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1. The role of active mRNA–ribosome dynamics and closing constriction in daughter chromosome separation in Escherichia coli

   https://doi.org/10.1073/pnas.2508100122  

   Amarasinghe, Chathuddasie I; Chang, Mu-Hung; Männik, Jaana; Retterer, Scott T; Lavrentovich, Maxim O; Männik, Jaan (November 2025, Proceedings of the National Academy of Sciences)

   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. 

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2. Motorized chromosome models of mitotic chromosome folding

   https://doi.org/10.1038/s41467-025-66025-y  

   Cao, Zhiyu; Du, Chaoqun; Hou, Zhonghuai; Wolynes, Peter G (December 2025, Nature Communications)

   During mitosis, near-spherical chromosomes reconfigure into rod-like structures to ensure their accurate segregation to daughter cells. We explore here, the interplay between the nonequilibrium activity of molecular motors in determining the chromosomal organization in mitosis and its characteristic symmetry-breaking events. We present a hybrid motorized chromosome model that highlights the distinct roles of condensin I and II in shaping mitotic chromosomes. Guided by experimental observations, the simulations suggest that condensin II facilitates large-scale scaffold formation, while condensin I is paramount in local helical loop arrangement. Together, these two distinct grappling motors establish the hierarchical helical structure characteristic of mitotic chromosomes, which exhibit striking local and, sometimes global, chirality and contribute to the robust mechanical properties of mitotic chromosomes. Accompanying the emergence of rigidity, the model provides mechanisms of forming defects, including perversions and entanglements, and shows how these may be partially resolved through condensin activity and topoisomerase action. This framework bridges coarse-grained energy landscape models of chromosome dynamics and non-equilibrium molecular dynamics, advancing the understanding of chromosome organization during cell division and beyond. 

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3. Dynamics of chromosome organization in a minimal bacterial cell

   https://doi.org/10.3389/fcell.2023.1214962  

   Gilbert, Benjamin R.; Thornburg, Zane R.; Brier, Troy A.; Stevens, Jan A.; Grünewald, Fabian; Stone, John E.; Marrink, Siewert J.; Luthey-Schulten, Zaida (August 2023, Frontiers in Cell and Developmental Biology)

   Computational models of cells cannot be considered complete unless they include the most fundamental process of life, the replication and inheritance of genetic material. By creating a computational framework to model systems of replicating bacterial chromosomes as polymers at 10 bp resolution with Brownian dynamics, we investigate changes in chromosome organization during replication and extend the applicability of an existing whole-cell model (WCM) for a genetically minimal bacterium, JCVI-syn3A, to the entire cell-cycle. To achieve cell-scale chromosome structures that are realistic, we model the chromosome as a self-avoiding homopolymer with bending and torsional stiffnesses that capture the essential mechanical properties of dsDNA in Syn3A. In addition, the conformations of the circular DNA must avoid overlapping with ribosomes identitied in cryo-electron tomograms. While Syn3A lacks the complex regulatory systems known to orchestrate chromosome segregation in other bacteria, its minimized genome retains essential loop-extruding structural maintenance of chromosomes (SMC) protein complexes (SMC-scpAB) and topoisomerases. Through implementing the effects of these proteins in our simulations of replicating chromosomes, we find that they alone are sufficient for simultaneous chromosome segregation across all generations within nested theta structures. This supports previous studies suggesting loop-extrusion serves as a near-universal mechanism for chromosome organization within bacterial and eukaryotic cells. Furthermore, we analyze ribosome diffusion under the influence of the chromosome and calculatein silicochromosome contact maps that capture inter-daughter interactions. Finally, we present a methodology to map the polymer model of the chromosome to a Martini coarse-grained representation to prepare molecular dynamics models of entire Syn3A cells, which serves as an ultimate means of validation for cell states predicted by the WCM. 

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4. The role of macromolecular crowders in the formation and compaction of the Escherichia coli nucleoid

   https://doi.org/10.1128/ecosalplus.esp-0002-2024  

   Männik, Jaan; Männik, Jaana; Amarasinghe, Chathuddasie; Chang, Mu-Hung; Lavrentovich, Maxim O (December 2025, EcoSal Plus)

   Espeli, Olivier (Ed.)

   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. 

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5. Role of Ribosomal Protein bS1 in Orthogonal mRNA Start Codon Selection

   https://doi.org/10.1021/acs.biochem.4c00688  

   Boyko, Kristina V; Bernstein, Rebecca A; Kim, Minji; Cate, Jamie HD (February 2025, Biochemistry)

   In many bacteria, the location of the mRNA start codon is determined by a short ribosome binding site sequence that base pairs with the 3′-end of 16S rRNA (rRNA) in the 30S subunit. Many groups have changed these short sequences, termed the Shine–Dalgarno (SD) sequence in the mRNA and the anti-Shine–Dalgarno (ASD) sequence in 16S rRNA, to create “orthogonal” ribosomes to enable the synthesis of orthogonal polymers in the presence of the endogenous translation machinery. However, orthogonal ribosomes are prone to SD-independent translation. Ribosomal protein bS1, which binds to the 30S ribosomal subunit, is thought to promote translation initiation by shuttling the mRNA to the ribosome. Thus, a better understanding of how the SD and bS1 contribute to start codon selection could help efforts to improve the orthogonality of ribosomes. Here, we engineered the Escherichia coli ribosome to prevent binding of bS1 to the 30S subunit and separate the activity of bS1 binding to the ribosome from the role of the mRNA SD sequence in start codon selection. We find that ribosomes lacking bS1 are slightly less active than wild-type ribosomes in vitro. Furthermore, orthogonal 30S subunits lacking bS1 do not have an improved orthogonality. Our findings suggest that mRNA features outside the SD sequence and independent of binding of bS1 to the ribosome likely contribute to start codon selection and the lack of orthogonality of present orthogonal ribosomes. 

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