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 researches highlighted intriguing DNA reactions at the early stage of conjugation that are important for plasmid establishment in the recipient cell, including prompt expression of anti-defense genes. However, genomics-based approaches to investigate plasmid establishment have been challenging, because identical, fully established plasmids in donor cells mask transconjugant-specific signals. To overcome this limitation, we developed a new EDTA method which exploits a donor mutant hypersensitive to hypoosmotic shock. It allows unprecedently quick and efficient Elimination of Donor population for Transconjugant Accumulation. RNAseq analysis revealed highly selective and robust induction of plasmid genes during early stage of conjugation. We experimentally identified +1 sites of 6 possible operons that are matched with ssDNA promoter predictions. We also showed gene expression program is altered in different recipient cells in which plasmid establishment is perturbed. As the EDTA method is straightforward and broadly applicable, it will further understanding of the plasmid establishment processes in new host cell, not only gene expression but also ss to double-stranded DNA conversion and plasmid circularization. Overall design: RNA-seq of recipient cells after 30 minutes or 60 minutes conjugation of pESBL and F plasmid