Cell Boundary Confinement Sets the Size and Position of the E. coli Chromosome

Abstract

22While the spatiotemporal structure of the genome is crucial to its biological function, many basic questions 23 remain unanswered on the morphology and segregation of chromosomes. Here, we experimentally show in 24 Escherichia coli that spatial confinement plays a dominant role in determining both the chromosome size 25 and position. In non-dividing cells with lengths up to 10 times normal, single chromosomes are observed 26 to expand more than 4 fold in size, an effect only modestly influenced by deletions of various nucleoid-27 associated proteins. Chromosomes show pronounced internal dynamics but exhibit a robust positioning 28 where single nucleoids reside strictly at mid-cell, while two nucleoids self-organize at ¼ and ¾ cell 29 positions. Molecular dynamics simulations of model chromosomes recapitulate these phenomena and 30 indicate that these observations can be attributed to depletion effects induced by cytosolic crowders. These 31 findings highlight boundary confinement as a key causal factor that needs to be considered for 32 understanding chromosome organization. 33 34 Key words 35 36

Direct observation of independently moving replisomes in Escherichia coli

“…As seen in Fig. 2a-c, cells placed at 40ºC maintained a single chromosome positioned at the center of the cell, as previously observed 16 . The intensity of the nucleoids was not homogeneous but showed a remarkable heterogeneity with a pronounced helical-like structure, as reported by Fisher et al 14 .…”

“…Cells can maintain a single chromosome, elongate, and reach longer sizes compared to normal rod-shaped cells ( Supplementary Fig. 1), with accordingly larger nucleoid sizes 16 , overcoming the diffraction limit to resolve separate foci. We use well-established monomeric fluorophores that avoid the problem that replisome co-localization may result due to attracting forces between fluorescent tags.…”

Cell morphology and nucleoid dynamics in dividing D. radiodurans

“…Many spatial features have been proposed as key determinants of pole-to-pole oscillations of Min in E. coli, such as the highly negative membrane curvature at the poles 46 , the longest axis of confinement 47 As bacterial nucleoids fill a large fraction of the total cell volume, a common hypothesis suggests that the cell size and shape may play a major role in chromosome positioning and sizing. It was recently proposed that cell shape and size were 'sensed' indirectly by chromosomes via the pressure exerted by depletion forces induced by cytosolic crowders 8,50 , which would promote the compact shape and central localization of nucleoids in cells. In the case of D. radiodurans, several studies have described its nucleoid as being highly condensed [26][27][28][29][30] , but this visual impression is in part due to the relatively large size of D. radiodurans cells that have a mean volume of ~2.6 µm 3 .…”

“…However, recent studies now indicate that additional factors, such as molecular crowding and depletion forces, also play important roles in determining the volume of the cell occupied by the nucleoid [5][6][7] , and suggest that cell shape and size may critically influence nucleoid organization 7,8 .…”

The effects of polydisperse crowders on the compaction of the Escherichia coli nucleoid

“…Although several NAPs, which can bridge (H-NS) and bend (HU, Fis, IHF) chromosomal DNA, are abundantly present in E. coli during its lag-phase growth (for review see (Dame et al, 2011, Dillon andDorman, 2010)) they appear to affect chromosome conformations only at the local scale, in regions spanning less than 300 kb (Lioy et al, 2018). Moreover, the removal of these binding proteins from the cell one at a time does not change the nucleoid size (Wu et al, 2019, Wang et al, 2013. The exception is a low-abundance of MatP that anchors the replication terminus region to the divisome (Espeli et al, 2012, Männik et al, 2016 and conveys different organization to this ~800 kb long domain (Mercier et al, 2008, Lioy et al, 2018.…”