Four-Dimensional Imaging of E. coli Nucleoid Organization and Dynamics in Living Cells

Abstract

Visualization of living E. coli nucleoids, defined by HupA-mCherry, reveals a discrete, dynamic helical ellipsoid. Three basic features emerge. (i) Nucleoid density efficiently coalesces into longitudinal bundles, giving a stiff, low DNA density ellipsoid. (ii) This ellipsoid is radially confined within the cell cylinder. Radial confinement gives helical shape and drives and directs global nucleoid dynamics, including sister segregation. (iii) Longitudinal density waves flux back and forth along the nucleoid, with 5–10% of density shifting within 5s, enhancing internal nucleoid mobility. Furthermore, sisters separate end-to-end in sequential discontinuous pulses, each elongating the nucleoid by 5–15%. Pulses occur at 20min intervals, at defined cell cycle times. This progression is mediated by sequential installation and release of programmed tethers, implying cyclic accumulation and relief of intra-nucleoid mechanical stress. These effects could comprise a chromosome-based cell cycle engine. Overall, the presented results suggest a general conceptual framework for bacterial nucleoid morphogenesis and dynamics.

Persistent super-diffusive motion of Escherichia coli chromosomal loci

“…Second, it is interesting to compare our observations with another recent finding of Fisher et al 13 : 'density waves' where a consistent fraction of the nucleoid mass is relocated in a few seconds. Some RCMs might be related to this phenomenon, since the crossover from subdiffusion to RCMs occurs around 10 s, in agreement with the timescale of 5 s emerging from the density waves and RCMs for some loci are consistent with a net average flow ( Fig.…”

“…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).…”

“…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.…”

“…This behaviour suggests the existence of energetic or entropic barriers for separation, likely in interplay with active force-generating processes. A recent imaging study 13 revealed further intriguing nucleoid density dynamics, measured by the dynamic intensity of a fluorescent fusion of the nucleoid-associated protein HU. Longitudinal 'density waves' appear to travel along the nucleoid, causing 5-10% density changes within 5 s. The same study found that sister chromosomes separate by a series of sequential discontinuous pulses, each elongating the nucleoid by 5-15%.…”

“…The cytosol is a highly crowded environment, which promotes the chromosome to be (semi-)collapsed due to osmotic self-attraction 9,10 . However, in vivo the compacted chromosome (nucleoid) maintains a well-defined shape and tends to occupy a significantly smaller volume than the cell, likely a result of the activity of numerous DNA-associated proteins with varying functionality [11][12][13][14] . The average mobility of loci is a function of chromosomal coordinate, suggesting spatially varying physical organization and/or noise levels due to transcription, translation or protein activity 4 .…”