Conjugative plasmids are the main vector for antimicrobial resistance dissemination in bacteria. These plasmids are transferred from donor to recipient bacteria as single-stranded DNA and have to subdue recipient replication machineries to enable their conversion to double-stranded DNA, while simultaneously avoiding recipient defence systems. Here, we developed new fluorophores and new reporters to image this ss-to-dsDNA conversion at the single-cell level. Our work reveals the existence of a replication roadblock that delays the conversion of the first ssDNA segment to enter recipient bacteria. This “leading” segment encodes an array of genes that likely function as anti-defence factors transcribed from single-stranded promoters, with the identified roadblock enabling sustained transcription from ssDNA, full neutralization of defence systems, therefore facilitating plasmid establishment in new host bacteria. ​

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