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ABSTRACT The exchange of bacterial extracellular vesicles facilitates molecular exchange between cells, including the horizontal transfer of genetic material. Given the implications of such transfer events on cell physiology and adaptation, some bacterial cells have likely evolved mechanisms to regulate vesicle exchange. Past work has identified mechanisms that influence the formation of extracellular vesicles, including the production of small molecules that modulate membrane structure; however, whether these mechanisms also modulate vesicle uptake and have an overall impact on the rate of vesicle exchange is unknown. Here, we show that membrane-binding molecules produced by microbes influence both the formation and uptake of extracellular vesicles and have the overall impact of increasing the vesicle exchange rate within a bacterial coculture. In effect, production of compounds that increase vesicle exchange rates encourage gene exchange between neighboring cells. The ability of several membrane-binding compounds to increase vesicle exchange was demonstrated. Three of these compounds, nisin, colistin, and polymyxin B, are antimicrobial peptides added at sub-inhibitory concentrations. These results suggest that a potential function of exogenous compounds that bind to membranes may be the regulation of vesicle exchange between cells. IMPORTANCE The exchange of bacterial extracellular vesicles is one route of gene transfer between bacteria, although it was unclear if bacteria developed strategies to modulate the rate of gene transfer within vesicles. In eukaryotes, there are many examples of specialized molecules that have evolved to facilitate the production, loading, and uptake of vesicles. Recent work with bacteria has shown that some small molecules influence membrane curvature and induce vesicle formation. Here, we show that similar compounds facilitate vesicle uptake, thereby increasing the overall rate of vesicle exchange within bacterial populations. The addition of membrane-binding compounds, several of them antibiotics at subinhibitory concentrations, to a bacterial coculture increased the rate of horizontal gene transfer via vesicle exchange. 

ABSTRACT Horizontal gene transfer is responsible for the exchange of many types of genetic elements, including plasmids. Properties of the exchanged genetic element are known to influence the efficiency of transfer via the mechanisms of conjugation, transduction, and transformation. Recently, an alternative general pathway of horizontal gene transfer has been identified, namely, gene exchange by extracellular vesicles. Although extracellular vesicles have been shown to facilitate the exchange of several types of plasmids, the influence of plasmid characteristics on genetic exchange within vesicles is unclear. Here, a set of different plasmids was constructed to systematically test the impact of plasmid properties, specifically, plasmid copy number, size, and origin of replication, on gene transfer in vesicles. The influence of each property on the production, packaging, and uptake of vesicles containing bacterial plasmids was quantified, revealing how plasmid properties modulate vesicle-mediated horizontal gene transfer. The loading of plasmids into vesicles correlates with the plasmid copy number and is influenced by characteristics that help set the number of plasmids within a cell, including size and origin of replication. Plasmid origin also has a separate impact on both vesicle loading and uptake, demonstrating that the origin of replication is a major determinant of the propensity of specific plasmids to transfer within extracellular vesicles. IMPORTANCE Extracellular vesicle formation and exchange are common within bacterial populations. Vesicles package multiple types of biomolecules, including genetic material. The exchange of extracellular vesicles containing genetic material facilitates interspecies DNA transfer and may be a promiscuous mechanism of horizontal gene transfer. Unlike other mechanisms of horizontal gene transfer, it is unclear whether characteristics of the exchanged DNA impact the likelihood of transfer in vesicles. Here, we systematically examine the influence of plasmid copy number, size, and origin of replication on the loading of DNA into vesicles and the uptake of DNA containing vesicles by recipient cells. These results reveal how each plasmid characteristic impacts gene transfer in vesicles and contribute to a greater understanding of the importance of vesicle-mediated gene exchange in the landscape of horizontal gene transfer.