Selective DNA delivery to specific members of assembled bacterial communities remains challenging. Bacterial conjugation enables efficient DNA delivery, but transfer to non-target recipients limits its specificity within mixed communities. Here, we repurpose plasmid entry exclusion (Eex) as a recipient-side gate to control conjugative DNA delivery. We demonstrate selective plasmid delivery to Eex-negative recipients within populations containing both Eex-expressing and Eex-negative cells. This recipient selectivity was maintained at increased cell densities and during prolonged mating. By combining RP4-type and F-type conjugation systems with their corresponding exclusion modules, we further directed DNA delivery from distinct donors to defined recipient populations. RP4-derived Eex also functioned in environmental bacteria, including Pseudomonas putida and Sphingobium japonicum, enabling recipient-specific exclusion within a multispecies mixture. Furthermore, repeated cycles of Eex-guided conjugation and selection altered community composition after assembly. These results establish entry exclusion as a recipient-side strategy for selective conjugative DNA delivery and, when combined with selection, for controlling the composition of assembled bacterial communities.
Kishida, K., Ogawa-Kishida, N., Stari, L., Ohtsubo, Y., Nagata, Y.
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