Strand replacement of plasmid R1162 and transport of MobA during conjugative transfer

R1162 is a broad-host range, mobilizable plasmid conferring resistance to streptomycin and sulfonamides. Efficient conjugative mobilization of R1162 requires three plasmid-encoded proteins: MobA, MobB and MobC. MobA binds plasmid DNA at the origin of transfer (oriT), nicks the subsequently transferred strand and ligates the ends of the strand after transfer into the recipient. The N-terminal region of this protein carries out this DNA processing. The C-terminal half is a primase required to initiate DNA synthesis at two single-stranded priming sites sites, oriL and oriR, during vegetative plasmid replication. The primase region of MobA is not necessary for DNA processing by the N-terminal part of the protein, however its role in strand replacement during conjugation is not clearly defined. This study demonstrates that R1162 can undergo multiple rounds of transfer from a single plasmid molecule. The presence of oriL increases the frequency of second-round transfer, presumably due to initiation of replacement strand synthesis at this site by R1162 primase in the donor. Priming at oriR by the primase region of MobA is required for efficient replacement strand synthesis in the recipient when the plasmid is transferred to Salmonella. When the plasmid is transferred into E. coli, the plasmid-encoded priming system is not required for strand replacement in the recipient, presumably due to a host-encoded mechanism capable of priming the transferred strand. Transport of MobA through the R751 conjugative pore was also investigated. The two domains of MobA can be transported to recipient cells independently of each other. However, MobB is required for the transport of either fragment. Two sites, named the R-site and the P-site, are located in the relaxase and primase domains of MobA, respectively, and make up part of the signals required for MobA transport. Unlike previously described type IV transport signals, domain structure is required for the MobA transport signals to be active.