Characteristics and significance of DNA binding activity of plasmid stabilization protein ParD from the broad host-range plasmid RK2.

Abstract

A region of the plasmid RK2 has been shown to stabilize plasmid replicons in a broad host-range manner. This region encodes two divergently transcribed operons: parCBA and parDE. The parCBA operon specifies a multimer resolution system, while the parDE operon alone is capable of stabilizing an RK2-derived minireplicon under defined growth conditions in several different Gram-negative bacteria. The observed autoregulation of the parDE operon is most likely the result of ParD protein binding within the PparDE region. The characteristics of ParD binding to this region and the role of such binding in plasmid stabilization were examined with purified ParD protein. The results indicate that the binding of a single dimer of ParD protein to the promoter region most likely blocks interaction of RNA polymerase holoenzyme with the promoter. DNase I protection experiments indicate that ParD binds to a discrete sequence of 48 base pairs in length. While the binding of ParD to PparDE is essential for proper regulation of expression of the ParD and ParE proteins in vivo, the analyses of binding properties of mutant ParD proteins suggest that binding to this region does not play a direct role in plasmid stabilization.