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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV130232	1/1	Escherichia coli	Bacteria	(d)(U)C Filtration	Schwechheimer C	2013	14%
Study summary Full title Synthetic effect between envelope stress and lack of outer membrane vesicle production in Escherichia coli All authors Schwechheimer C, Kuehn MJ Journal J Bacteriol Abstract Outer membrane vesicles (OMVs) are composed of outer membrane and periplasmic components and are ubi (show more...)Outer membrane vesicles (OMVs) are composed of outer membrane and periplasmic components and are ubiquitously secreted by Gram-negative bacteria. OMVs can disseminate virulence factors for pathogenic bacteria as well as serve as an envelope stress response. From a transposon mutant screen for OMV phenotypes, it was discovered that an nlpA mutant of Escherichia coli produces fewer OMVs than the wild type, whereas a degP mutant produces higher levels of OMVs. NlpA is an inner-membrane-anchored lipoprotein that has a minor role in methionine import. DegP is a periplasmic chaperone/protease for misfolded envelope proteins that is critical when cells are heat shocked. To reveal how these proteins contribute to OMV production, the mutations were combined and the double mutant analyzed. The ?nlpA ?degP strain displayed a high-temperature growth defect that corresponded to the production of fewer OMVs than produced by the ?degP strain. This phenotype also pertained to other undervesiculation mutations in a ?degP background. The hypovesiculation phenotype of ?nlpA in the wild-type strain as well as in the degP deletion strain was found to be a stationary-phase phenomenon. The periplasm of the ?nlpA ?degP strain was determined to contain significantly more protein in stationary phase than the wild type. Additionally, misfolded DegP substrate outer membrane porins were detected in ?degP mutant-derived OMVs. These data suggest that an accumulation of envelope proteins resulting from decreased vesiculation was toxic and contributed to the growth defect. We conclude that OMV production contributes to relieve the envelope of accumulated toxic proteins and that NlpA plays an important role in the production of vesicles in stationary phase. (hide) EV-METRIC 14% (56th 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 Bacteria Sample origin NAY Focus vesicles OMV 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 (d)(U)C Filtration Adj. k-factor 147.2 (pelleting) Protein markers EV: non-EV: Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Escherichia coli Sample Type Bacteria Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type TLA100.3 Pelleting: adjusted k-factor 147.2 Filtration steps 0.45µm > x > 0.22µm,

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EV-TRACK ID	EV130232
species	Escherichia coli
sample type	Bacteria
condition	NAY
separation protocol	(d)(U)C Filtration
Exp. nr.	1
EV-METRIC %	14