Dokument

Summary:
Faithful chromosome replication and segregation are essential for every living cell and must be tightly coordinated with other cell cycle events such as cell division. Our knowledge about prokaryotic chromosome dynamics is based on studies of only a few model organisms that divide by binary fission and are mostly characterized by a rod-like morphology. To broaden our insight into bacterial chromosome segregation, our lab has recently started to analyze chromosome dynamics in the marine alphaproteobacterium Hyphomonas neptunium, which divides by budding at the tip of the stalk and uses its stalk as a reproductive structure. This mode of reproduction distinguishes H. neptunium from so far studied model organisms and renders it an exciting candidate for the study of chromosome dynamics, since the duplicated chromosome must transit the stalk to reach the newly generated daughter cell. Recent work has revealed that the H. neptunium chromosome is segregated in a unique two-step process. At first, one of the duplicated origins is segregated within the mother cell, possibly in a ParABS-dependent manner, and remains at the stalked mother cell pole until a visible bud has formed at the tip of the stalk. In a second step, it is then segregated through the stalk into the bud. Several lines of evidence suggest that the transport through the stalk is mediated by a novel, yet unidentified, segregation mechanism. Commonly, chromosome replication and segregation occur concomitantly in bacteria. However, this two-step segregation mechanism implies a temporal uncoupling of chromosome replication and segregation through the stalk, reminiscent of eukaryotic mitosis. In this work, we analyzed the role of the ParABS system in chromosome segregation of H. neptunium. The ParABS system was shown to be essential for cell viability and chromosome segregation. Impairment of ParA functioning leads to morphological alterations and incomplete origin segregation within the mother cell and, consequently, hampers chromosome segregation through the stalk. This shows that the ParABS system mediates origin segregation within the mother cell. It also implies that chromosome segregation within the mother cell and through the stalk are sequential processes. Furthermore, we analyzed the role of PopZ and SMC in H. neptunium, since these proteins were shown to be involved in chromosome segregation in other bacteria. PopZ localizes to the pole opposite the stalk in the newly generated bud and appears to play only a minor role in the positioning of the ParABS partitioning machinery. SMC seems to be essential in H. neptunium and shows a similar localization pattern as ParB. Determination of the location of seven genomic loci in new-born cells revealed that the chromosome shows a longitudinal arrangement with the origin located at the flagellated pole and the terminus at the opposite cell pole. The other loci are arranged between both cell poles in a linear order that correlates with their position on the genomic map. Moreover, analysis of chromosome dynamics indicates that the ParB/parS complex is the region to be segregated first within the mother cell and also through the stalk, emphasizing its central role in the segregation process. As mentioned above, the observed two-step chromosome segregation mechanism suggested a temporal uncoupling of chromosome replication and its segregation through the stalk. To investigate the coordination between these two processes in more detail, we followed replisome dynamics by fluorescence labeling of different replisome components. The replication machinery shows a dynamic localization within the mother cell: in cells that are most likely at the swarmer-to-stalked cell transition as well as in stalked cells, it assembles at the pole opposite the (future) stalk and moves, via midcell, close to the stalked cell pole, where it disassembles again. This localization pattern is consistent with the observed location of the origin and terminus region. Furthermore, the replisomes appear to track independently along the two chromosome arms. Co-localization of ParB (origin) and DnaN (replisome) revealed that a large part of the chromosome is replicated before its segregation through the stalk commences, indicating that these processes are partially temporally uncoupled. Collectively, these observations expand our insight into chromosome dynamics in H. neptunium and suggest that it combines previously described segregation mechanisms, such as the ParABS system, with a novel segregation mechanism that awaits discovery.

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