Tskhay, F.; Huang, H.; Starke, R.; de la Cruz Barron, M.; Garcillan-Barcia, M. P.; Berendonk, T. U.; Worrich, A.; Klümper, U.

Plasmids are key drivers of horizontal gene transfer, yet their dissemination is not limited to conjugation. Extracellular vesicles (EVs) can transport plasmid DNA, but the factors governing plasmid incorporation into EVs remain poorly understood. Here, we tested whether principles of conjugative plasmid transfer, including plasmid mobility type and plasmid-plasmid interactions, extend to EV-mediated export. Using a conjugative plasmid (pKJK5) and a mobilizable plasmid (RSF1010) in two Gram-negative hosts, we quantified plasmid incorporation into EVs under single- and dual-plasmid conditions. When present individually, the conjugative plasmid was preferentially incorporated into EVs, exceeding RSF1010 by 10-23-fold despite its lower intracellular abundance. Under co-residence, this pattern reversed: RSF1010 became enriched by 13-39-fold, while pKJK5 was reduced by 2-7-fold. Consequently, EV-associated plasmid cargo shifted to RSF1010 dominance, deviating strongly from the expected 10-fold higher pKJK5 cargo if a stochastic model based on intracellular abundance and single-plasmid conditions were applicable. We propose that mobilizable plasmids under coexistence exploit conjugative plasmid transfer machinery to access membrane-associated sites, increasing their likelihood of incorporation into EVs. Our findings demonstrate that plasmid-plasmid interactions reshape EV cargo and identify a previously unrecognized mechanism that may influence extracellular gene transfer potential in microbial communities.