The role of the Bacillus subtilis ParABS system in DNA replication and segregation

Abstract

The partitioning of low-copy number plasmids into daughter cells requires the plasmid encoded ParA and ParB proteins acting on centromeric sites termed parS. Bioinformatics approaches have shown that bacterial chromosome also encodes the ParABS system (Livny et al., 2007). In B. subtilis the majority of the chromosomally encoded parS sites are located near the replication origin region where they are bound by Spo0J (ParB), forming a set of nucleoprotein complexes that colocalizes with oriC (Breier and Grossman, 2007; Graham et al., 2014; Lin and Grossman, 1998; Livny et al., 2007; Murray et al., 2006). SMC complex (condensin) localization at oriC is dependent on Spo0J and is required for chromosome origin segregation (Gruber and Errington, 2009; Sullivan et al., 2009; Wang et al., 2014b). In addition Spo0J regulates Soj (ParA), which in turn controls the master DNA replication initiation protein DnaA (Murray and Errington, 2008; Scholefield et al., 2012; Scholefield et al., 2011). Therefore, I hypothesized that the localization of parS sites proximal to the replication origin might be important for the regulation of Soj by Spo0J. In this thesis I have constructed a range of genetically modified strains that differ in the number and location of parS sites. I have found that a single Spo0J:parS nucleoprotein complex is necessary and sufficient for effective regulation of Soj activity, and that this regulation is independent of the genetic context of the parS site. In contrast chromosome organization and origin region segregation were affected when Spo0J (and presumably condensin) was redistributed to ectopic locations. Finally, I provide evidence that Soj interacts with Spo0J:parS nucleoprotein complexes to promote DNA segregation and dictate chromosome orientation. I propose a model that integrates the roles of 3 Soj as a regulator of DNA replication initiation and chromosome origin segregation.