Definition of a minimal plasmid stabilization system from the broad-host-range plasmid RK2

The stable inheritance of the broad-host-range plasmid RK2 is due at least in part to functions within a region located at coordinates 32.8 to 35.9 kb, termed the RK2 par locus. This locus encodes four previously identified genes in two operons (parCBA and parD; M. Gerlitz, O. Hrabak, and H. Schwab, J. Bacteriol. 172:6194-6203, 1990, and R. C. Roberts, R. Burioni, and D. R. Helinski, J. Bacteriol. 172:6204-6216, 1990). The parCBA operon is functional in resolving plasmid multimers to monomers. Analysis of the plasmid stabilization capacity of deletions within this region, however, indicates that this multimer resolution operon is required for stabilization only in certain Escherichia coli strains and under specific growth conditions. The deletion analysis further allowed a redefinition of the minimal functional region as 790 bp in length, consisting of the parD gene (243 bp) and its promoter as well as sequences downstream of parD. This minimal region stabilizes an RK2-derived minireplicon in several different gram-negative bacteria and, at least in E. coli, in a vector-independent manner. By insertional mutagenesis, both the parD gene and downstream (3') regions were found to be required for plasmid stabilization. The downstream DNA sequence contained an open reading frame which was subsequently shown by transcriptional and translational fusions to encode a protein with a predicted size of 11,698 Da, designated ParE. Since the parDE operon requires the presence of the parCBA operon for efficient stabilization under certain growth conditions, the potential role of multimer resolution in plasmid stabilization was tested by substituting the ColE1 cer site for the parCBA operon. While the cer site did function to resolve plasmid multimers, it was not sufficient to restore stabilization activity to the parDE operon under growth conditions that require the parCBA operon for plasmid stability. This suggests that plasmid stabilization by the RK2 par locus relies on a complex mechanism, representing a multifaceted stabilization system of which multimer resolution is a conditionally dispensable component, and that the function(s) encoded by the parDE operon is essential.