Incorporation of Plasmid DNA Into Bacterial Membrane Vesicles by Peptidoglycan Defects in Escherichia coli

Abstract

Membrane vesicles (MVs) are released by various prokaryotes and play a role in the delivery of various cell-cell interaction factors. Recent studies have determined that these vesicles are capable of functioning as mediators of horizontal gene transfer. Outer membrane vesicles (OMVs) are a type of MV that is released by Gram-negative bacteria and primarily composed of outer membrane and periplasm components; however, it remains largely unknown why DNA is contained within OMVs. Our study aimed to understand the mechanism by which DNA that is localized in the cytoplasm is incorporated into OMVs in Gram-negative bacteria. We compared DNA associated with OMVs using Escherichia coli BW25113 cells harboring the non-conjugative, non-mobilized, and high-copy plasmid pUC19 and its hypervesiculating mutants that included ΔnlpI, ΔrseA, and ΔtolA. Plasmid copy per vesicle was increased in OMVs derived from ΔnlpI, in which peptidoglycan (PG) breakdown and synthesis are altered. When supplemented with 1% glycine to inhibit PG synthesis, both OMV formation and plasmid copy per vesicle were increased in the wild type. The bacterial membrane condition test indicated that membrane permeability was increased in the presence of glycine at the late exponential phase, in which cell lysis did not occur. Additionally, quick-freeze deep-etch and replica electron microscopy observations revealed that outer-inner membrane vesicles (O-IMVs) are formed in the presence of glycine. Thus, two proposed routes for DNA incorporation into OMVs under PG-damaged conditions are suggested. These routes include DNA leakage due to increased membrane permeation and O-IMV formation. Additionally, our findings contribute to a greater understanding of the vesicle-mediated horizontal gene transfer that occurs in nature and the utilization of MVs for DNA cargo.

Unravelling the DNA sequences carried by Streptomyces coelicolor membrane vesicles

“… 12 demonstrated, for the first time, the association of DNA with MVs released by Gram-positive bacteria belonging to the Ruminococcus genus, and the capability of these MVs to restore a specific metabolic activity in mutant strains 12 . Moreover, MVs have been reported to transfer plasmids between different strains 13 , 14 and DNA cargo into eukaryotic host cells 15 .…”

Reductions in bacterial viability stimulate the production of Extra-intestinal Pathogenic Escherichia coli (ExPEC) cytoplasm-carrying Extracellular Vesicles (EVs)

“…in bacterial OMVs remained largely unexplained [ 20 , 34 , 38 , 75 ]. There are very few studies on how nucleic acids and cytoplasmic proteins pass through the bacterial inner membrane and are packaged into OMVs [ 76 ] ( Fig 15A ). It is possible that cytoplasm-carrying vesicles are not bona fide OMVs.…”

The Role of Bacterial Membrane Vesicles in Human Health and Disease

“…In addition to short term resistance, we now have reason to believe that MVs promote the development of long-term adaptive resistance to antibiotics via the horizontal transfer of resistance genes. Recently, it was reported that glycine induced peptidoglycan defects in E. coli caused the bacteria to secrete MVs which carried plasmid DNA ( Aktar et al, 2021 ). Though the peptidoglycan defects described in this paper arose from glycine, these findings beg the question: do antibiotics or other environmental influences which impact the peptidoglycan layer also favor the production of plasmid carrying MVs?…”