We constructed a transfer system consisting of two compatible multicopy plasmids carrying the transfer regions Tral and Tra2 of the broad-host-range IncP plasmid RP4. In this system, the plasmid containing the Tral 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 (IncPa) and R751 (IncPj3) are interchangeable, facilitating mobilization of the plasmid carrying the RP4 Tral 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 (kiIB). 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.The conjugative transfer system of the promiscuous plasmid RP4 consists of two distinct regions, Tral and Tra2, separated by the Par (partitioning)/Mrs (multimer resolution system) region, the fiwA locus, IS8, and the kanamycin resistance gene (aphA). Tral, containing the origin of transfer (oriT), encodes functions involved in generating the single strand to be transferred and also includes the primase genes (59; for reviews see references 19 and 60). However, functions encoded by Tra2 have not yet been characterized extensively. Genetic approaches by transposon mutagenesis and complementation studies located the Tra2 region between the genes trbB (kiIB) andfiwA (3,4,37,51 acting replication function), encoding the replication protein and the Par/Mrs region, specifying a multimer resolution system (17,41). Plasmids containing the minimal functional Tra2 region could only be maintained stably when the korA-korB operon was present in trans, implying that KorA and KorB are involved in regulating expression of the transfer loci of Tra2. MATERUILS AND METHODSStrains, phages, and plasmids. E. coli HB101 (8) and S17-1 (47) were used as hosts for plasmids, and the nalidixic acid-resistant strain HB101 Nxr was used as a recipient for filter matings. Cells were grown in YT medium (33) buffered with 25 mM 3-(N-morpholino)propanesulfonic acid (sodium salt, pH 8.0) and supplemented with 0.1% glucose and 25 ,ug of thiamine-hydrochloride per ml. When appropriate, antibiotics...

Bacterial conjugation, in which DNA is transferred from one bacterium to another, was first reported in 1946 and found to be mediated by the F factor. Although the F and RK2/RP4 prototypic plasmids can mediate the transfer of DNA from bacteria to yeast, there has been no evidence of classical bacterial conjugation to higher eukaryotes. Here, I present evidence of such transfer, using Escherichia coli, the RK2 plasmid system and Chinese hamster ovary CHO K1 cells.

The IncP antibiotic-resistance plasmids transfer to a broad range of bacterial species. The RK2 origin of DNA transfer (oriT) consists of a 250-base-pair segment including the single-stranded cleavage site (nic) needed to generate the DNA strand believed to be transferred. Deletion derivatives and a bank of hydroxylamine-generated oriT mutants were screened for loss of transferability. DNA regions flanking both sides of nic are required for optimal transfer of the oriT clone. Of the chemically induced mutants, critical base-pair changes that dramatically reduced transfer frequency were found in a 10-base-pair region adjacent to nic. Relaxation (nicking) assays performed with these point mutants using protein-DNA complexes reconstituted in vitro revealed a correlation between DNA nicking and transfer frequency. Base-pair changes within the proximal arm of an inverted repeat upstream from the nick site resulted in reduced binding of the essential transfer protein TraJ and correspondingly reduced transfer frequencies. The results support a model of relaxosome formation involving at least two essential proteins: TraI and TraJ. The nick region defined by the point mutants was located in a segment known to be nearly identical in the related plasmid R751. This sequence was also found to be highly conserved in both border junctions of the transfer DNA (T-DNA) of plant tumor-inducing plasmids of Agrobacterium tumefaciens, indicating a relationship between IncP-mediated broad-host-range bacterial conjugation and T-DNA transfer to plants.

Data from prokaryotic replicative and conjugative systems, which interrelate DNA processing events initiated by a site-specific nick, are reviewed. While the replicative systems have been established in accordance with the rolling circle replication model, the mechanism of conjugative replication has not been elucidated experimentally. We summarize data involving random point mutagenesis of the RK2 transfer origin (oriT), which yielded relaxation-deficient and transfer-deficient derivatives having mutations exclusively in a 10bp region defined as the nick region. Features of the RK2 (IncP) nick region, including the DNA sequence, nick site position, and 5' covalent attachment of the nicking protein, have striking parallels in other systems involving nicking and mobilization of single-stranded DNA from a supercoiled substrate. These other systems include T-DNA transfer occurring in Agrobacterium tumefaciens Ti plasmid-mediated tumorigenesis in plants, and the rolling circle replication of plasmids of Gram-positive bacteria and of phi X174-like bacteriophage. The structural and functional similarities suggest that IncP conjugative replication, originating at the oriT, and T-DNA transfer replication, originating at the T-DNA border, produce continuous strands via a rolling circle-type replication.

Although the broad-host-range IncP plasmids can vegetatively replicate in diverse gram-negative bacteria, the development of shuttle vector systems has established that the host range for IncP plasmid conjugative transfer is greater than the range of bacteria that sustain IncP replicons. Towards understanding IncP plasmid conjugation and the connection between IncP conjugation and Agrobacterium tumefaciens T-DNA transfer to plants, two sets of mutants were generated in the larger transfer region (Tral) of the IncPa plasmid RK2. Mutagenesis strategies were chosen to minimize transcriptional polar effects. Mutant Tral clones were mapped, sequenced, and processed to reconstruct 49.5-kb Tra2-containing plasmid derivatives in order to assay for transfer activity and IncP plasmid-specific phage sensitivity. Focusing on the activities of the gene products of traF and traG in Escherichia coli, we found that mutations in traF abolished transfer activity and rendered the host cells phage resistant and mutations in traG abolished transfer activity but had no effect on phage sensitivity. Complementation of these mutant derivatives with corresponding trans-acting clones carrying traF or traG restored transfer activity and, in the case of the traF mutant, the phage sensitivity of the host cell. We conclude that in E. coli, both TraF and TraG are essential for IncP plasmid transfer and that TraF is necessary (but not sufficient) for donor-specific phage sensitivity, and sequencing data suggest that both TraF and TraG are membrane spanning.The remarkable broad-host-range transfer properties of the IncP plasmids have stimulated investigation of the basic mechanism of conjugation as well as the use of these plasmids as tools for genetic manipulation of a wide variety of bacteria. The IncP plasmids can replicate in diverse gram-negative organisms, but the host range defined by conjugative transfer ability is considerably greater than the range of vegetative replication proficiency (15,19,50). With the construction of shuttle vector systems consisting of more than one host-specific replication system and the IncP transfer system, plasmid derivatives have been shown to transfer from gram-negative bacteria to highly divergent gram-negative organisms (17, 30, 52), to acid-fast bacteria (26), to gram-positive bacteria (47), and to yeast (22). In addition, significant similarities have recently been described between the IncP conjugation system and the T-DNA and vir loci of Agrobacterium tumefaciens tumor-inducing (Ti) plasmids that promote DNA transfer to plants (49, 56). These similarities are underscored by the recent demonstration that theA. tumefaciens Vir region can mobilize the small non-selftransmissible IncQ plasmids (2) among agrobacteria in a manner similar to the established IncP mobilization of these IncQ plasmids (11).Unlike the F plasmid, in which the conjugative transfer genes are organized into a single contiguous operon (51), the IncP plasmids have a more complicated arrangement of their transfer systems. In the IncPa ...