Characterisation of an in vivo system for nicking at the origin of conjugal DNA transfer of the sex factor F

Abstract

Transfer of the F plasmid between conjugating Escherichia coli cells has been assumed to require endonucleolytic cleavage at a specific site ( oriT) on a specific strand of the F molecule. Using a lambda transducing phage which contains oriT we have detected this nicking process in vivo. Nicking of DNA occurred in the strand that included the “transferred” F strand and at a location within the transducing segment consistent with all previous genetic and restriction enzyme cleavage data on the position of oriT in F. Genetic study of the nicking process using F lac tra − point and deletion mutants, and also λ tra phages which carried various parts of the transfer region, indicated that the products of two transfer operon genes, traY and the previously unidentified gene traZ, were directly involved in nicking at oriT. The product of traJ was also required for nicking, but the possibility that this was solely due to the regulatory function of the traJ product could not be excluded. The plasmid specificities of oriT, traY and traZ between F and the related F-like plasmids R1-19 and R100-1 were investigated using the λoriT nicking system, and shown to be consistent with those determined in genetic complementation tests. The differences in specificity observed imply that the oriT sequence of F differs from those of R1-19 and R100-1. The products of the traM and traI genes are known to be required for the initiation of DNA transfer; their possible roles in modulating the activity of the traY Z endonuclease are discussed.