Escherichia coli sister chromosome separation includes an abrupt global transition with concomitant release of late-splitting intersister snaps

Abstract

The basis for segregation of sister chromosomes in bacteria is not established. We show here that two discrete~150-kb regions, both located early in the right replichore, exhibit prolonged juxtaposition of sister loci, for 20 and 30 min, respectively, after replication. Flanking regions, meanwhile, separate. Thus, the two identified regions comprise specialized late-splitting intersister connections or snaps. Sister snap loci separate simultaneously in both snap regions, concomitant with a major global nucleoid reorganization that results in emergence of a bilobed nucleoid morphology. Split snap loci move rapidly apart to a separation distance comparable with one-half the length of the nucleoid. Concomitantly, at already split positions, sister loci undergo further separation to a comparable distance. The overall consequence of these and other effects is that thus far replicated sister chromosomes become spatially separated (individualized) into the two nucleoid lobes, while the terminus region (and likely, all unreplicated portions of the chromosome) moves to midcell. These and other findings imply that segregation of Escherichia coli sister chromosomes is not a smooth continuous process but involves at least one and likely, two major global transition(s). The presented patterns further suggest that accumulation of internal intranucleoid forces and constraining of these forces by snaps play central roles in global chromosome dynamics. They are consistent with and supportive of our previous proposals that individualization of sisters in E. coli is driven primarily by internally generated pushing forces and is directly analogous to sister individualization at the prophase to prometaphase transition of the eukaryotic cell cycle.E. coli chromosome | chromosome segregation | bacterial nucleoid I n bacteria, sister chromosomes segregate concomitant with DNA replication. We previously examined sister chromosome relationships over time in the Escherichia coli cell cycle (1). High temporal resolution (5-10 min) was achieved using synchronous populations (2). Sister relationships at individual loci were evaluated as in other studies. Also, uniquely, at a larger scale, whole nucleoid disposition and morphology were defined. This analysis identified a discrete transition, occurring part way through the replication process, in which the nucleoid becomes bilobed. This morphological change is accompanied by reciprocal repositioning of the replication origin (oriC) and terminus region (ter) and by strongly delayed splitting of one particular locus located near the replication origin (gln). These coordinate effects pointed to global reorganization of the nucleoid. We proposed that this reorganization resulted in spatial segregation of sister chromosomes and that it is analogous to the prophase to prometaphase transition of the eukaryotic cell cycle. We further proposed that, in both cases, sister individualization results from internally generated forces, more specifically, mechanical pushing effects (1, 3).Other models for seg...

Persistent super-diffusive motion of Escherichia coli chromosomal loci

“…Such effects were first reported by Fisher and co-workers 13,16 , who, for example, observed that the splitting of the gln locus is accompanied by rapid, asymmetric separation of sister loci, indicating that the basis for the movement is accumulated stress within the nucleoid. More recently, they proposed that programmed intranucleoid 'tethers' (for example, by nucleoidassociated bridging proteins like H-NS or Fis, or by condensins like MukB), accumulate in inter-segment linkages, which, in combination with changes in supercoiling state, tend to put the chromosome in an unfavourable condition, promoting eventual catastrophic release of tethers.…”

“…Additionally, such global rearrangements are expected to also affect loci that are not within the main moving domains. Considering the average trajectories of the 1-min frame separation data, we did not find direct evidence of the discrete transitions in the cell cycle 13,16 , likely because our technique, . Cells were imaged and segmented every minute, in order to reconstruct the segregation cycle, and the data were synchronized by the time at which the replicated sister loci were resolved optically (origin of times in these plots).…”

“…11). Equally, Ori2 is within the domain that is affected by programmed release of oriC sisters 16,22 and ensuing replisome splitting, which occurs a few minutes later. The motions observed at Ori2 could reflect these effects.…”

“…They can be connected to a set of different reported phenomena, some of which may be more or less frequent for different chromosomal regions, or even loci-specific. First, RCMs could be related to 'snaps' 13,16 associated with the segregation dynamics. For example, at the so-called 'T2' transition, one sister domain is reported to move half the length of the cell in a few minutes.…”

“…If segregation were a smooth process, it would appear as a drift over the underlying short-timescale subdiffusion. However, it has been reported that the spatial separation of sister chromosomes is not a continuous process but rather occurs as a small number of rapid chromosomal rearrangements 16,17 . This behaviour suggests the existence of energetic or entropic barriers for separation, likely in interplay with active force-generating processes.…”