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ABSTRACT The chromosomal DNA ofEscherichia coliis approximately a thousand times longer than the linear dimensions of the cell it occupies. Nevertheless, it fills only about one-half of the cytosolic volume of the cell. The volume pervaded by the chromosomal DNA is known as nucleoid. The nucleoid is a ribosome-depleted region that behaves as a distinct liquid-like phase within the cytosol. In most bacteria, includingE. coli, which lack membrane-enclosed organelles, the phase separation between the nucleoid and the ribosome-rich cytosolic fraction represents the most prominent organizational principle of the cell’s cytosolic interior. This review explores the mechanisms driving nucleoid phase separation, including the roles of DNA-binding proteins, supercoiling, and active DNA looping. Recent studies highlight macromolecular crowding as the dominant factor governing this spatial organization. The main focus of this review is on experimental and theoretical works—ranging fromin vitroandin vivostudies to polymer physics-based models—that elucidate how macromolecular crowding drives nucleoid phase formation and regulates DNA compactionin E. coli. 

Abstract The mechanisms by which two sister chromosomes separate and partition into daughter cells in bacteria remain poorly understood. A recent theoretical model has proposed that out-of-equilibrium processes associated with mRNA–ribosome (polysome) dynamics play a significant role in this process. Here we investigate the role of ribosomal dynamics on nucleoid segregation and separation inEscherichia coliusing high-throughput fluorescence microscopy in microfluidic devices. We compare our experimental observations with predictions from a reaction-diffusion model that includes the interactions among ribosomal subunits, polysomes, and chromosomal DNA. Our results show that the non-equilibrium behavior of mRNA and ribosomes causes them to aggregate at the midcell and this process contributes to the separation of the two daughter chromosomes. However, this effect is considerably weaker than that predicted by the model. Rather than relying solely on active mRNA–ribosome dynamics, our data suggest that the closing division septum via steric interactions and potentially entropic forces between two DNA strands coupled to cell elongation act as additional mechanisms to ensure faithful partitioning of the nucleoids to two daughter cells. SignificanceThe mitotic spindle separates chromosomes in eukaryotic cells, but bacteria lack this structure. It remains unclear how bacterial chromosomes partition prior to cell division. It has been hypothesized that non-equilibrium dynamics of polysomes, that is mRNA–ribosome complexes, actively drive the separation of bacterial chromosomes. Using quantitative microscopy combined with computational modeling, we show that polysome dynamics significantly contribute to chromosome segregation inEscherichia colibut this process does not constitute the sole mechanism. Our findings suggest the closing division septum via steric interactions and potentially entropic forces between two DNA strands act as additional mechanisms. 

The mechanisms by which two sister chromosomes separate and partition into daughter cells in bacteria remain poorly understood. A recent theoretical model has proposed that out-of-equilibrium central dogma reactions involving mRNA and ribosomes play a significant role in this process. Here, we test this idea in theEscherichia colimodel system using high-throughput fluorescence microscopy in microfluidic devices. We compare our experimental observations with predictions from a reaction–diffusion model that includes central dogma-related reactions and excluded volume interactions between ribosomal subunits, polysomes, and chromosomal DNA. Our results show that the nonequilibrium reactions of ribosomes cause them to aggregate at the midcell, and this process facilitates the separation of the two daughter chromosomes. However, the observed effects are weaker in live cells than our one-dimensional reaction–diffusion model predicts. Rather than relying solely on active mRNA–ribosome dynamics, our data suggest that the closing division septum via steric interactions and potentially entropic forces between two DNA strands coupled to cell elongation act as additional mechanisms to ensure faithful partitioning of the nucleoids to two daughter cells. 

Data Analysis Codes for article: Determining the rate-limiting processes for cell division in Escherichia coli; (Nat. Comm. 2024)