title: A mechanistic understanding of bacterial chromosome organisation
title: Ein mechanistisches Verständnis der bakteriellen Chromosomenorganisation
titleType: AlternativeTitle
Creators: name: Sadhir, Ismath
Publisher: name: Philipps-Universität Marburg
Year: 2023
Subjects: Life sciences
Biowissenschaften, Biologie
Chromosome organisation
microfluidics
Escherichia coli
origin of replication
terminus of replication

description: Chromosomes serve as repositories of genetic information, crucial to the functionality of all living organisms. Bacteria, in their simplicity, offer an ideal model to investigate the intricacies of chromosomal dynamics. Despite their lack of compartmentalisation, bacterial chromosomes display spatiotemporal organization like eukaryotic cells. However, understanding the mechanisms of bacterial chromosome organization and segregation, particularly in the extensively studied model organism, Escherichia coli, remains incomplete.
In E. coli, like most bacteria, the origin of replication is the first part of the chromosome to be replicated and segregated. While centromere-like sequences aiding origin segregation have been identified in other bacteria, none have been discovered in E. coli. The SMC complex, which the origin is attracted to and co-localizes with throughout the cell cycle, has been identified as important for positioning and segregation. However, a sequence identity facilitating this interaction has yet to be determined.
Our study began with comprehensive genomic screening to identify sequences that might aid origin positioning in E. coli. Our hypothesis was motivated by the observation that specific chromosomal regions, when present within unstable low-copy plasmids, lead to their active positioning and maintenance within the population. We found that two loci in the E. coli genome, near the spoT and seqA genes, confer a moderate degree of stability to an unstable plasmid. The stability effect of the spoT locus is strain-specific, while the seqA locus demonstrates stability that is consistent across different strains. Due to time constraints, the precise mechanism for the latter remains unidentified. Our findings at this stage support the established view of the absence of a centromere-like sequence for origin positioning in the E. coli genome.
While the origin of replication has been the focus of many studies, the terminus region—the last chromosomal segment to undergo replication and segregation—also plays a vital role in successful completion of cell cycle, ensuring the equal distribution of genetic material between daughter cells. In slow-growing E. coli cells, the terminus region transitions from the new pole at birth to the midcell during the cell cycle. Unlike other chromosomal regions, it maintains the midcell positioning for the majority of the cell cycle, even after its duplication. Understanding the behavior of the terminus region can provide insights into the broader aspects of bacterial chromosome organization and segregation.
Using a high-throughput single-cell approach, we tracked tens of thousands of cell cycles to quantitatively analyse the transition of the terminus region from the new pole to midcell and investigated its relationship to various cell cycle events. We found that terminus centralisation, a rapid discrete event, is closely associated with the completion of origin segregation, even in the absence of its linkage to the divisome. This revealed a previously unexplored relationship between origin and terminus. Additionally, we found that E. coli exhibits a longitudinal-like chromosome organization even under slow growth conditions.
In conclusion, our research significantly advances our understanding of chromosome organization in E. coli and paves the way for the application of our methodologies to studying chromosomal organization in other bacterial species.