Contribution of different segments of the par region to stable maintenance of the broad-host-range plasmid RK2

A 3.2-kb region of the broad-host-range plasmid RK2 has been shown to encode a highly efficient plasmid maintenance system that functions in a vector-independent manner. This region, designated par, consists of two divergently arranged operons: parCBA and parDE. The 0.7-kb parDE operon promotes plasmid stability by a postsegregational killing mechanism that ensures that plasmid-free daughter cells do not survive after cell division. The 2.3-kb parCBA operon encodes a site-specific resolvase protein (ParA) and its multimer resolution site (res) and two proteins (ParB and ParC) whose functions are as yet unknown. It has been proposed that the parCBA operon encodes a plasmid partitioning system (M. Gerlitz, O. Hrabak, and H. Schwabb, J. Bacteriol. 172:6194-6203, 1990; R. C. Roberts, R. Burioni, and D. R. Helinski, J. Bacteriol. 172:6204-6216, 1990). To further define the role of this region in promoting the stable maintenance of plasmid RK2, the parCBA and parDE operons separately and the intact (parCBA/DE) par region (3.2 kb) were reintroduced into an RK2 plasmid deleted for par and assayed for plasmid stability in two Escherichia coli strains (MC1061K and MV10delta lac). The intact 3.2-kb region provided the highest degree of stability in the two strains tested. The ability of the parCBA or parDE region alone to promote stable maintenance in the E. coli strains was dependent on the particular strain and the growth temperature. Furthermore, the insertion of the ColE1 cer site into the RK2 plasmid deleted for the par region failed to stabilize the plasmid in the MC1061K strain, indicating that the multimer resolution activity encoded by parCBA is not by itself responsible for the stabilization activity observed for this operon. To examine the relative contributions of postsegregational cell killing and a possible partitioning function encoded by the intact 3.2-kb par region, stability assays were carried out with ParD provided in trans by a compatible (R6K) minireplicon to prevent postsegregational killing. In E. coli MV10delta lac, postsegregational killing appeared to be the predominant mechanism for stabilization since the presence of ParD substantially reduced the stability of plasmids carrying either the 3.2- or 0.7-kb region. However, in the case of E. coli MC1061K, the presence of ParD in trans did not result in a significant loss of stabilization by the 3.2-kb region, indicating that the putative partitioning function was largely responsible for RK2 maintenance. To examine the basis for the apparent differences in postsegregational killing between the two E. coli strains, transformation assays were carried out to determine the relative sensitivities of the strains to the ParE toxin protein. Consistent with the relatively small contribution of the postsegregational killing to plasmid stabilization in MC1061K, we found that this strain was substantially more resistant to killing by ParE in comparison to E. coli MV10delta lac. A transfer-deficient mutant of thepar-deleted plasmid was constructed for the stable maintenance studies. This plasmid was found to be lost from E. coli MV10delta lac at a rate three times greater than the rate for the transfer-proficient plasmid, suggesting that conjugation can also play a significant role in the maintenance of plasmid RK2.