Dissection of the ATPase active site of P1 ParA reveals multiple active forms essential for plasmid partition.

ABSTRACT

The segregation, or partition, of bacterial plasmids is driven by the action of plasmid-encoded partition ATPases, which work to position plasmids inside the cell. The most common type of partition ATPase, generally called ParA, is represented by the P1 plasmid ParA protein. ParA interacts with P1 ParB (the site-specific DNA binding protein that recognizes the parS partition site), and interacts with the bacterial chromosome via an ATP-dependent nonspecific DNA binding activity. ParA also regulates expression of the par genes by acting as a transcriptional repressor. ParA requires ATP for multiple steps and in different ways during the partition process. Here, we analyze the properties of mutations in P1 ParA that are altered in a key lysine in the Walker A motif of the ATP binding site. Four different residues at this position (Lys, Glu, Gln, Arg) result in four different phenotypes in vivo. We focus particularly on the arginine substitution (K122R) because it results in a worse-than-null and dominant-negative phenotype called ParPD. We show that ParAK122R binds and hydrolyzes ATP, although the latter activity is reduced compared with wild-type. ParAK122R interacts with ParB, but the consequences of the interaction are damaged. The ability of ParB to stimulate the ATPase activity of ParA in vitro and its repressor activity in vivo is defective. The K122R mutation specifically damages the disassembly of ParA-ParB-DNA partition complexes, which we believe explains the ParPD phenotype in vivo.