Abstract
Escherichia coli produces extracellular vesicles called outer membrane vesicles (OMVs) by releasing a part of its outer membrane. We previously reported that the combined deletion of nlpI and mlaE, related to envelope structure and phospholipid accumulation in the outer leaflet of the outer membrane, respectively, resulted in the synergistic increase of OMV production. In this study, the analysis of ΔmlaEΔnlpI cells using quick-freeze, deep-etch electron microscopy (QFDE-EM) revealed that plasmolysis occurred at the tip of the long axis in cells and that OMVs formed from this tip. Plasmolysis was also observed in the single-gene knockout mutants ΔnlpI and ΔmlaE. This study has demonstrated that plasmolysis was induced in the hypervesiculating mutant E. coli cells. Furthermore, intracellular vesicles and multilamellar OMV were observed in the ΔmlaEΔnlpI cells. Meanwhile, the secretion of recombinant green fluorescent protein (GFP) expressed in the cytosol of the ΔmlaEΔnlpI cells was more than 100 times higher than that of WT and ΔnlpI, and about 50 times higher than that of ΔmlaE in the OMV fraction, suggesting that cytosolic components were incorporated into outer-inner membrane vesicles (OIMVs) and released into the extracellular space. Additionally, QFDE-EM analysis revealed that ΔmlaEΔnlpI sacculi contained many holes noticeably larger than the mean radius of the peptidoglycan (PG) pores in wild-type (WT) E. coli. These results suggest that in ΔmlaEΔnlpI cells, cytoplasmic membrane materials protrude into the periplasmic space through the peptidoglycan holes and are released as OIMVs.
Highlights
Outer membrane vesicles (OMVs) are nanosized, spherical, bilayered membranous structures with a diameter of 20–250 nm
There was a deviation depending on the measurement method, it was again confirmed that the OMV production was significantly increased in ΔmlaEΔnlpI
The observation of ΔmlaEΔnlpI cells using QFDE-EM revealed that plasmolysis occurred at the tip of the long axis of the cells and that OMVs formed from this tip
Summary
Outer membrane vesicles (OMVs) are nanosized, spherical, bilayered membranous structures with a diameter of 20–250 nm. Other studies showed phospholipid accumulation in the outer leaflet of the outer membrane causes the dysfunction associated with disruption of VacJ ( known as MlaA) and Yrb, which encodes an ATP-binding cassette transport system, increasing OMV production in Gram-negative bacteria Haemophilus influenzae and Vibrio cholerae (Roier et al, 2016). This observation confirms a genome-wide assessment of OMV production in E. coli that demonstrated the increase in vesiculation in the mlaA (vacJ) and mlaE (yrbE) mutants (Kulp et al, 2015). Since the OMV production by ΔmlaEΔnlpI was 30 times that of the WT strain, a 500-fold increase in OmpW-GFP secretion by ΔmlaEΔnlpI could not be fully explained by an increase in OMV production
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