Abstract
The production of outer membrane vesicles by Gram-negative bacteria has been well documented; however, the mechanism behind the biogenesis of these vesicles remains unclear. Here a high-throughput experimental method and systems-scale analysis was conducted to determine vesiculation values for the whole genome knockout library of Escherichia coli mutant strains (Keio collection). The resultant dataset quantitatively recapitulates previously observed phenotypes and implicates nearly 150 new genes in the process of vesiculation. Gene functional and biochemical pathway analyses suggest that mutations that truncate outer membrane structures such as lipopolysaccharide and enterobacterial common antigen lead to hypervesiculation, whereas mutants in oxidative stress response pathways result in lower levels. This study expands and refines the current knowledge regarding the cellular pathways required for outer membrane vesiculation in E. coli.
Highlights
Outer membrane vesicles (OMVs), derived from both pathogenic and commensal Gram-negative bacteria, are formed from the budding and release of the outer membrane (OM), leading to the formation of lipid-encapsulated spheres that range from 20–200 nm in diameter [1]
To gain global insight into the mechanism of OMV production in bacteria, we designed experimental and analytical methods to test a wide variety of mutant strains in a high-throughput, cost-effective manner (Fig 1A)
To rule out low vesiculation values that were due to growth impairments or high vesiculation values that were due to lysis, growth of strains across the collection were monitored in liquid medium in a plate reader under conditions matching those used for the vesiculation assay (Fig 1C, S2 Table)
Summary
Outer membrane vesicles (OMVs), derived from both pathogenic and commensal Gram-negative bacteria, are formed from the budding and release of the outer membrane (OM), leading to the formation of lipid-encapsulated spheres that range from 20–200 nm in diameter [1]. Periplasmic cargo is encapsulated in the lumen of OMVs, protecting it from degradative environmental factors [2, 3]. Membrane-bound cargo is found on the surface of OMVs, including outer membrane proteins (OMPs) [4], lipopolysaccharide (LPS) [5], and in some cases, quorum signaling molecules [6]. Because OMVs contain biologically active proteins, it is no surprise that they partake in a multitude of biological functions, including the delivery of toxins during infection [7,8,9], biofilm nucleation [10, 11], defense against antimicrobials [12], PLOS ONE | DOI:10.1371/journal.pone.0139200. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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