Selective elimination of donor bacteria enables global profiling of plasmid gene expression at early stages of conjugation.

Conjugative plasmids are a major driving force for the dissemination of antimicrobial resistance. During conjugation, plasmid DNA is transferred from the donor cell as a single-stranded (ss) linear molecule. Recent research has highlighted intriguing DNA reactions at the early stages of conjugation that are important for plasmid establishment in the recipient cell, including prompt expression of antidefense genes. However, genomics-based approaches to investigate plasmid establishment have been challenging because the identical fully established plasmids in donor cells mask transconjugant-specific signals. To overcome this limitation, we developed a new method, ED-TA, which exploits a donor mutant hypersensitive to hypoosmotic shock. ED-TA allows unprecedently quick and efficient Elimination of Donor population for Transconjugant Analysis. RNA-seq analyses revealed highly selective and robust induction of plasmid genes during the early stages of conjugation. We experimentally identified transcription start sites of six possible operons that are matched with ssDNA promoter predictions. We also showed that transconjugant gene expression is altered in recipients with perturbed plasmid establishment. As the ED-TA method is straightforward and broadly applicable, it will further our understanding of plasmid establishment processes in the new host cell, including not only gene expression but also ss to double-stranded DNA conversion and plasmid circularization.