[3S04a] 11. The Society for Biotechnology - Part 2

3:10 PM - 3:30 PM JST(6:10 AM - 6:30 AM UTC)

[3SBT-11] Construction of a Hypervesiculating Escherichia coli Strain and Analysis of Enhanced Vesicle Production Mechanisms

○Yoshihiro Ojima¹ (1. Osaka Metropolitan University (Japan))

Keywords:

outer membrane vesicle,Escherichia coli,peptidoglycan,quick-freeze deep-etch replica electron microscopy

Outer membrane vesicles (OMVs) are extracellular vesicles with diameters of 20–250 nm released from Gram-negative bacteria. Recent studies have revealed that secreted OMVs fuse with surrounding cells to deliver nucleic acids and signaling molecules, thereby playing important roles in microbial interactions such as horizontal gene transfer and cell-to-cell communication. Among Gram-negative bacteria, Escherichia coli is widely used as an industrial host. However, OMV production in laboratory strains of E. coli is insufficient for biotechnological applications. Therefore, we constructed a hypervesiculating strain of E. coli through genetic modification and analyzed the mechanisms underlying enhanced OMV production.
We constructed E. coli K-12 strains carrying combined deletions of genes involved in cell wall synthesis. Among them, a double-gene deletion strain (ΔmlaEΔnlpI) exhibited the highest OMV production, approximately 30-fold higher than that of WT(1,2). It is thought that deletion of the nlpI gene decreases lipoprotein–peptidoglycan crosslinking, whereas phospholipid accumulation in the outer leaflet of the outer membrane is caused by deletion of mlaE. Observation of ΔmlaEΔnlpI cells by quick-freeze, deep-etch electron microscopy (QFDE-EM) revealed that periplasmic space increased at one pole of the longitudinal axis and that OMVs were formed from this site(3). Intracellular vesicles and multilamellar OMVs were also observed in ΔmlaEΔnlpI cells. In addition, secretion of recombinant GFP expressed in the cytosol of ΔmlaEΔnlpI cells was more than 100-fold higher than that of WT in the OMV fraction, suggesting that cytosolic components were incorporated into outer–inner membrane vesicles (OIMVs) and released into the extracellular space. QFDE-EM revealed that the peptidoglycan contained numerous holes. These results suggest that, in ΔmlaEΔnlpI cells, cytoplasmic membrane materials protrude into the periplasmic space through these peptidoglycan holes and are released as OIMVs.
We also constructed a ΔmlaEΔnlpI in the probiotic strain Nissle 1917 (EcN)(4). The ΔmlaEΔnlpI of EcN exhibited higher OMV production than the parental strain. QFDE-EM revealed that periplasmic space increased in the elongated cells and that the peptidoglycan contained a greater number of peptidoglycan holes throughout the entire structure(4).
We have also reported an alternative mechanism underlying hypervesiculation in the ΔrodZ(5). RodZ forms complexes with actin protein MreB and peptidoglycan (PG) synthase, and plays an important role in determining the cell shape. The ΔrodZ produced 50-fold more vesicles than the WT strain. QFDE-EM revealed that ΔrodZ cells were spherical, and that a subset of cells (approximately 7% of the total population) exhibited aberrant surface structures, including budding vesicles, dented surfaces, or curved surface patterns. Compared with WT cells, ΔrodZ cells displayed holes in the peptidoglycan layer and an increased cell volume. Vesicle production in the ΔrodZ is promoted by surface budding and cell bursting resulting from osmotic sensitivity caused by incomplete peptidoglycan structure.
References(1)Biotechnol Prog, 34(1), 51-57 (2018); (2)Biotechnol Bioeng, 117(3), 701-709 (2020); (3)Front Microbiol, 12, 706525 (2021); (4)PLoS One, 19(4), e0301613 (2024); (5)Front Microbiol, 15, 1400434 (2024)