DIFFERENT RELATIVE IMPORTANCE OF THE PAR OPERONS AND THE EFFECT OF CONJUGAL TRANSFER ON THE MAINTENANCE OF INTACT PROMISCUOUS PLASMID RK2

The par region of the broad-host-range, IncP alpha plasmid RK2 has bee n implicated as a stability determinant by its ability to enhance the maintenance of mini-RK2 plasmids or heterologous replicons in a growin g population of host cells. The region consists of two operons: parCBA , which encodes a multimer resolution system, and parDE, which specifi es a postsegregational response mechanism that is toxic to plasmidless segregants. To assess the importance of this region to the stable mai ntenance of the complete RK2 plasmid in different hosts, we used the v ector-mediated excision (VEX) deletion system to specifically remove t he entire par region or each operon separately from an otherwise intac t RK2 plasmid carrying a lacZ marker. The par region was found to be i mportant to stable maintenance of RK2lac (pRK2526) in Escherichia call and five other gram negative hosts (Agrobacterium tumefaciens, Azotob acter vinelandii, Acinetobacter calcoaceticus, Caulobacter crescentus, and Pseudomonas aeruginosa). However, the relative importance of the parCBA and parDE operons varied from host to host. Deletion of parDE h ad no effect on the maintenance of pRK2526 in A. calcoaceticus, but it severely reduced pRK2526 maintenance in A. vinelandii and resulted in significant instability in the other hosts. Deletion of parCBA did no t alter pRK2526 stability in E. coli, A. tumefaciens, or A. vinelandii but severely reduced plasmid maintenance in A. calcoaceticus and P. a eruginosa. In the latter two hosts and C. crescentus, the Delta parCBA mutant caused a notable reduction in growth rate in the absence of se lection for the plasmid, indicating that instability resulting from th e absence of parCBA may trigger the postsegregational response mediate d by parDE. We also examined the effect of the conjugal transfer syste m on RK2 maintenance in E. coli. Transfer-defective traJ and traG muta nts of pRK2526 were stably maintained in rapidly growing broth culture s. On solid medium, which should be optimal for IncP-mediated conjugat ion, colonies from cells containing the pRK2526 tra mutants displayed significant numbers of white (Lac(-)) sectors on X-Gal bromo-4-chloro- 3-indolyl-beta-D-galactopyranoside) plates, whereas sectors appeared r arely in colonies from tra(+) plasmid-containing cells. Both the traJ and traG mutations further reduced the maintenance of the already unst able Delta par derivative. Thus, these experiments with defined mutati ons in an intact RK2 plasmid have revealed (i) that the par region all ows RK2 to adapt to the different requirements for stable maintenance in various hosts and (ii) that conjugal transfer can contribute to the maintenance of RK2 in a growing population, particularly under condit ions that are favorable to RK2 transfer.