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Possessing only essential genes, a minimal cell can reveal mechanisms and processes that are critical for the persistence and stability of life^1,2. Here we report on how an engineered minimal cell^3,4contends with the forces of evolution compared with theMycoplasma mycoidesnon-minimal cell from which it was synthetically derived. Mutation rates were the highest among all reported bacteria, but were not affected by genome minimization. Genome streamlining was costly, leading to a decrease in fitness of greater than 50%, but this deficit was regained during 2,000 generations of evolution. Despite selection acting on distinct genetic targets, increases in the maximum growth rate of the synthetic cells were comparable. Moreover, when performance was assessed by relative fitness, the minimal cell evolved 39% faster than the non-minimal cell. The only apparent constraint involved the evolution of cell size. The size of the non-minimal cell increased by 80%, whereas the minimal cell remained the same. This pattern reflected epistatic effects of mutations inftsZ, which encodes a tubulin-homologue protein that regulates cell division and morphology^5,6. Our findings demonstrate that natural selection can rapidly increase the fitness of one of the simplest autonomously growing organisms. Understanding how species with small genomes overcome evolutionary challenges provides critical insights into the persistence of host-associated endosymbionts, the stability of streamlined chassis for biotechnology and the targeted refinement of synthetically engineered cells^2,7–9.

 

The bacterial strain JCVI-syn3.0 stands as the first example of a living organism with a minimized synthetic genome, derived from the Mycoplasma mycoides genome and chemically synthesized in vitro. Here, we report the experimental evolution of a syn3.0- derived strain. Ten independent replicates were evolved for several hundred generations, leading to growth rate improvements of > 15%. Endpoint strains possessed an average of 8 mutations composed of indels and SNPs, with a pronounced C/G- > A/T transversion bias. Multiple genes were repeated mutational targets across the independent lineages, including phase variable lipoprotein activation, 5 distinct; nonsynonymous substitutions in the same membrane transporter protein, and inactivation of an uncharacterized gene. Transcriptomic analysis revealed an overall tradeoff reflected in upregulated ribosomal proteins and downregulated DNA and RNA related proteins during adaptation. This work establishes the suitability of synthetic, minimal strains for laboratory evolution, providing a means to optimize strain growth characteristics and elucidate gene functionality. 

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.

 

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. 

This article written in the popular journal The New Yorker takes a look at how the world's first minimal bacterial cell, which was made by a team from the J. Craig Venter Institute (JCVI) in 2016 has been used by both JCVI biologists and many other research groups to investigate the first principles of cellular life. The author, James Somers, writes much of the article using information obtained in his extended interview with JCVI scientist and minimal cell creator John Glass. The article introduces the public to ideas in synthetic biology, evolution, and the history of modern biology.