P1 ParA interacts with the P1 partition complex at parS and an ATP-ADP switch controls ParA activities

Abstract

The partition system of P1 plasmids is composed of two proteins, ParA and ParB, and a cis-acting site parS. parS is wrapped around ParB and Escherichia coli IHF protein in a higher order nucleoprotein complex called the partition complex. ParA is an ATPase that autoregulates the expression of the par operon and has an essential but unknown function in the partition process. In this study we demonstrate a direct interaction between ParA and the P1 partition complex.

Stoichiometry of P1 Plasmid Partition Complexes

“…These interactions required magnesium in the gel and running buffer. Under these conditions, the two discrete complexes, corresponding to I ϩ B1 and I ϩ B2, are not observed, although the ParB complex migrates more slowly with increasing ParB concentrations (24). While the experimental conditions are not identical, it is possible, for example, that ParA is recruited only to a partition complex that contains two ParB dimers (complex I ϩ B2 or higher), perhaps explaining a requirement for high concentrations of ParB.…”

“…We reported recently that ParA interacts with ParB on the partition complex (24). The nature of this interaction is dependent on ParB concentration.…”

“…In Situ OP-Cu Footprinting-In situ footprinting assays were performed as described by Sigman et al (26) with the modifications reported previously (24). Briefly, protein-DNA complexes were separated by gel electrophoresis, and the entire gel was treated with a solution containing OP-Cu to cleave the DNA (24).…”

“…Briefly, protein-DNA complexes were separated by gel electrophoresis, and the entire gel was treated with a solution containing OP-Cu to cleave the DNA (24). Complexes were visualized by autoradiography and excised from the gel.…”

Towards understanding the molecular basis of bacterial DNA segregation

“…It has since been shown that ParA assembles onto the partition complex at parS and that this interaction is completely dependent on ATP, whereas the repressor activity of ParA prefers the ADP-bound form. These observations have led to the proposal that an ATP-ADP switch controls ParA entry into two separate pathways in which ParA plays separate roles (Bouet & Funnell 1999).…”