The origin of greater‐than‐unit‐length plasmids generated during bacterial conjugation

Summary In Gram‐negative bacteria, the general mechanism of conjugal plasmid transfer, which is probably similar for many different groups of plasmids, involves the transfer of a single plasmid DNA strand with 5′ to 3′ polarity. Transfer is initiated by nicking of the duplex DNA at a particular site, i.e. the origin of transfer ( oriT ). We constructed plasmids containing two directly repeated copies of oriT , derived from the broad‐host‐range plasmid R1162 and flanking the lac operator. The number of lacO copies in the plasmid after transfer could be determined from the colour of transconjugant colonies on medium containing X‐Gal. When the oriTs were mutated to prevent initiation and termination of transfer at the same oriTs , almost all of the transconjugant cells contained greater‐than‐unit‐length plasmids with two copies of lacO and three copies of oriT. We show that these molecules were generated by an intramolecular, conjugation dependent mechanism unlikely to depend solely on a pre‐existing population of circular or linear multimers in donor cells. We propose that the greater‐than‐unit‐length molecules were instead generated by a rolling‐circle mechanism of DNA replication.