Partitioning of Chromosomal DNA during Establishment of Cellular Asymmetry

Separation of daughter chromosomes occurs prior to cell division in nearly all organisms. In eukaryotes, the importance of properly orchestrating chromosome segregation and cytokinesis is highlighted by the existence of checkpoints to arrest the cell cycle when chromosomes are damaged or incompletely segregated. In bacteria, chromosome separation and cytokinesis are regulated both temporally and spatially, so that, even during rapid exponential growth, daughter chromosomes are well separated prior to the onset of division. Bacterial chromosome segregation appears to consist of two distinct steps: the rapid movement of the duplicated origins apart from one another (9, 11, 38, 40) and the separate condensation of the replicated daughter chromosomes (12, 19, 35). This process results in vegetative bacterial chromosomes having a bilobed structure during replication, with the daughter chromosomes being well separated prior to the onset of cytokinesis, so the path of the invaginating septum appears free of chromosomal DNA (4, 14). When chromosome segregation fails, as it does in mutants defective in chromosome partitioning or condensation, the chromosomes can be sheared during daughter cell separation (24, 34, 39, 47).

How, then, are bacterial chromosomes prepared for asymmetrically positioned division events, such as that which occurs during the sporulation pathway of Bacillus subtilis? At the onset of sporulation, the chromosome is reorganized into a rod-like structure (the axial filament), which is readily distinguished from the bilobed chromosome of vegetative cells (31). Next, a septum is formed close to one cell pole, trapping the origin-proximal region of the chromosome in the smaller daughter cell (the forespore), while the remainder is translocated across the septum after division (43, 44). This translocation event requires the SpoIIIE protein (1, 43), without which the forespore receives only the origin-proximal 30% of a chromosome, while the remainder is located in the mother cell (44, 45). Thus, in the B. subtilis sporulation pathway, the relative order of division and chromosome segregation is reversed, with division occurring prior to chromosome segregation, rather than after.

Little is known about the architecture of the forespore chromosome before the sporulation septum is synthesized. Is the region of the invaginating septum cleared of DNA prior to asymmetric septation, as during vegetative growth, or is the septum required to partition the chromosome? Previous studies have supported the latter proposal, largely due to the failure to observe partitioned axial filaments in the absence of septation (45). The presence of a partitioned axial filament with a condensed region of the chromosome near one cell pole was therefore thought to indicate that a sporulation septum had formed (13, 21, 29, 45), implying that chromosome partitioning was a consequence of cell division. However, these studies preceded reliable methods to simultaneously observe the asymmetrically positioned sporulation septum and the chromosomes (27), leaving open the possibility that individual chromosomes are reorganized into two separate domains, creating a gap to accommodate the invaginating septum. Here, we address this issue using time-lapse deconvolution microscopy together with fluorescent membrane stains that allow visualization of septa and nucleic acid stains to reveal chromosome structure. We demonstrate that, during sporulation, the bacterial chromosome is partitioned into two domains of unequal size prior to asymmetric cell division. This asymmetric chromosome condensation event is independent of cell division protein FtsZ, providing evidence for an FtsZ-independent reorganization of cellular architecture that serves to prepare the bacterium for asymmetric cell division.

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