Real-time visualisation of the intracellular dynamics of conjugative plasmid transfer.

Conjugation is a contact-dependent mechanism for the transfer of plasmid DNA between bacterial cells, which contributes to the dissemination of antibiotic resistance. Here, we use live-cell microscopy to visualise the intracellular dynamics of conjugative transfer of F-plasmid in E. coli, in real time. We show that the transfer of plasmid in single-stranded form (ssDNA) and its subsequent conversion into double-stranded DNA (dsDNA) are fast and efficient processes that occur with specific timing and subcellular localisation. Notably, the ssDNA-to-dsDNA conversion determines the timing of plasmid-encoded protein production. The leading region that first enters the recipient cell carries single-stranded promoters that allow the early and transient synthesis of leading proteins immediately upon entry of the ssDNA plasmid. The subsequent conversion into dsDNA turns off leading gene expression, and activates the expression of other plasmid genes under the control of conventional double-stranded promoters. This molecular strategy allows for the timely production of factors sequentially involved in establishing, maintaining and disseminating the plasmid.