AUTHOR=Ojima Yoshihiro , Toda Kaho , Sawabe Tomomi , Kumazoe Yuki , Tahara Yuhei O. , Miyata Makoto , Azuma Masayuki 

TITLE=Budding and explosive membrane vesicle production by hypervesiculating Escherichia coli strain ΔrodZ

JOURNAL=Frontiers in Microbiology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2024.1400434

DOI=10.3389/fmicb.2024.1400434

ISSN=1664-302X

ABSTRACT=<p><italic>Escherichia coli</italic> produces extracellular vesicles called outer membrane vesicles. In this study, we investigated the mechanism underlying the hypervesiculation of deletion mutant Δ<italic>rodZ</italic> of <italic>E. coli</italic>. RodZ forms supramolecular complexes with actin protein MreB and peptidoglycan (PG) synthase, and plays an important role in determining the cell shape. Because <italic>mreB</italic> is an essential gene, an expression-repressed strain (<italic>mreB</italic><sup>R3</sup>) was constructed using CRISPRi, in which the expression of <italic>mreB</italic> decreased to 20% of that in the wild-type (WT) strain. In shaken-flask culture, the Δ<italic>rodZ</italic> strain produced &gt;50 times more vesicles than the WT strain. The <italic>mreB</italic>-repressed strain <italic>mreB</italic><sup>R3</sup> showed eightfold higher vesicle production than the WT. Δ<italic>rodZ</italic> and <italic>mreB</italic><sup>R3</sup> cells were observed using quick-freeze replica electron microscopy. As reported in previous studies, Δ<italic>rodZ</italic> cells were spherical (WT cells are rod-shaped). Some Δ<italic>rodZ</italic> cells (around 7% in total) had aberrant surface structures, such as budding vesicles and dented surfaces, or curved patterns on the surface. Holes in the PG layer and an increased cell volume were observed for Δ<italic>rodZ</italic> and <italic>mreB</italic><sup>R3</sup> cells compared with the WT. In conditions of osmotic support using sucrose, the OD<sub>660</sub> value of the Δ<italic>rodZ</italic> strain increased significantly, and vesicle production decreased drastically, compared with those in the absence of sucrose. This study first clarified that vesicle production by the <italic>E. coli</italic> Δ<italic>rodZ</italic> strain is promoted by surface budding and a burst of cells that became osmotically sensitive because of their incomplete PG structure.</p>