The mating pilus of E. coli pED208 acts as a conduit for ssDNA during horizontal gene transfer

Bacterial conjugation, a process of horizontal gene transfer, plays a key role in promoting the spread of antimicrobial resistance among human pathogens. The mechanism of conjugation involves the development of a conjugative pilus that forms a physical bridge between two bacterial cells and the subsequent unidirectional transfer of single-stranded DNA (ssDNA) complexed with a protein from the donor to the recipient cell. Atomic structures exist for many of the components of the type IV secretion system (T4SS), responsible for the nucleoprotein secretion, but little is known about the events preceding gene transfer, specifically what is the extent of the participation of the conjugative pilus in ssDNA transfer? There has been a longstanding debate about whether its main role is to bring a donor and a recipient cell into physical juxtaposition and form a mating junction that allows for ssDNA transfer via the T4SS machinery complex or whether ssDNA is actually transferred through the lumen of the pilus. Here, through a combination of maleimide labeling of the conjugative pilus and SeqA-YFP labeling of the transferred ssDNA, we visualize the process of bacterial conjugation in real time. We discover that the conjugative pilus is capable of transferring the ssDNA at a distance, between physically separated cells, and thus conclude that a physical mating junction is not essential for conjugative gene transfer.IMPORTANCE Bacteria are constantly exchanging DNA, which constitutes horizontal gene transfer. While some of these occurs by a non-specific process called natural transformation, some occurs by a specific mating between a donor and a recipient cell. In specific conjugation, the mating pilus is extended from the donor cell to make contact with the recipient cell, but whether DNA is actually transferred through this pilus or by another mechanism involving the type IV secretion system complex without the pilus has been an open question. Using Escherichia coli, we show that DNA can be transferred through this pilus between a donor and a recipient cell that has not established a tight mating junction, providing a new picture for the role of this pilus.