RESUMEN

Computational models of cells cannot be considered complete unless they include the most fundamental process of life, the replication of genetic material. In a recent study, we presented a computational framework to model systems of replicating bacterial chromosomes as polymers at 10 bp resolution with Brownian dynamics. This approach was used to 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 modeled the chromosome as a self-avoiding homopolymer with bending and torsional stiffnesses that capture the essential mechanical properties of dsDNA in Syn3A. Additionally, the polymer interacts with ribosomes distributed according to cryo-electron tomograms of Syn3A. The polymer model was further augmented by computational models of loop extrusion by structural maintenance of chromosomes (SMC) protein complexes and topoisomerase action, and the modeling and analysis of multi-fork replication states.

Asunto(s)

Cromosomas Bacterianos , Replicación del ADN , Cromosomas Bacterianos/genética , ADN Bacteriano/genética , Bacterias/genética

RESUMEN

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 calculate in silico chromosome 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.

RESUMEN

The ultimate microscope, directed at a cell, would reveal the dynamics of all the cell's components with atomic resolution. In contrast to their real-world counterparts, computational microscopes are currently on the brink of meeting this challenge. In this perspective, we show how an integrative approach can be employed to model an entire cell, the minimal cell, JCVI-syn3A, at full complexity. This step opens the way to interrogate the cell's spatio-temporal evolution with molecular dynamics simulations, an approach that can be extended to other cell types in the near future.

RESUMEN

A complete description of the state of the cell requires knowledge of its size, shape, components, intracellular reactions, and interactions with its environment-all of these as a function of time and cell growth. Adding to this list is the need for theoretical models and simulations that integrate and help to interpret this daunting amount of experimental data. It seems like an overwhelming list of requirements, but progress is being made on many fronts. In this review, we discuss the current challenges and problems in obtaining sufficient information about each aspect of a dynamical whole-cell model (DWCM) for simple and well-studied bacterial systems.

Asunto(s)

Modelos Teóricos , Estructura Molecular

RESUMEN

We present a whole-cell fully dynamical kinetic model (WCM) of JCVI-syn3A, a minimal cell with a reduced genome of 493 genes that has retained few regulatory proteins or small RNAs. Cryo-electron tomograms provide the cell geometry and ribosome distributions. Time-dependent behaviors of concentrations and reaction fluxes from stochastic-deterministic simulations over a cell cycle reveal how the cell balances demands of its metabolism, genetic information processes, and growth, and offer insight into the principles of life for this minimal cell. The energy economy of each process including active transport of amino acids, nucleosides, and ions is analyzed. WCM reveals how emergent imbalances lead to slowdowns in the rates of transcription and translation. Integration of experimental data is critical in building a kinetic model from which emerges a genome-wide distribution of mRNA half-lives, multiple DNA replication events that can be compared to qPCR results, and the experimentally observed doubling behavior.

Asunto(s)

Células/citología , Simulación por Computador , Adenosina Trifosfato/metabolismo , Ciclo Celular/genética , Proliferación Celular/genética , Células/metabolismo , Replicación del ADN/genética , Regulación de la Expresión Génica , Imagenología Tridimensional , Cinética , Lípidos/química , Redes y Vías Metabólicas , Metaboloma , Anotación de Secuencia Molecular , Nucleótidos/metabolismo , Termodinámica , Factores de Tiempo

RESUMEN

JCVI-syn3A is a genetically minimal bacterial cell, consisting of 493 genes and only a single 543 kbp circular chromosome. Syn3A's genome and physical size are approximately one-tenth those of the model bacterial organism Escherichia coli's, and the corresponding reduction in complexity and scale provides a unique opportunity for whole-cell modeling. Previous work established genome-scale gene essentiality and proteomics data along with its essential metabolic network and a kinetic model of genetic information processing. In addition to that information, whole-cell, spatially-resolved kinetic models require cellular architecture, including spatial distributions of ribosomes and the circular chromosome's configuration. We reconstruct cellular architectures of Syn3A cells at the single-cell level directly from cryo-electron tomograms, including the ribosome distributions. We present a method of generating self-avoiding circular chromosome configurations in a lattice model with a resolution of 11.8 bp per monomer on a 4 nm cubic lattice. Realizations of the chromosome configurations are constrained by the ribosomes and geometry reconstructed from the tomograms and include DNA loops suggested by experimental chromosome conformation capture (3C) maps. Using ensembles of simulated chromosome configurations we predict chromosome contact maps for Syn3A cells at resolutions of 250 bp and greater and compare them to the experimental maps. Additionally, the spatial distributions of ribosomes and the DNA-crowding resulting from the individual chromosome configurations can be used to identify macromolecular structures formed from ribosomes and DNA, such as polysomes and expressomes.