For all organisms, segregation and proper intracellular localization of DNA are essential processes in ensuring faithful inheritance of genetic material. In prokaryotes, several different mechanisms have developed for efficiently moving chromosomal DNA to proper cellular locations prior to cell division, and the same holds true for bacterial plasmids. Low-copy-number plasmids and bacterial chromosomes encode active partition systems to ensure their inheritance within a bacterial cell population. One of the well-studied models of partition is that of the P1 plasmid in E. coli. The partition system encoded by the P1 plasmid is known as parABS - ParA is the partition ATPase, ParB is the partition site binding protein and parS is the partition site. The goal of this thesis was to investigate the nucleoprotein complexes essential in the P1 plasmid partition reaction. First, I examined how a single ParB dimer can bind its complicated arrangement of recognition motifs in parS to initiate the partition reaction. I then characterized a novel ParA interaction with the host nucleoid that is critical for proper P1 plasmid dynamics in vivo. Finally, I demonstrate how ParA can act as an adaptor between the nucleoid and the partition complex; effectively allowing the plasmid to use the nucleoid as a track for its intracellular movement and localization. My thesis work provides evidence towards a model that explains the P1 plasmid partition mechanism.

For all organisms, segregation and proper intracellular localization of DNA are essential processes in ensuring faithful inheritance of genetic material. In prokaryotes, several different mechanisms have developed for efficiently moving chromosomal DNA to proper cellular locations prior to cell division, and the same holds true for bacterial plasmids. Low-copy-number plasmids and bacterial chromosomes encode active partition systems to ensure their inheritance within a bacterial cell population. One of the well-studied models of partition is that of the P1 plasmid in E. coli. The partition system encoded by the P1 plasmid is known as parABS - ParA is the partition ATPase, ParB is the partition site binding protein and parS is the partition site. The goal of this thesis was to investigate the nucleoprotein complexes essential in the P1 plasmid partition reaction. First, I examined how a single ParB dimer can bind its complicated arrangement of recognition motifs in parS to initiate the partition reaction. I then characterized a novel ParA interaction with the host nucleoid that is critical for proper P1 plasmid dynamics in vivo. Finally, I demonstrate how ParA can act as an adaptor between the nucleoid and the partition complex; effectively allowing the plasmid to use the nucleoid as a track for its intracellular movement and localization. My thesis work provides evidence towards a model that explains the P1 plasmid partition mechanism.

P1 prophage is stably maintained in E. coli as a low-copy-number plasmid. Stable maintenance of P1 is dependent on the function of the plasmid encoded partition system, parABS. ParA is the partition ATPase, ParB is the partition-site binding protein, and parS is the partition site. The concerted action of these proteins results in dynamic movement of the plasmid over the bacterial nucleoid, which results in its stable maintenance. Plasmid movement has been proposed to be caused by interactions between parS bound ParB and nucleoid bound ParA. In this thesis, I have identified a complex of ParA, ParB, and DNA that is capable of promoting plasmid stability. ParA, ParB, DNA interactions required the ATP bound conformation of ParA. The ParA-ParB-DNA complex was dynamically regulated by nucleotide hydrolysis, which promoted complex disassembly. Complex formation resulted from the cooperative binding of ParA and ParB to DNA. ParA-ParB and ParB-DNA interactions were both necessary for complex formation. ParA-ParB-DNA complex size was regulated by ParB stimulation of ParA-ATP hydrolysis. Microscopy demonstrated that complexes resulted in the association of multiple DNA molecules due to protein binding. The properties of complex assembly, dynamics, and DNA grouping lead me to propose a model where associations between ParA bound to the bacterial nucleoid and the partition complex mediated plasmid movement and localization.

P1 prophage is stably maintained in E. coli as a low-copy-number plasmid. Stable maintenance of P1 is dependent on the function of the plasmid encoded partition system, parABS. ParA is the partition ATPase, ParB is the partition-site binding protein, and parS is the partition site. The concerted action of these proteins results in dynamic movement of the plasmid over the bacterial nucleoid, which results in its stable maintenance. Plasmid movement has been proposed to be caused by interactions between parS bound ParB and nucleoid bound ParA. In this thesis, I have identified a complex of ParA, ParB, and DNA that is capable of promoting plasmid stability. ParA, ParB, DNA interactions required the ATP bound conformation of ParA. The ParA-ParB-DNA complex was dynamically regulated by nucleotide hydrolysis, which promoted complex disassembly. Complex formation resulted from the cooperative binding of ParA and ParB to DNA. ParA-ParB and ParB-DNA interactions were both necessary for complex formation. ParA-ParB-DNA complex size was regulated by ParB stimulation of ParA-ATP hydrolysis. Microscopy demonstrated that complexes resulted in the association of multiple DNA molecules due to protein binding. The properties of complex assembly, dynamics, and DNA grouping lead me to propose a model where associations between ParA bound to the bacterial nucleoid and the partition complex mediated plasmid movement and localization.