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The Escherichia coli chromosome is a dynamic molecule, exhibiting choreographed reorganizations across spatial scales. The chromosome is involved in a number of essential processes, such as DNA replication and chromosome segregation. These processes ensure that there are at least two copies of the genetic material at cell division, one for each daughter cell to inherit. However, maintaining a cycle-dependent chromosome organization is no small feat. To understand how the chromosome organization is regulated over the cell cycle and to understand the functional importance of the chromosome structure, we investigated the dynamics and intracellular positioning of various chromosomal loci in live E. coli using fluorescence microscopy. 

Our efforts to understand when and where in the cell chromosomal loci are replicated were based on fluorescently labeling a chromosomal locus and a subunit of the replisome in the same cell. With this labeling strategy, we followed the intracellular positioning of the replisome and various loci relative to each other, as well as their short-time-scale movements. We found that as loci were replicated their short-time-scale movements slowed down momentarily. Mapping the short-time-scale movements over different intracellular positions showed a clear repositioning of several loci towards the replisome to be replicated, which led us to conclude that the chromosome moves to the replisome during DNA replication. 

To investigate the three-dimensional positioning of chromosomal loci, we performed time-lapse imaging of E. coli strains with fluorescently labeled loci using a microscope with an astigmatic fluorescence emission path. To determine the 3D coordinates of the emitters, we developed a neural network-based algorithm trained on simulated images of E. coli cells with fluorescent foci. Applying this neural network to different loci showed distinct 3D localization patterns over the cell cycle. 

To study the 3D chromosome organization, we imaged a collection of 83 E. coli strains, each with a different locus label. Using in situ genotyping of barcode sequences to determine each strain’s identity, we mapped 3D localization phenotypes to multiple chromosomal loci in a single experiment. We captured known longitudinal chromosome reorganization, as well as radial localization patterns that had not been observed previously. Finally, we used the experimental location distributions to inform a polymer model of the chromosome, which showed how Mbp-sized domains form dynamically over the cell cycle. Using this approach to study the 3D chromosome organization in live E. coli, we hope to gain further insights into the regulation and functional importance of the chromosome structure.