The conjugation system of the IncPoa plasmid RK2/RP4 is encoded by transfer regions designated Tral, Tra2, and Tra3. The Tral core region, cloned on plasmid pDG4A22, consists of the origin of transfer (oriT) and 2.6 kilobases of flanking DNA providing IncPa plasmid-specific functions that allow pDG4A22 to be mobilized by the heterologous IncPI8 plasmid R751. TnS insertions in pDG4A22 define a minimal 2.2-kilobase region required for plasmid-specific transfer of oriT. The Tral core contains the traJ and traK genes as well as an 18-kilodalton open reading frame downstream of traj. The traj and traK genes were shown to be required for transfer by complementation of inserts within these genes. Genetic evidence for the role of the 18-kilodalton open reading frame in transfer was obtained, although this protein has not been detected in cell lysates. These studies indicate that at least three transfer proteins are involved in plasmid-specific interactions at oriT.The broad-host-range conjugation systems of IncP plasmids are able to mediate DNA transfer into a wide variety of bacterial cells (9). The detailed molecular mechanisms of the transfer process are not understood. The general model of conjugative DNA transfer involves nicking of a single plasmid DNA strand at the origin of transfer (oril) sequence and subsequent transfer of the nicked strand to the recipient (17). Conjugal DNA synthesis in the donor and recipient cells reconstitutes the daughter plasmid molecules. In support of this model, the site-specific, single-strand nick induced in vitro by the IncP plasmid RK2/RP4 relaxation complex maps within the minimal oriT sequence defined by genetic studies (5-7). In addition, IncP plasmids encode DNA primases that function in conjugation to ensure efficient priming of complementary-strand synthesis in the recipient, thus promoting the broad-host-range transfer properties of these plasmids (11, 13).The protein-DNA interactions at oriT that initiate the transfer process are plasmid specific. Conjugation systems do not generally recognize oriT sequences from unrelated plasmids, and small nonconjugative plasmids such as ColEl and R1162/RSF1010 encode specific mobilization proteins to allow their oriT sequence to be transferred (1, 3, 4, 16). Even within the IncP group of plasmids, the transfer systems of the a and 13 subgroups (2, 12, 18) have diverged such that R751 (IncP,B) cannot mobilize the RK2/RP4 oriT sequence in the absence of specific RK2/RP4 transfer gene products (5, 18). The RK2/RP4 and R751 oriT regions have overall structural similarities but significant sequence differences, which account for this specificity of oriT transfer (9, 14). We have used R751 mobilization of the RK2 oriT as an assay to identify RK2/RP4 transfer genes and products that interact specifically with oriT.Previous results have shown that R751 can mobilize pDG4, which contains 12 kilobases (kb) of RK2 counterclockwise from the EcoRI site, but cannot transfer pDG5, which contains a 760-base-pair HaeII oriT fragment (18). Subsequently,...