mechanism for such differences based on vesicle disruption. We found that in wild strains of B. subtilis, surfactin disrupted vesicles while in laboratory strains harbouring a mutation in the gene sfp, vesicles accumulated in the culture supernatant. Surfactin not only lysed B. subtilis vesicles, but also vesicles from Bacillus anthracis, indicating a mechanism that crossed species boundaries. To our knowledge, this is the first time a gene and a mechanism has been identified in the active disruption of extracellular vesicles and subsequent release of vesicular cargo in Gram-positive bacteria. We also identify a new mechanism of action for surfactin. (hide) EV-METRIC 57% (95th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Bacteria Sample origin DNF Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + (d)(U)C + Filtration Protein markers EV: non-EV: Proteomics yes TEM measurements 137.69999999999999 Show all info Study aim Biogenesis/Sorting Sample Species Bacillus subtilis Sample Type Bacteria Separation Method Differential ultracentrifugation centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 60 Density gradient Only used for validation of main results 1 Density medium Iodixanol Lowest density fraction 10 Highest density fraction 30 Orientation Bottom-up Filtration steps > 0.45 µm, 0.22µm or 0.2µm Characterization: Protein analysis Characterization: Particle analysis DLS EM EM-type transmission EM/ immune EM Proteïns GXM Image type Close-up, Wide-field

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