DISSECTION OF INCP CONJUGATIVE PLASMID TRANSFER - DEFINITION OF THE TRANSFER REGION TRA2 BY MOBILIZATION OF THE TRA1 REGION IN TRANS

We constructed a transfer system consisting of two compatible multicopy plasmids carrying the transfer regions Tra1 and Tra2 of the broad-host-range IncP plasmid RP4. In this system, the plasmid containing the Tra1 region with the origin of transfer (oriT) was transferred, whereas additional functions essential for the conjugative process were provided from the Tra2 plasmid in trans. The Tra2 region, as determined for matings between Escherichia coli cells, maps between coordinates 18.03 and 29.26 kb of the RP4 standard map. The section of Tra2 required for mobilization of the plasmid RSF1010 (IncQ) and the propagation of bacteriophages Pf3 and PRD1 appears to be the same as that needed for RP4 transfer. Tra2 regions of RP4 (IncP-alpha) and R751 (Incp-beta) are interchangeable, facilitating mobilization of the plasmid carrying the RP4 Tra1 region. The transfer frequencies of both systems are similar. Transcription of Tra2 proceeds clockwise relative to the standard map of RP4 and is probably initiated at a promoter region located upstream of trbB (kilB). From this promoter region the trfA operon and the Tra2 operon are likely to be transcribed divergently. A second potential promoter has been located immediately upstream of trbB (kilB). Plasmids encoding the functional Tra2 region can only be maintained stably in host cells in the presence of the RP4 regulation region carrying the korA-korB operon or part of it. This indicates the involvement of RP4 key regulatory functions that apparently are active not only in the control of replication but also in conjugation.