T4SS-mediated transfer of 89K PAI in S. suis 2 1677 linearized with a restriction endonuclease (ScaI or NdeI) and then co-incubated with the protein components of interest. The predicted oriT nick on each of these specifically linearized fragments is shown in Fig. 5C . As expected, three distinct bands corresponding to the predicted sizes (based on nicking at the predicted oriT site) were detected on an alkaline agarose gel, in which the dsDNAs were denatured into single strands (Fig. 5D ). The C-terminally truncated MobA89K protein showed a comparable nicking activity to that of the full-length protein. In all cases, the largest fragment corresponded to the fulllength plasmid ssDNA, and the different sizes of the two additional bands with higher mobility indicated sitespecific cleavage of one of the plasmid strands. Indeed, the sum of the sizes of these two fragments equals one full-length linear ssDNA plasmid. These results, together with the supercoiled plasmid DNA cleavage data described above, demonstrated that the in vitro nicking reaction was dependent on the presence of both MobA89K and MobC89K, and the MobA89K-dependent oriT nicking activity resides within the N-terminal 258-aa relaxase domain of MobA89K.