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

<jats:title>ABSTRACT</jats:title> <jats:p> Outer 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 <jats:named-content content-type="genus-species">Acinetobacter baylyi</jats:named-content> as a model system. OMVs were extracted from bacterial cultures and tested for the ability to vector gene transfer into populations of <jats:named-content content-type="genus-species">Escherichia coli</jats:named-content> and <jats:named-content content-type="genus-species">A. baylyi</jats:named-content> , including naturally transformation-deficient mutants of <jats:named-content content-type="genus-species">A. baylyi</jats:named-content> . 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 anti-fluorescein 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 <jats:named-content content-type="genus-species">A. baylyi</jats:named-content> and <jats:named-content content-type="genus-species">E. coli</jats:named-content> at transfer frequencies ranging from 10 <jats:sup>−6</jats:sup> to 10 <jats:sup>−8</jats:sup> , with transfer efficiencies of approximately 10 <jats:sup>3</jats:sup> and 10 <jats:sup>2</jats:sup> per μg 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 <jats:named-content content-type="genus-species">A. baylyi</jats:named-content> 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 <jats:named-content content-type="genus-species">A. baylyi</jats:named-content> . </jats:p>