More Like this

1. Generating Chromosome Geometries in a Minimal Cell From Cryo-Electron Tomograms and Chromosome Conformation Capture Maps

   https://doi.org/10.3389/fmolb.2021.644133  

   Gilbert, Benjamin R.; Thornburg, Zane R.; Lam, Vinson; Rashid, Fatema-Zahra M.; Glass, John I.; Villa, Elizabeth; Dame, Remus T.; Luthey-Schulten, Zaida (July 2021, Frontiers in Molecular Biosciences)

   null (Ed.)

   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. 

   more » « less
2. Traditional protocols and optimization methods lead to absent expression in a mycoplasma cell-free gene expression platform

   https://doi.org/10.1093/synbio/ysac008  

   Sakai, Andrei; Deich, Christopher R; Nelissen, Frank H; Jonker, Aafke J; Bittencourt, Daniela M; Kempes, Christopher P; Wise, Kim S; Heus, Hans A; Huck, Wilhelm T; Adamala, Katarzyna P; et al (February 2022, Synthetic Biology)

   Abstract Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a Mycoplasma bacterium-based CFE system using lysates of the genome-minimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative Mycoplasma capricolum (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of Escherichia coli. Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of in vitro transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease (RNase) activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNase activity yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned E. coli cell-free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNase activity. However, it is not clear which gene encodes the RNase. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field. Graphical Abstract 

   more » « less
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. 

   more » « less
4. Molecular dynamics simulation of an entire cell

   https://doi.org/10.3389/fchem.2023.1106495  

   Stevens, Jan A.; Grünewald, Fabian; van Tilburg, P. A.; König, Melanie; Gilbert, Benjamin R.; Brier, Troy A.; Thornburg, Zane R.; Luthey-Schulten, Zaida; Marrink, Siewert J. (January 2023, Frontiers in Chemistry)

   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. 

   more » « less
5. Metabolite Damage and Damage Control in a Minimal Genome

   https://doi.org/10.1128/mbio.01630-22  

   Haas, Drago; Thamm, Antje M.; Sun, Jiayi; Huang, Lili; Sun, Lijie; Beaudoin, Guillaume A.; Wise, Kim S.; Lerma-Ortiz, Claudia; Bruner, Steven D.; Breuer, Marian; et al (July 2022, mBio)

   Johnson, Michael David (Ed.)

   ABSTRACT Analysis of the genes retained in the minimized Mycoplasma JCVI-Syn3A genome established that systems that repair or preempt metabolite damage are essential to life. Several genes known to have such functions were identified and experimentally validated, including 5-formyltetrahydrofolate cycloligase, coenzyme A (CoA) disulfide reductase, and certain hydrolases. Furthermore, we discovered that an enigmatic YqeK hydrolase domain fused to NadD has a novel proofreading function in NAD synthesis and could double as a MutT-like sanitizing enzyme for the nucleotide pool. Finally, we combined metabolomics and cheminformatics approaches to extend the core metabolic map of JCVI-Syn3A to include promiscuous enzymatic reactions and spontaneous side reactions. This extension revealed that several key metabolite damage control systems remain to be identified in JCVI-Syn3A, such as that for methylglyoxal. IMPORTANCE Metabolite damage and repair mechanisms are being increasingly recognized. We present here compelling genetic and biochemical evidence for the universal importance of these mechanisms by demonstrating that stripping a genome down to its barest essentials leaves metabolite damage control systems in place. Furthermore, our metabolomic and cheminformatic results point to the existence of a network of metabolite damage and damage control reactions that extends far beyond the corners of it that have been characterized so far. In sum, there can be little room left to doubt that metabolite damage and the systems that counter it are mainstream metabolic processes that cannot be separated from life itself. 

   more » « less