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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230700	1/2	Vibrio mediterranei	AK1	(d)(U)C DG Filtration UF	Li J	2016	57%
Study summary Full title Outer membrane vesicles containing signalling molecules and active hydrolytic enzymes released by a coral pathogen Vibrio shilonii AK1. All authors Li J, Azam F, Zhang S Journal Environ Microbiol Abstract Production and release of outer-membrane vesicles (OMVs) is known in many bacteria including human p (show more...)Production and release of outer-membrane vesicles (OMVs) is known in many bacteria including human pathogens. To date, OMV release has not been reported in coral-associated bacteria. We discovered that Vibrio shilonii AK1, a well-studied coral pathogen, produces OMVs in culture. Transmission electron microscopy showed that V. shilonii cultures release two types of vesicles, with a single membrane or two membranes, as well as vesicle chain-like morphotype in purified vesicle fraction. No significant difference was observed in the amount of OMVs produced by cultures grown at 20°C or 30°C. OMV proteomic analysis, never before done in a coral isolate, showed that a large number of low abundance proteins were exclusively detected in OMVs released by 20°C cultures. Further, the OMVs purified from AK1 cultures grown at both 20°C and 30°C carry N-acylhomoserine lactone quorum sensing signals, as well as alkaline phosphatase, lipase and chitinase activities. Our results show that V. shilonii OMVs are conduits of signalling molecules, active enzymes and other proteins to its environment. These findings suggest important ecophysiological roles of OMVs in coral reef environment. We discuss the importance of OMV release for V. shilonii fitness and propose several hypotheses as well as a conceptual model. (hide) EV-METRIC 57% (92nd 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. For the quantitative method, the reporting of measured EV concentration is expected. 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 Cell culture supernatant Sample origin Grown at 20°C Focus vesicles outer membrane vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Ultrafiltration Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) not specified Show all info Study aim Function Sample Species Vibrio mediterranei Sample Type Cell culture supernatant EV-producing cells AK1 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type P27A Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 9 Lowest density fraction 0% Highest density fraction 45% Total gradient volume, incl. sample (mL) not specified Sample volume (mL) 1.3 Orientation Bottom-up Speed (g) 111132 Duration (min) 360 Fraction volume (mL) not specified Fraction processing Centrifugation Pelleting: volume per fraction not spec Pelleting: rotor type P90AT Pelleting: speed (g) 100000 Pelleting: adjusted k-factor TDB Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per 4 l of starting sample Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 20-120

	EV230700	2/2	Vibrio mediterranei	AK1	(d)(U)C DG Filtration UF	Li J	2016	57%
Study summary Full title Outer membrane vesicles containing signalling molecules and active hydrolytic enzymes released by a coral pathogen Vibrio shilonii AK1. All authors Li J, Azam F, Zhang S Journal Environ Microbiol Abstract Production and release of outer-membrane vesicles (OMVs) is known in many bacteria including human p (show more...)Production and release of outer-membrane vesicles (OMVs) is known in many bacteria including human pathogens. To date, OMV release has not been reported in coral-associated bacteria. We discovered that Vibrio shilonii AK1, a well-studied coral pathogen, produces OMVs in culture. Transmission electron microscopy showed that V. shilonii cultures release two types of vesicles, with a single membrane or two membranes, as well as vesicle chain-like morphotype in purified vesicle fraction. No significant difference was observed in the amount of OMVs produced by cultures grown at 20°C or 30°C. OMV proteomic analysis, never before done in a coral isolate, showed that a large number of low abundance proteins were exclusively detected in OMVs released by 20°C cultures. Further, the OMVs purified from AK1 cultures grown at both 20°C and 30°C carry N-acylhomoserine lactone quorum sensing signals, as well as alkaline phosphatase, lipase and chitinase activities. Our results show that V. shilonii OMVs are conduits of signalling molecules, active enzymes and other proteins to its environment. These findings suggest important ecophysiological roles of OMVs in coral reef environment. We discuss the importance of OMV release for V. shilonii fitness and propose several hypotheses as well as a conceptual model. (hide) EV-METRIC 57% (92nd 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. For the quantitative method, the reporting of measured EV concentration is expected. 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 Cell culture supernatant Sample origin Grown at 30°C Focus vesicles outer membrane vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Ultrafiltration Protein markers EV: None non-EV: None Proteomics yes EV density (g/ml) not specified Show all info Study aim Function Sample Species Vibrio mediterranei Sample Type Cell culture supernatant EV-producing cells AK1 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type P27A Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 9 Lowest density fraction 0% Highest density fraction 45% Total gradient volume, incl. sample (mL) not specified Sample volume (mL) 1.3 Orientation Bottom-up Speed (g) 111132 Duration (min) 360 Fraction volume (mL) not specified Fraction processing Centrifugation Pelleting: volume per fraction not spec Pelleting: rotor type P90AT Pelleting: speed (g) 100000 Pelleting: adjusted k-factor TDB Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per 4 l of starting sample Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 20-120

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EV-TRACK ID	EV230700
species	Vibrio mediterranei
sample type	Cell culture
cell type	AK1

Study summary

Study data

Study summary

Study data