Gene Transfer Potential of Outer Membrane Vesicles of Acinetobacter baylyi and Effects of Stress on Vesiculation

Abstract

dOuter membrane vesicles (OMVs) are continually released from a range of bacterial species. Numerous functions of OMVs, including the facilitation of horizontal gene transfer (HGT) processes, have been proposed. In this study, we investigated whether OMVs contribute to the transfer of plasmids between bacterial cells and species using Gram-negative Acinetobacter baylyi as a model system. OMVs were extracted from bacterial cultures and tested for the ability to vector gene transfer into populations of Escherichia coli and A. baylyi, including naturally transformation-deficient mutants of A. baylyi. Anti-double-stranded DNA (anti-dsDNA) antibodies were used to determine the movement of DNA into OMVs. We also determined how stress affected the level of vesiculation and the amount of DNA in vesicles. OMVs were further characterized by measuring particle size distribution (PSD) and zeta potential. Transmission electron microscopy (TEM) and immunogold labeling were performed using antifluorescein isothiocyanate (anti-FITC)-conjugated antibodies and anti-dsDNA antibodies to track the movement of FITC-labeled and DNA-containing OMVs. Exposure to OMVs isolated from plasmid-containing donor cells resulted in HGT to A. baylyi and E. coli at transfer frequencies ranging from 10 ؊6 to 10 ؊8 , with transfer efficiencies of approximately 10 3 and 10 2 per g of vesicular DNA, respectively. Antibiotic stress was shown to affect the DNA content of OMVs as well as their hydrodynamic diameter and zeta potential. Morphological observations suggest that OMVs from A. baylyi interact with recipient cells in different ways, depending on the recipient species. Interestingly, the PSD measurements suggest that distinct size ranges of OMVs are released from A. baylyi.

A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus

“…Packaging of DNA into OMVs has been noted historically as a mechanism facilitating horizontal transfer of antibiotic resistance genes between Gram-negative bacteria (24–26). Recent studies also report that RNA molecules are present in OMVs in marine ecosystems and in pathogenic Vibrio cholerae OMVs (27–29).…”

Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions

“…Typically, OMV-associated DNA is thought to be indiscriminately and externally bound to the vesicle. However, in a study investigating the capacity of Acinetobacter baylyi OMVs to enable horizontal gene transfer to A. baylyi and E. coli cells 93 , the location of DNA was carefully tracked using an antibody against double-stranded DNA. This analysis revealed that the DNA translocated from the cytoplasm into the periplasm and subsequently into OMVs, and that successful gene transfer required competence proteins in the recipient cell, suggesting that the OMVs lysed before DNA uptake by the recipient.…”

Growth phase-specific evolutionary benefits of natural transformation in Acinetobacter baylyi

“…Stress included growth above the optimum temperature (Fulsundar et al, 2014), growth in minimal medium and extended periods in stationary and death phase between serial dilutions (90 h passages). We demonstrate for the first time growth phase-dependent costs and benefits of natural transformation in bacteria.…”