Chromosomensegregation in Hyphomonas neptunium - Das Kamel und das Nadelöhr

Abstract The generation of progeny is the ultimate goal of every living cell. To achieve this aim, proper replication and segregation of the DNA is essential. Our current knowledge about chromosome segregation in bacteria derives from a few well-studied model organisms which divide by symmetric or asymmetric binary fission. In order to expand our knowledge in this field we established a new model organism, Hyphomonas neptunium that reproduces by budding off new offspring from the tip of a reproductive stalk structure (Abbildung 1). Its close relationship to the established model system Caulobacter crescentus enabled us to develop a broad spectrum of methods. This study investigates the mechanism of chromosome segregation in H. neptunium. In vivo localization studies of the chromosomal origin region revealed that the centromere-like region defined by the ParBHNE/parSHNE-complex is segregated in two discrete steps. One of the two centromere-like regions initially moves to the stalked pole of the mother cell. Once bud formation initiates, the centromere-like region then moves through the stalk and is tethered to the flagellated pole of the bud cell. To gather further information about chromosome segregation, two systems for the in vivo localization of chromosomal regions were established in H. neptunium: the FROS- and the parBSYp-System. An analysis of the dynamics of chromosome segregation revealed that the centromere-like region is segregated before the origin of replication, which resides at a distance of ~570 kb near the dnaA gen. So H. neptunium is the first bacterium to be investigated in which chromosome replication and segregation appear to be uncoupled. A comparative analysis of closely related species indicated that the origin regions of stalked budding bacteria display major organizational differences. Before the ParBHNE/parSHNE-complex moves through the stalk, the stalk is DNA free. This observation suggests that H. neptunium possesses a second segregation mechanism since the generic ParA-based segregation system requires chromosomal DNA as interaction partner. To shed light on the mechanism of chromosome segregation, I characterized the ParA homologs of H. neptunium with fluorescent fusions and deletion mutants. The results of localization and mutational analyses indicate that besides the generic chromosomal partitioning protein ParA HNE3561 (ParAHNE) HNE0708 may also be involved in the segregation process. The deletion of HNE0708 caused a pleiotropic phenotype. The mutant cells exhibited DNA accumulations in the stalks and displayed small bubble-like structures at the stalk tips. Furthermore, mothercells showed an elevated number of origin regions, suggesting a defect in the regulation of replication initiation of origin segregation. HNE3561 (ParAHNE) colocalized with ParBHNE in the mother cell. However, it localized earlier to the stalk tip than ParBHNE and was never observed in the stalk. This observation supported our hypothesis that a second segregation mechanism drives the movement of the origin region through the stalk. Collectively, these results suggest a two-step chromosome segregation process with the centromere-like ParBHNE/parSHNE-complex acting as a central anchor point for the segregation machinery.