Direct imaging of the circular chromosome in a live bacterium

Abstract

Although the physical properties of chromosomes, including their morphology, mechanics, and dynamics are crucial for their biological function, many basic questions remain unresolved. Here we directly image the circular chromosome in live E. coli with a broadened cell shape. We find that it exhibits a torus topology with, on average, a lower-density origin of replication and an ultrathin flexible string of DNA at the terminus of replication. At the single-cell level, the torus is strikingly heterogeneous, with blob-like Mbp-size domains that undergo major dynamic rearrangements, splitting and merging at a minute timescale. Our data show a domain organization underlying the chromosome structure of E. coli , where MatP proteins induce site-specific persistent domain boundaries at Ori/Ter, while transcription regulators HU and Fis induce weaker transient domain boundaries throughout the genome. These findings provide an architectural basis for the understanding of the dynamic spatial organization of bacterial genomes in live cells.

Direct observation of independently moving replisomes in Escherichia coli

“…4b-d). We found that the Left-Ori-Right orientation of chromosomes that was reported earlier in rod-shaped E.coli cells 21,22,23 was preserved in these widened cell too. We observed the origin of replication of single nucleoids to duplicate near the midcell location ( Fig.…”

“…Since MreB is essential for maintaining the rod shape of E.coli cells 19 , the addition of A22 (which inhibits the MreB polymerisation) results in cells that are significantly wider than normal rod-shaped cells 10,2021 . Our earlier work showed that cells treated with A22 stay physiologically healthy and active, while the single nucleoid adopts a toroidal configuration within the larger available cell volume 21 . Importantly, these cells allow to follow the dynamics of the replisomes and the nucleoid upon re-initiating replication.…”

“…To better understand the nucleoid rearrangements, in particular the segregation of chrosmomes, in the replication process during the cell cycle, we imaged an E.coli strain with a HU-mYpet labelled chromosome and ori1 and ter3 FROS arrays 21 with widefield and SIM microscopy ( Fig. 4;…”

The SMC complex, MukBEF, organizes theEscherichia colichromosome by forming an axial core

“…S7). This is consistent with the observation that different genetic markers in ter can localize to distant regions of the same cell (17), with Hi-C data (18), and with analysis of cells with increased volume (16). We summarize the dimensions of MukBEF axial filaments in Fig.…”

“…MukBEF axial cores were also observed in rifampicin-treated cells (reduced molecular crowding) and in cells of increased volume after treatment with A22 ( Fig. S4) (16).…”