Characterization of the stable maintenance properties of the par region of broad-host-range plasmid RK2

Abstract

A 3.2-kb fragment encoding five genes, parCBA/DE, in two divergently transcribed operons promotes stable maintenance of the replicon of the broad-host-range plasmid RK2 in a vector-independent manner in Escherichia coli. The parDE operon has been shown to contribute to stabilization through the postsegregational killing of plasmid-free daughter cells, while the parCBA operon encodes a resolvase, ParA, that mediates the resolution of plasmid multimers through site-specific recombination. To date, evidence indicates that multimer resolution alone does not play a significant role in RK2 stable maintenance by the parCBA operon in E. coli. It has been proposed, instead, that the parCBA region encodes an additional stability mechanism, a partition system, that ensures that each daughter cell receives a plasmid copy at cell division. However, studies carried out to date have not directly determined the plasmid stabilization activity of the parCBA operon alone. An assessment was made of the relative contributions of postsegregational killing (parDE) and the putative partitioning system (parCBA) to the stabilization of mini-RK2 replicons in E. coli. Mini-RK2 replicons carrying either the entire 3.2-kb (parCBA/DE) fragment or the 2.3-kb parCBA region alone were found to be stably maintained in two E. coli strains tested. The stabilization found is not due to resolution of multimers. The stabilizing effectiveness of parCBA was substantially reduced when the plasmid copy number was lowered, as in the case of E. coli cells carrying a temperature-sensitive mini-RK2 replicon grown at a nonpermissive temperature. The presence of the entire 3.2-kb region effectively stabilized the replicon, however, under both low-and high-copy-number conditions. In those instances of decreased plasmid copy number, the postsegregational killing activity, encoded by parDE, either as part of the 3.2-kb fragment or alone played the major role in the stabilization of mini-RK2 replicons within the growing bacterial population. Our findings indicate that the parCBA operon functions to stabilize by a mechanism other than cell killing and resolution of plasmid multimers, while the parDE operon functions solely to stabilize plasmids by cell killing. The relative contribution of each system to stabilization depends on plasmid copy number and the particular E. coli host.

Plasmid RK2 toxin protein ParE: purification and interaction with the ParD antitoxin protein

“…Previous work has demonstrated that the 0.7-kb region, a segment of the 3.2-kb region which encodes the entire parDE operon, stabilizes plasmids via a growth inhibition or cellkilling mechanism (38,46). These results suggested that ParE is a toxin, while ParD is an antitoxin protein that neutralizes the activity of ParE.…”

“…These studies further indicated that ParD and ParE are proteins of a toxin-antitoxin system analogous to those found on plasmids F and R1, with ParE acting as the toxic component. Stabilization of plasmids in E. coli by the parDE operon has been shown to be accompanied by growth inhibition and filamentation of plasmid-free segregants (38,46). In addition, plasmids carrying the parDE operon can be destabilized by providing ParD in trans from a compatible plasmid that is maintained by antibiotic selection (38).…”

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

“…The res site is found within a 100-bp region between the two promoters P parCBA and P parDE . Earlier work using mini-RK2 replicons indicated that multimer resolution activity specified by the parCBA operon by itself does not account for the level of stabilization observed (29,31,35). ParB has been shown to be…”

“…ParB has been shown to be an endonuclease, but its contribution to the stable maintenance provided by the parCBA operon is not known. It has been proposed that the ParA, ParB, and ParC proteins make up an active partition complex (8,29,31,35).…”

“…RK2 contains a 3.2-kb region designated par which has been shown to provide stable maintenance of RK2 and mini-RK2 replicons (8,29,31,32,34,35). The par region contains five genes in two divergent operons (8,29,35).…”