Recognition of oriT for DNA processing at termination of a round of conjugal transfer.

Conjugal transfer of plasmid DNA is terminated when the transferred strand, linearized at the 38 base-pair origin of transfer (oriT), is recircularized. For the plasmid R1162, it is the protein MobA, covalently linked to the linear strand, that rejoins the ends by a reversible transesterification reaction. We have identified from those oligonucleotides with a partially degenerate oriT base sequence, subpopulations bound by MobA that undergo transesterification, and support efficient termination of conjugal transfer. Two domains of oriT, a ten base-pair inverted repeat and an adjacent TAA, are required for tight binding by the protein, whereas the location of the dinucleotide YG determines the site of strand cleavage. The results indicate that capture of MobA by oriT, and subsequent processing of the DNA for termination, are determined by different sequence motifs within this locus.

Acquisition of resistance genes by the IncQ plasmid R1162 is limited by its high copy number and lack of a partitioning mechanism.

R1162 is a representative member of the broad-host-range IncQ group of multicopy plasmids. Lower-copy-number derivatives of R1162 were constructed in vitro and shown to be unstable, indicating that partitioning of plasmid copies at cell division is due to random distribution and not to an active partitioning mechanism. However, the normal copy number of R1162 reduces cell fitness during growth in broth and favors the emergence of unstable, lower-copy-number variants. As a result, plasmid-borne antibiotic resistance genes active at a low copy number eventually result in plasmid loss during periods of no selection. We argue that the maintenance of R1162 in a population requires a gene that is selected only at high levels. This reduces the potential for acquiring genes from other R factors and could explain the limited variety of antibiotic resistance genes contained by naturally occurring IncQ plasmids.

Replication of a plasmid lacking the normal site for initiation of one strand.

The origin of replication of the plasmid R1162 contains an initiation site for the synthesis of each DNA strand. When one of these sites (oriL) is deleted, synthesis on the corresponding strand is no longer initiated efficiently in vitro by the R1162-encoded replication proteins, and the plasmid is no longer stably maintained in the cell. However, in vivo the two strands of the plasmid duplex molecule are active at a similar level as templates for DNA synthesis, and newly synthesized copies of each strand are incorporated into daughter molecules at a similar rate. No secondary, strong initiation sites on the delta oriL strand were detected in the region of the origin. The delta oriL plasmid induces the SOS response, and this is important for plasmid maintenance even in a recombination-proficient strain. Our results indicate that an SOS-induced host system can maintain an R1162 derivative lacking one of its initiation sites.