Dissecting the role of the partition factor ParF in TP228 plasmid segregation

Antibiotic resistance is one of the most significant health concerns for the public. Bacterial antibiotic resistance can occur due to the presence of drug resistant plasmids; therefore understanding the mechanism of how these plasmids are passed from one generation to the next is of high importance. Low copy number plasmids utilise partition cassettes in order to ensure faithful segregation at cell division. This work dissects the multidrug resistance plasmid TP228, which contains the parFGH partition cassette. parH is a cis-acting like centromere site, ParG is a centromere DNA binding protein and ParF is a Walker-type ATPase that assembles into extended filaments upon ATP binding. Recent structural data has shown that ParF forms dimer-of-dimer units, which create the building blocks of the filaments. ParF also binds non-specifically to DNA in vitro and associates with the nucleoid in vivo. In the presence of the complete partition system, parFGH, ParF oscillates from one pole of the nucleoid to the other leading to correct positioning of the plasmids. In this study, the role that ParF plays in driving the accurate segregation of the TP228 plasmid is dissected. A range of in vitro biochemical assays and in vivo microscopy experiments have been used to study ParF DNA binding, assembly into higher order structures and ParF localisation. A triple mutant, harbouring changes at the dimer-dimer interface of ParF was shown to be unable to undergo ATP-dependent assembly into higher order structures, confirming the importance of this interface in this process. Mutants were also constructed at the monomer-monomer interface and investigated in this study. Residues were identified that were thought to be important in ns-DNA binding and a range of assays were carried out to determine the roles played. Conventional fluorescence and super resolution microscopy enabled a greater insight into the segregation dynamics of the TP228 partition proteins. Overall the results demonstrated that both ATP and DNA regulate the dynamic ParF higher order structures and this underpins plasmid segregation. Based on these findings, a new model for TP228 plasmid segregation is proposed.