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You searched for: EV230711 (EV-TRACK ID)

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Results list Study Tree
Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
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
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV230711 1/14 Escherichia coli RP437 (d)(U)C
DG
Filtration 		Aschtgen MS 2016 43%

Study summary

Full title
Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.
All authors
Aschtgen MS, Lynch JB, Koch E, Schwartzman J, McFall-Ngai M, Ruby E
Journal
J Bacteriol
Abstract
Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fisc (show more...)Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis/ however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. (hide)
EV-METRIC
43% (81st 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
Control condition
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
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
RP437
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 90 Ti
Pelleting: speed (g)
173000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
not specified
Lowest density fraction
20%
Highest density fraction
55%
Total gradient volume, incl. sample (mL)
not specified
Sample volume (mL)
not spec
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
not specified
Fraction processing
None
Filtration steps
Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm
Characterization: Protein analysis
None
Protein Concentration Method
Fluorometric assay
Characterization: Lipid analysis
Yes
EV230711 2/14 Escherichia coli RP437 (d)(U)C
DG
Filtration 		Aschtgen MS 2016 43%

Study summary

Full title
Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.
All authors
Aschtgen MS, Lynch JB, Koch E, Schwartzman J, McFall-Ngai M, Ruby E
Journal
J Bacteriol
Abstract
Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fisc (show more...)Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis/ however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. (hide)
EV-METRIC
43% (81st 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
flrA mutant
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
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Escherichia coli
Sample Type
Cell culture supernatant
EV-producing cells
RP437
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 90 Ti
Pelleting: speed (g)
173000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
not specified
Lowest density fraction
20%
Highest density fraction
55%
Total gradient volume, incl. sample (mL)
not specified
Sample volume (mL)
not spec
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
not specified
Fraction processing
None
Filtration steps
Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm
Characterization: Protein analysis
None
Protein Concentration Method
Fluorometric assay
Characterization: Lipid analysis
Yes
EV230711 3/14 Vibrio cholerae O395N1 (d)(U)C
DG
Filtration 		Aschtgen MS 2016 43%

Study summary

Full title
Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.
All authors
Aschtgen MS, Lynch JB, Koch E, Schwartzman J, McFall-Ngai M, Ruby E
Journal
J Bacteriol
Abstract
Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fisc (show more...)Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis/ however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. (hide)
EV-METRIC
43% (81st 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
Control condition
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
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Vibrio cholerae
Sample Type
Cell culture supernatant
EV-producing cells
O395N1
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 90 Ti
Pelleting: speed (g)
173000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
not specified
Lowest density fraction
20%
Highest density fraction
55%
Total gradient volume, incl. sample (mL)
not specified
Sample volume (mL)
not spec
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
not specified
Fraction processing
None
Filtration steps
Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm
Characterization: Protein analysis
None
Protein Concentration Method
Fluorometric assay
Characterization: Lipid analysis
Yes
EV230711 4/14 Vibrio cholerae O395N1 (d)(U)C
DG
Filtration 		Aschtgen MS 2016 43%

Study summary

Full title
Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.
All authors
Aschtgen MS, Lynch JB, Koch E, Schwartzman J, McFall-Ngai M, Ruby E
Journal
J Bacteriol
Abstract
Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fisc (show more...)Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis/ however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. (hide)
EV-METRIC
43% (81st 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
flrA mutant
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
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Vibrio cholerae
Sample Type
Cell culture supernatant
EV-producing cells
O395N1
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 90 Ti
Pelleting: speed (g)
173000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
not specified
Lowest density fraction
20%
Highest density fraction
55%
Total gradient volume, incl. sample (mL)
not specified
Sample volume (mL)
not spec
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
not specified
Fraction processing
None
Filtration steps
Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm
Characterization: Protein analysis
None
Protein Concentration Method
Fluorometric assay
Characterization: Lipid analysis
Yes
EV230711 5/14 Vibrio fischeri ES114 (d)(U)C
DG
Filtration 		Aschtgen MS 2016 43%

Study summary

Full title
Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.
All authors
Aschtgen MS, Lynch JB, Koch E, Schwartzman J, McFall-Ngai M, Ruby E
Journal
J Bacteriol
Abstract
Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fisc (show more...)Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis/ however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. (hide)
EV-METRIC
43% (81st 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
Control condition
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
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Vibrio fischeri
Sample Type
Cell culture supernatant
EV-producing cells
ES114
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 90 Ti
Pelleting: speed (g)
173000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
not specified
Lowest density fraction
20%
Highest density fraction
55%
Total gradient volume, incl. sample (mL)
not specified
Sample volume (mL)
not spec
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
not specified
Fraction processing
None
Filtration steps
Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm
Characterization: Protein analysis
None
Protein Concentration Method
Fluorometric assay
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
EM
EM-type
Transmission­-EM
Image type
Wide-field
EV230711 6/14 Vibrio fischeri ES114 (d)(U)C
DG
Filtration 		Aschtgen MS 2016 43%

Study summary

Full title
Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.
All authors
Aschtgen MS, Lynch JB, Koch E, Schwartzman J, McFall-Ngai M, Ruby E
Journal
J Bacteriol
Abstract
Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fisc (show more...)Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis/ however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. (hide)
EV-METRIC
43% (81st 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
HS (DM73) mutant
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
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Vibrio fischeri
Sample Type
Cell culture supernatant
EV-producing cells
ES114
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 90 Ti
Pelleting: speed (g)
173000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
not specified
Lowest density fraction
20%
Highest density fraction
55%
Total gradient volume, incl. sample (mL)
not specified
Sample volume (mL)
not spec
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
not specified
Fraction processing
None
Filtration steps
Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm
Characterization: Protein analysis
None
Protein Concentration Method
Fluorometric assay
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
None
EV230711 7/14 Vibrio fischeri ES114 (d)(U)C
DG
Filtration 		Aschtgen MS 2016 43%

Study summary

Full title
Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.
All authors
Aschtgen MS, Lynch JB, Koch E, Schwartzman J, McFall-Ngai M, Ruby E
Journal
J Bacteriol
Abstract
Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fisc (show more...)Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis/ however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. (hide)
EV-METRIC
43% (81st 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
cheA mutant
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
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Vibrio fischeri
Sample Type
Cell culture supernatant
EV-producing cells
ES114
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 90 Ti
Pelleting: speed (g)
173000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
not specified
Lowest density fraction
20%
Highest density fraction
55%
Total gradient volume, incl. sample (mL)
not specified
Sample volume (mL)
not spec
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
not specified
Fraction processing
None
Filtration steps
Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm
Characterization: Protein analysis
None
Protein Concentration Method
Fluorometric assay
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
None
EV230711 8/14 Vibrio fischeri ES114 (d)(U)C
DG
Filtration 		Aschtgen MS 2016 43%

Study summary

Full title
Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.
All authors
Aschtgen MS, Lynch JB, Koch E, Schwartzman J, McFall-Ngai M, Ruby E
Journal
J Bacteriol
Abstract
Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fisc (show more...)Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis/ however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. (hide)
EV-METRIC
43% (81st 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
flrA mutant
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
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Vibrio fischeri
Sample Type
Cell culture supernatant
EV-producing cells
ES114
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 90 Ti
Pelleting: speed (g)
173000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
not specified
Lowest density fraction
20%
Highest density fraction
55%
Total gradient volume, incl. sample (mL)
not specified
Sample volume (mL)
not spec
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
not specified
Fraction processing
None
Filtration steps
Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm
Characterization: Protein analysis
None
Protein Concentration Method
Fluorometric assay
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
None
EV230711 9/14 Vibrio fischeri ES114 (d)(U)C
DG
Filtration 		Aschtgen MS 2016 43%

Study summary

Full title
Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.
All authors
Aschtgen MS, Lynch JB, Koch E, Schwartzman J, McFall-Ngai M, Ruby E
Journal
J Bacteriol
Abstract
Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fisc (show more...)Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis/ however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. (hide)
EV-METRIC
43% (81st 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
motB1 mutant
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
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Vibrio fischeri
Sample Type
Cell culture supernatant
EV-producing cells
ES114
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 90 Ti
Pelleting: speed (g)
173000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
not specified
Lowest density fraction
20%
Highest density fraction
55%
Total gradient volume, incl. sample (mL)
not specified
Sample volume (mL)
not spec
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
not specified
Fraction processing
None
Filtration steps
Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm
Characterization: Protein analysis
None
Protein Concentration Method
Fluorometric assay
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
None
EV230711 10/14 Vibrio fischeri ES114 (d)(U)C
DG
Filtration 		Aschtgen MS 2016 43%

Study summary

Full title
Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.
All authors
Aschtgen MS, Lynch JB, Koch E, Schwartzman J, McFall-Ngai M, Ruby E
Journal
J Bacteriol
Abstract
Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fisc (show more...)Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis/ however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. (hide)
EV-METRIC
43% (81st 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
ompC1 mutant
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
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Vibrio fischeri
Sample Type
Cell culture supernatant
EV-producing cells
ES114
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 90 Ti
Pelleting: speed (g)
173000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
not specified
Lowest density fraction
20%
Highest density fraction
55%
Total gradient volume, incl. sample (mL)
not specified
Sample volume (mL)
not spec
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
not specified
Fraction processing
None
Filtration steps
Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm
Characterization: Protein analysis
None
Protein Concentration Method
Fluorometric assay
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230711 11/14 Vibrio fischeri ES114 (d)(U)C
DG
Filtration 		Aschtgen MS 2016 43%

Study summary

Full title
Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.
All authors
Aschtgen MS, Lynch JB, Koch E, Schwartzman J, McFall-Ngai M, Ruby E
Journal
J Bacteriol
Abstract
Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fisc (show more...)Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis/ however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. (hide)
EV-METRIC
43% (81st 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
ompC2 mutant
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
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Vibrio fischeri
Sample Type
Cell culture supernatant
EV-producing cells
ES114
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 90 Ti
Pelleting: speed (g)
173000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
not specified
Lowest density fraction
20%
Highest density fraction
55%
Total gradient volume, incl. sample (mL)
not specified
Sample volume (mL)
not spec
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
not specified
Fraction processing
None
Filtration steps
Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm
Characterization: Protein analysis
None
Protein Concentration Method
Fluorometric assay
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230711 12/14 Vibrio fischeri ES114 (d)(U)C
DG
Filtration 		Aschtgen MS 2016 43%

Study summary

Full title
Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.
All authors
Aschtgen MS, Lynch JB, Koch E, Schwartzman J, McFall-Ngai M, Ruby E
Journal
J Bacteriol
Abstract
Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fisc (show more...)Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis/ however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. (hide)
EV-METRIC
43% (81st 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
ompU mutant
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
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Vibrio fischeri
Sample Type
Cell culture supernatant
EV-producing cells
ES114
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 90 Ti
Pelleting: speed (g)
173000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
not specified
Lowest density fraction
20%
Highest density fraction
55%
Total gradient volume, incl. sample (mL)
not specified
Sample volume (mL)
not spec
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
not specified
Fraction processing
None
Filtration steps
Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm
Characterization: Protein analysis
None
Protein Concentration Method
Fluorometric assay
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
None
EV230711 13/14 Vibrio fischeri ES114 (d)(U)C
DG
Filtration 		Aschtgen MS 2016 43%

Study summary

Full title
Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.
All authors
Aschtgen MS, Lynch JB, Koch E, Schwartzman J, McFall-Ngai M, Ruby E
Journal
J Bacteriol
Abstract
Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fisc (show more...)Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis/ however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. (hide)
EV-METRIC
43% (81st 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
waaL mutant
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
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Vibrio fischeri
Sample Type
Cell culture supernatant
EV-producing cells
ES114
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 90 Ti
Pelleting: speed (g)
173000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
not specified
Lowest density fraction
20%
Highest density fraction
55%
Total gradient volume, incl. sample (mL)
not specified
Sample volume (mL)
not spec
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
not specified
Fraction processing
None
Filtration steps
Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm
Characterization: Protein analysis
None
Protein Concentration Method
Fluorometric assay
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
None
EV230711 14/14 Vibrio parahaemolyticus KNH1 (d)(U)C
DG
Filtration 		Aschtgen MS 2016 43%

Study summary

Full title
Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.
All authors
Aschtgen MS, Lynch JB, Koch E, Schwartzman J, McFall-Ngai M, Ruby E
Journal
J Bacteriol
Abstract
Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fisc (show more...)Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis/ however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. (hide)
EV-METRIC
43% (81st 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
Control condition
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
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
not specified
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Vibrio parahaemolyticus
Sample Type
Cell culture supernatant
EV-producing cells
KNH1
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 90 Ti
Pelleting: speed (g)
173000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
not specified
Lowest density fraction
20%
Highest density fraction
55%
Total gradient volume, incl. sample (mL)
not specified
Sample volume (mL)
not spec
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
not specified
Fraction processing
None
Filtration steps
Between 0.22 and 0.45 µm/ 0.2 or 0.22 µm
Characterization: Protein analysis
None
Protein Concentration Method
Fluorometric assay
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
None
1 - 14 of 14
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV230711
species
Escherichia
coli
Escherichia
coli
Vibrio
cholerae
Vibrio
cholerae
Vibrio
fischeri
Vibrio
fischeri
Vibrio
fischeri
Vibrio
fischeri
Vibrio
fischeri
Vibrio
fischeri
Vibrio
fischeri
Vibrio
fischeri
Vibrio
fischeri
Vibrio
parahaemolyticus
sample type
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
cell type
RP437
RP437
O395N1
O395N1
ES114
ES114
ES114
ES114
ES114
ES114
ES114
ES114
ES114
KNH1
condition
Control
condition
flrA
mutant
Control
condition
flrA
mutant
Control
condition
HS
(DM73)
mutant
cheA
mutant
flrA
mutant
motB1
mutant
ompC1
mutant
ompC2
mutant
ompU
mutant
waaL
mutant
Control
condition
separation protocol
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
dUC/
Density
gradient/
Filtration
Exp. nr.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
EV-METRIC %
43
43
43
43
43
43
43
43
43
43
43
43
43
43