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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230273	1/4	Buttiauxella agrestis	JCM1090	DG (d)(U)C Filtration	Takaki K	2020	71%
Study summary Full title Multilamellar and Multivesicular Outer Membrane Vesicles Produced by a Buttiauxella agrestis Mutant. All authors Takaki K, Tahara YO, Nakamichi N, Hasegawa Y, Shintani M, Ohkuma M, Miyata M, Futamata H, Tashiro Y Journal Appl Environ Microbiol Abstract Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important (show more...)Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important roles in various biological functions. Released vesicles are not uniform in shape, size, or characteristics, and little is known about this diversity of OMVs. Here, we show that deletion of , which encodes a part of the Tol-Pal system, leads to the production of multiple types of vesicles and increases overall vesicle production in the high-vesicle-forming type strain JCM 1090. The Δ mutant produced small OMVs and multilamellar/multivesicular OMVs (M-OMVs) as well as vesicles with a striking similarity to the wild type. M-OMVs, previously undescribed, contained triple-lamellar membrane vesicles and multiple vesicle-incorporating vesicles. Ultracentrifugation enabled the separation and purification of each type of OMV released from the Δ mutant, and visualization by quick-freeze deep-etch and replica electron microscopy indicated that M-OMVs are composed of several lamellar membranes. Visualization of intracellular compartments of Δ mutant cells showed that vesicles were accumulated in the broad periplasm, which is probably due to the low linkage between the outer and inner membranes attributed to the Tol-Pal defect. The outer membrane was invaginating inward by wrapping a vesicle, and the precursor of M-OMVs existed in the cell. Thus, we demonstrated a novel type of bacterial OMV and showed that unconventional processes enable the Δ mutant to form unique vesicles. Membrane vesicle (MV) formation has been recognized as a common mechanism in prokaryotes, and MVs play critical roles in intercellular interaction. However, a broad range of MV types and their multiple production processes make it difficult to gain a comprehensive understanding of MVs. In this work, using vesicle separation and electron microscopic analyses, we demonstrated that diverse types of outer membrane vesicles (OMVs) were released from an engineered strain, JCM 1090 Δ mutant. We also discovered a previously undiscovered type of vesicle, multilamellar/multivesicular outer membrane vesicles (M-OMVs), which were released by this mutant using unconventional processes. These findings have facilitated considerable progress in understanding MV diversity and expanding the utility of MVs in biotechnological applications. (hide) EV-METRIC 71% (96th 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 Other/ 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 Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Buttiauxella agrestis Sample Type Cell culture supernatant EV-producing cells JCM1090 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: rotor type P45AT Pelleting: speed (g) 150000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: rotor type P45AT Pelleting: speed (g) 150000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis None Protein Concentration Method BCA Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 140 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 20-150

	EV230273	2/4	Buttiauxella agrestis	JCM1090	DG (d)(U)C Filtration	Takaki K	2020	57%
Study summary Full title Multilamellar and Multivesicular Outer Membrane Vesicles Produced by a Buttiauxella agrestis Mutant. All authors Takaki K, Tahara YO, Nakamichi N, Hasegawa Y, Shintani M, Ohkuma M, Miyata M, Futamata H, Tashiro Y Journal Appl Environ Microbiol Abstract Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important (show more...)Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important roles in various biological functions. Released vesicles are not uniform in shape, size, or characteristics, and little is known about this diversity of OMVs. Here, we show that deletion of , which encodes a part of the Tol-Pal system, leads to the production of multiple types of vesicles and increases overall vesicle production in the high-vesicle-forming type strain JCM 1090. The Δ mutant produced small OMVs and multilamellar/multivesicular OMVs (M-OMVs) as well as vesicles with a striking similarity to the wild type. M-OMVs, previously undescribed, contained triple-lamellar membrane vesicles and multiple vesicle-incorporating vesicles. Ultracentrifugation enabled the separation and purification of each type of OMV released from the Δ mutant, and visualization by quick-freeze deep-etch and replica electron microscopy indicated that M-OMVs are composed of several lamellar membranes. Visualization of intracellular compartments of Δ mutant cells showed that vesicles were accumulated in the broad periplasm, which is probably due to the low linkage between the outer and inner membranes attributed to the Tol-Pal defect. The outer membrane was invaginating inward by wrapping a vesicle, and the precursor of M-OMVs existed in the cell. Thus, we demonstrated a novel type of bacterial OMV and showed that unconventional processes enable the Δ mutant to form unique vesicles. Membrane vesicle (MV) formation has been recognized as a common mechanism in prokaryotes, and MVs play critical roles in intercellular interaction. However, a broad range of MV types and their multiple production processes make it difficult to gain a comprehensive understanding of MVs. In this work, using vesicle separation and electron microscopic analyses, we demonstrated that diverse types of outer membrane vesicles (OMVs) were released from an engineered strain, JCM 1090 Δ mutant. We also discovered a previously undiscovered type of vesicle, multilamellar/multivesicular outer membrane vesicles (M-OMVs), which were released by this mutant using unconventional processes. These findings have facilitated considerable progress in understanding MV diversity and expanding the utility of MVs in biotechnological applications. (hide) EV-METRIC 57% (91st 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 TolB mutant Focus vesicles Other/ 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 Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Buttiauxella agrestis Sample Type Cell culture supernatant EV-producing cells JCM1090 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: rotor type P45AT Pelleting: speed (g) 150000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: rotor type P45AT Pelleting: speed (g) 150000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes Translucent band Characterization: Protein analysis None Protein Concentration Method BCA Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 75 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 20-150

	EV230273	3/4	Buttiauxella agrestis	JCM1090	DG (d)(U)C Filtration	Takaki K	2020	57%
Study summary Full title Multilamellar and Multivesicular Outer Membrane Vesicles Produced by a Buttiauxella agrestis Mutant. All authors Takaki K, Tahara YO, Nakamichi N, Hasegawa Y, Shintani M, Ohkuma M, Miyata M, Futamata H, Tashiro Y Journal Appl Environ Microbiol Abstract Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important (show more...)Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important roles in various biological functions. Released vesicles are not uniform in shape, size, or characteristics, and little is known about this diversity of OMVs. Here, we show that deletion of , which encodes a part of the Tol-Pal system, leads to the production of multiple types of vesicles and increases overall vesicle production in the high-vesicle-forming type strain JCM 1090. The Δ mutant produced small OMVs and multilamellar/multivesicular OMVs (M-OMVs) as well as vesicles with a striking similarity to the wild type. M-OMVs, previously undescribed, contained triple-lamellar membrane vesicles and multiple vesicle-incorporating vesicles. Ultracentrifugation enabled the separation and purification of each type of OMV released from the Δ mutant, and visualization by quick-freeze deep-etch and replica electron microscopy indicated that M-OMVs are composed of several lamellar membranes. Visualization of intracellular compartments of Δ mutant cells showed that vesicles were accumulated in the broad periplasm, which is probably due to the low linkage between the outer and inner membranes attributed to the Tol-Pal defect. The outer membrane was invaginating inward by wrapping a vesicle, and the precursor of M-OMVs existed in the cell. Thus, we demonstrated a novel type of bacterial OMV and showed that unconventional processes enable the Δ mutant to form unique vesicles. Membrane vesicle (MV) formation has been recognized as a common mechanism in prokaryotes, and MVs play critical roles in intercellular interaction. However, a broad range of MV types and their multiple production processes make it difficult to gain a comprehensive understanding of MVs. In this work, using vesicle separation and electron microscopic analyses, we demonstrated that diverse types of outer membrane vesicles (OMVs) were released from an engineered strain, JCM 1090 Δ mutant. We also discovered a previously undiscovered type of vesicle, multilamellar/multivesicular outer membrane vesicles (M-OMVs), which were released by this mutant using unconventional processes. These findings have facilitated considerable progress in understanding MV diversity and expanding the utility of MVs in biotechnological applications. (hide) EV-METRIC 57% (91st 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 TolB mutant Focus vesicles Other/ 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 Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Buttiauxella agrestis Sample Type Cell culture supernatant EV-producing cells JCM1090 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: rotor type P45AT Pelleting: speed (g) 150000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: rotor type P45AT Pelleting: speed (g) 150000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes Upper band Characterization: Protein analysis None Protein Concentration Method BCA Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 125 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 20-150

	EV230273	4/4	Buttiauxella agrestis	JCM1090	DG (d)(U)C Filtration	Takaki K	2020	57%
Study summary Full title Multilamellar and Multivesicular Outer Membrane Vesicles Produced by a Buttiauxella agrestis Mutant. All authors Takaki K, Tahara YO, Nakamichi N, Hasegawa Y, Shintani M, Ohkuma M, Miyata M, Futamata H, Tashiro Y Journal Appl Environ Microbiol Abstract Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important (show more...)Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important roles in various biological functions. Released vesicles are not uniform in shape, size, or characteristics, and little is known about this diversity of OMVs. Here, we show that deletion of , which encodes a part of the Tol-Pal system, leads to the production of multiple types of vesicles and increases overall vesicle production in the high-vesicle-forming type strain JCM 1090. The Δ mutant produced small OMVs and multilamellar/multivesicular OMVs (M-OMVs) as well as vesicles with a striking similarity to the wild type. M-OMVs, previously undescribed, contained triple-lamellar membrane vesicles and multiple vesicle-incorporating vesicles. Ultracentrifugation enabled the separation and purification of each type of OMV released from the Δ mutant, and visualization by quick-freeze deep-etch and replica electron microscopy indicated that M-OMVs are composed of several lamellar membranes. Visualization of intracellular compartments of Δ mutant cells showed that vesicles were accumulated in the broad periplasm, which is probably due to the low linkage between the outer and inner membranes attributed to the Tol-Pal defect. The outer membrane was invaginating inward by wrapping a vesicle, and the precursor of M-OMVs existed in the cell. Thus, we demonstrated a novel type of bacterial OMV and showed that unconventional processes enable the Δ mutant to form unique vesicles. Membrane vesicle (MV) formation has been recognized as a common mechanism in prokaryotes, and MVs play critical roles in intercellular interaction. However, a broad range of MV types and their multiple production processes make it difficult to gain a comprehensive understanding of MVs. In this work, using vesicle separation and electron microscopic analyses, we demonstrated that diverse types of outer membrane vesicles (OMVs) were released from an engineered strain, JCM 1090 Δ mutant. We also discovered a previously undiscovered type of vesicle, multilamellar/multivesicular outer membrane vesicles (M-OMVs), which were released by this mutant using unconventional processes. These findings have facilitated considerable progress in understanding MV diversity and expanding the utility of MVs in biotechnological applications. (hide) EV-METRIC 57% (91st 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 TolB mutant Focus vesicles Other/ 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 Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Buttiauxella agrestis Sample Type Cell culture supernatant EV-producing cells JCM1090 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: rotor type P45AT Pelleting: speed (g) 150000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: rotor type P45AT Pelleting: speed (g) 150000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes Lower band Characterization: Protein analysis None Protein Concentration Method BCA Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 145 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 20-150

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EV-TRACK ID	EV230273
species	Buttiauxella agrestis
sample type	Cell culture
cell type	JCM1090
condition	Control condition	TolB mutant	TolB mutant	TolB mutant
separation protocol	Density gradient/ dUC/ Filtration	Density gradient/ dUC/ Filtration	Density gradient/ dUC/ Filtration	Density gradient/ dUC/ Filtration
EV subtype	NA	Translucent band	Upper band	Lower band
Exp. nr.	1	2	3	4
EV-METRIC %	71	57	57	57