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

Outer membrane vesicles (OMVs) are continually released from a range of bacterial species. Numerous functions of OMVs, in-cluding 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 distribu-tion (PSD) and zeta potential. Transmission electronmicroscopy (TEM) and immunogold labeling were performed using anti-fluorescein isothiocyanate (anti-FITC)-conjugated antibodies and anti-dsDNA antibodies to track the movement of FITC-la-beled 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 106 to 108, with transfer efficiencies of approximately 103 and 102 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 differ-ent ways, depending on the recipient species. Interestingly, the PSDmeasurements suggest that distinct size ranges of OMVs are released from A. baylyi