Chromosome organisation and segregation during sporulation in Bacillus subtilis

Abstract

In times of nutrient scarcity, Bacillus subtilis can form highly resistant spores. Sporulation is a complex differentiation process requiring the coordinated differential expression of hundreds of genes in the smaller prespore and larger mother cell. The critical morphological events in sporulation include a conformational change of the chromosomes and polar anchoring of the chromosome origins that are normally located at quarter cell positions. This is followed by asymmetric cell division that bisects the prespore chromosome, and the subsequent transfer of this chromosome into the spore (Lewis et al., 1994; Wu and Errington, 1998; Ben-Yehuda and Losick, 2002; Ben-Yehuda et al., 2003b; Wu and Errington, 2003). Since these events are critical, there are dedicated systems to ensure their success. This work explores aspects of these processes. In this thesis, I began by attempting to map the precise chromosomal boundaries at the site of the asymmetric septum. For this I performed chromatin-affinity-purification and also developed a methylase-based method. Next, I examined several novel proteins involved in capturing the DNA at the cell pole. This included deciphering the genetic hierarchy and localisation patterns of the newly implicated proteins (MinD and ComN), and confirmed the reported functional redundancy between chromosome capture machineries. Finally my work explored the role played by Soj (ParA) in chromosome movement and origin capture at the cell pole using high resolution microscopy. Soj is a member of the chromosomally encoded B. subtilis ParABS system, and has been long implicated in controlling chromosome segregation and DNA replication initiation (Ireton et al., 1994; Wu and Errington, 2003; Murray and Errington, 2008). My work has demonstrated that the ATPase function of Soj is not necessary for origin capture, but simply the Soj-ATP monomer is proficient. Based on these data, I propose two models for Soj function in polar origin capture during sporulation.