Adaptive evolution of a minimal organism with a synthetic genome.

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.

Metabolite Damage and Damage Control in a Minimal Genome.

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.

Traditional protocols and optimization methods lead to absent expression in a mycoplasma cell-free gene expression platform.

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.

Fundamental behaviors emerge from simulations of a living minimal cell.

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.

Genetic requirements for cell division in a genomically minimal cell.

Genomically minimal cells, such as JCVI-syn3.0, offer a platform to clarify genes underlying core physiological processes. Although this minimal cell includes genes essential for population growth, the physiology of its single cells remained uncharacterized. To investigate striking morphological variation in JCVI-syn3.0 cells, we present an approach to characterize cell propagation and determine genes affecting cell morphology. Microfluidic chemostats allowed observation of intrinsic cell dynamics that result in irregular morphologies. A genome with 19 genes not retained in JCVI-syn3.0 generated JCVI-syn3A, which presents morphology similar to that of JCVI-syn1.0. We further identified seven of these 19 genes, including two known cell division genes, ftsZ and sepF, a hydrolase of unknown substrate, and four genes that encode membrane-associated proteins of unknown function, which are required together to restore a phenotype similar to that of JCVI-syn1.0. This result emphasizes the polygenic nature of cell division and morphology in a genomically minimal cell.

Essential metabolism for a minimal cell.

JCVI-syn3A, a robust minimal cell with a 543 kbp genome and 493 genes, provides a versatile platform to study the basics of life. Using the vast amount of experimental information available on its precursor, Mycoplasma mycoides capri, we assembled a near-complete metabolic network with 98% of enzymatic reactions supported by annotation or experiment. The model agrees well with genome-scale in vivo transposon mutagenesis experiments, showing a Matthews correlation coefficient of 0.59. The genes in the reconstruction have a high in vivo essentiality or quasi-essentiality of 92% (68% essential), compared to 79% in silico essentiality. This coherent model of the minimal metabolism in JCVI-syn3A at the same time also points toward specific open questions regarding the minimal genome of JCVI-syn3A, which still contains many genes of generic or completely unclear function. In particular, the model, its comparison to in vivo essentiality and proteomics data yield specific hypotheses on gene functions and metabolic capabilities; and provide suggestions for several further gene removals. In this way, the model and its accompanying data guide future investigations of the minimal cell. Finally, the identification of 30 essential genes with unclear function will motivate the search for new biological mechanisms beyond metabolism.

One way that researchers can test whether they understand a biological system is to see if they can accurately recreate it as a computer model. The more they learn about living things, the more the researchers can improve their models and the closer the models become to simulating the original. In this approach, it is best to start by trying to model a simple system. Biologists have previously succeeded in creating 'minimal bacterial cells'. These synthetic cells contain fewer genes than almost all other living things and they are believed to be among the simplest possible forms of life that can grow on their own. The minimal cells can produce all the chemicals that they need to survive ­ in other words, they have a metabolism. Accurately recreating one of these cells in a computer is a key first step towards simulating a complete living system. Breuer et al. have developed a computer model to simulate the network of the biochemical reactions going on inside a minimal cell with just 493 genes. By altering the parameters of their model and comparing the results to experimental data, Breuer et al. explored the accuracy of their model. Overall, the model reproduces experimental results, but it is not yet perfect. The differences between the model and the experiments suggest new questions and tests that could advance our understanding of biology. In particular, Breuer et al. identified 30 genes that are essential for life in these cells but that currently have no known purpose. Continuing to develop and expand models like these to reproduce more complex living systems provides a tool to test current knowledge of biology. These models may become so advanced that they could predict how living things will respond to changing situations. This would allow scientists to test ideas sooner and make much faster progress in understanding life on Earth. Ultimately, these models could one day help to accelerate medical and industrial processes to save lives and enhance productivity.

Tuning Gene Activity by Inducible and Targeted Regulation of Gene Expression in Minimal Bacterial Cells.

Functional genomics studies in minimal mycoplasma cells enable unobstructed access to some of the most fundamental processes in biology. Conventional transposon bombardment and gene knockout approaches often fail to reveal functions of genes that are essential for viability, where lethality precludes phenotypic characterization. Conditional inactivation of genes is effective for characterizing functions central to cell growth and division, but tools are limited for this purpose in mycoplasmas. Here we demonstrate systems for inducible repression of gene expression based on clustered regularly interspaced short palindromic repeats-mediated interference (CRISPRi) in Mycoplasma pneumoniae and synthetic Mycoplasma mycoides, two organisms with reduced genomes actively used in systems biology studies. In the synthetic cell, we also demonstrate inducible gene expression for the first time. Time-course data suggest rapid kinetics and reversible engagement of CRISPRi. Targeting of six selected endogenous genes with this system results in lowered transcript levels or reduced growth rates that agree with lack or shortage of data in previous transposon bombardment studies, and now produces actual cells to analyze. The ksgA gene encodes a methylase that modifies 16S rRNA, rendering it vulnerable to inhibition by the antibiotic kasugamycin. Targeting the ksgA gene with CRISPRi removes the lethal effect of kasugamycin and enables cell growth, thereby establishing specific and effective gene modulation with our system. The facile methods for conditional gene activation and inactivation in mycoplasmas open the door to systematic dissection of genetic programs at the core of cellular life.

Minimal Cells-Real and Imagined.

A minimal cell is one whose genome only encodes the minimal set of genes necessary for the cell to survive. Scientific reductionism postulates the best way to learn the first principles of cellular biology would be to use a minimal cell in which the functions of all genes and components are understood. The genes in a minimal cell are, by definition, essential. In 2016, synthesis of a genome comprised of only the set of essential and quasi-essential genes encoded by the bacterium Mycoplasma mycoides created a near-minimal bacterial cell. This organism performs the cellular functions common to all organisms. It replicates DNA, transcribes RNA, translates proteins, undergoes cell division, and little else. In this review, we examine this organism and contrast it with other bacteria that have been used as surrogates for a minimal cell.

Design and synthesis of a minimal bacterial genome.

We used whole-genome design and complete chemical synthesis to minimize the 1079-kilobase pair synthetic genome of Mycoplasma mycoides JCVI-syn1.0. An initial design, based on collective knowledge of molecular biology combined with limited transposon mutagenesis data, failed to produce a viable cell. Improved transposon mutagenesis methods revealed a class of quasi-essential genes that are needed for robust growth, explaining the failure of our initial design. Three cycles of design, synthesis, and testing, with retention of quasi-essential genes, produced JCVI-syn3.0 (531 kilobase pairs, 473 genes), which has a genome smaller than that of any autonomously replicating cell found in nature. JCVI-syn3.0 retains almost all genes involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions. JCVI-syn3.0 is a versatile platform for investigating the core functions of life and for exploring whole-genome design.

Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling.

The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ∼ 10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.

Rescue of mutant fitness defects using in vitro reconstituted designer transposons in Mycoplasma mycoides.

With only hundreds of genes contained within their genomes, mycoplasmas have become model organisms for precise understanding of cellular processes, as well as platform organisms for predictable engineering of microbial functions for mission-critical applications. Despite the availability of "whole genome writing" in Mycoplasma mycoides, some traditional methods for genetic engineering are underdeveloped in mycoplasmas. Here we demonstrate two facile transposon-mediated approaches for introducing genes into the synthetic cell based on M. mycoides. The marker-less approach involves preparing a fragment containing only a small genomic region of interest with flanking transposase-binding sites, followed by in vitro transposase loading and introduction into the cells. The marker-driven approach involves cloning an open reading frame (ORF) of interest into a vector containing a marker for mycoplasma transformation, as well as sites for transposase loading and random genomic integration. An innovative feature of this construct is to use a single promoter to express the transformation marker and the introduced ORF. The marker-driven approach can be conveniently applied to any exogenous or synthetic gene without any information on the effect of the gene on the strain, whereas the marker-less approach requires that the fragment has a recognizable effect. Using the marker-less method, we found that a region containing the nusG gene rescues a slow growth phenotype of a strain containing a larger deletion encompassing this gene. Using the marker-driven approach, we better defined this finding, thereby establishing that nusG is required for a normal growth rate in synthetic M. mycoides. These methods are suitable for complementation tests to identify genes responsible for assorted functions lacking in deletion mutants. These approaches are also expected to facilitate rapid testing of various natural and engineered genes or gene clusters from numerous sources in M. mycoides.

Complete genome sequences of Mycoplasma leachii strain PG50T and the pathogenic Mycoplasma mycoides subsp. mycoides small colony biotype strain Gladysdale.

Mycoplasma mycoides subsp. mycoides small colony biotype (SC) is the high-consequence animal pathogen causing contagious bovine pleuropneumonia. We report the complete genome sequences of the pathogenic strain M. mycoides subsp. mycoides SC Gladysdale and a close phylogenetic relative, Mycoplasma leachii PG50(T), another bovine pathogen of the M. mycoides phylogenetic clade.

Complete genome sequence of Mycoplasma bovis type strain PG45 (ATCC 25523).

This complete and fully assembled genome sequence of Mycoplasma bovis type strain PG45 is the first available for this species and offers a framework for comparison with additional pathogenic isolates. The single circular chromosome of 1,003,404 bp reveals multiple gene sets and mechanisms involved in variable expression of surface antigens and the incursion of numerous and assorted mobile elements, despite its reduced size.

A versatile palindromic amphipathic repeat coding sequence horizontally distributed among diverse bacterial and eucaryotic microbes.

BACKGROUND: Intragenic tandem repeats occur throughout all domains of life and impart functional and structural variability to diverse translation products. Repeat proteins confer distinctive surface phenotypes to many unicellular organisms, including those with minimal genomes such as the wall-less bacterial monoderms, Mollicutes. One such repeat pattern in this clade is distributed in a manner suggesting its exchange by horizontal gene transfer (HGT). Expanding genome sequence databases reveal the pattern in a widening range of bacteria, and recently among eucaryotic microbes. We examined the genomic flux and consequences of the motif by determining its distribution, predicted structural features and association with membrane-targeted proteins. RESULTS: Using a refined hidden Markov model, we document a 25-residue protein sequence motif tandemly arrayed in variable-number repeats in ORFs lacking assigned functions. It appears sporadically in unicellular microbes from disparate bacterial and eucaryotic clades, representing diverse lifestyles and ecological niches that include host parasitic, marine and extreme environments. Tracts of the repeats predict a malleable configuration of recurring domains, with conserved hydrophobic residues forming an amphipathic secondary structure in which hydrophilic residues endow extensive sequence variation. Many ORFs with these domains also have membrane-targeting sequences that predict assorted topologies; others may comprise reservoirs of sequence variants. We demonstrate expressed variants among surface lipoproteins that distinguish closely related animal pathogens belonging to a subgroup of the Mollicutes. DNA sequences encoding the tandem domains display dyad symmetry. Moreover, in some taxa the domains occur in ORFs selectively associated with mobile elements. These features, a punctate phylogenetic distribution, and different patterns of dispersal in genomes of related taxa, suggest that the repeat may be disseminated by HGT and intra-genomic shuffling. CONCLUSIONS: We describe novel features of PARCELs (Palindromic Amphipathic Repeat Coding ELements), a set of widely distributed repeat protein domains and coding sequences that were likely acquired through HGT by diverse unicellular microbes, further mobilized and diversified within genomes, and co-opted for expression in the membrane proteome of some taxa. Disseminated by multiple gene-centric vehicles, ORFs harboring these elements enhance accessory gene pools as part of the "mobilome" connecting genomes of various clades, in taxa sharing common niches.

Distinctive repertoire of contingency genes conferring mutation- based phase variation and combinatorial expression of surface lipoproteins in Mycoplasma capricolum subsp. capricolum of the Mycoplasma mycoides phylogenetic cluster.

The generation of surface variation among many divergent species of Mollicutes (mycoplasmas) occurs through stochastic expression patterns of diverse lipoprotein genes. The size and wide distribution of such variable gene sets in minimal (approximately 0.6- to 1.4-Mb) mycoplasmal genomes suggest their key role in the adaptation and survival of these wall-less monoderms. Diversity through variable genes is less clearly established among phylogenetically similar mycoplasmas, such as the Mycoplasma mycoides cluster of ruminant pathogens, which vary widely in host range and pathobiology. Using (i) genome sequences from two members of this clade, Mycoplasma capricolum subsp. capricolum and M. mycoides subsp. mycoides small colony biotype (SC), (ii) antibodies to specific peptide determinants of predicted M. capricolum subsp. capricolum gene products, and (iii) analysis of the membrane-associated proteome of M. capricolum subsp. capricolum, a novel set of six genes (vmcA to vmcF) expressing distinct Vmc (variable M. capricolum subsp. capricolum) lipoproteins is demonstrated. These occur at two separate loci in the M. capricolum subsp. capricolum genome, which shares striking overall similarity and gene synteny with the M. mycoides subsp. mycoides SC genome. Collectively, Vmc expression is noncoordinate and combinatorial, subject to a single-unit insertion/deletion in a 5' flanking dinucleotide repeat that governs expression of each vmc gene. All vmc genes share modular regions affecting expression and membrane translocation. In contrast, vmcA to vmcD genes at one locus express surface proteins with highly structured size-variable repeating domains, whereas vmcE to vmcF genes express products with short repeats devoid of predicted structure. These genes confer a distinctive, dynamic surface architecture that may represent adaptive differences within this important group of pathogens as well as exploitable diagnostic targets.

Molecular genetic analysis of ICEF, an integrative conjugal element that is present as a repetitive sequence in the chromosome of Mycoplasma fermentans PG18.

Mycoplasma genomes contain compact gene sets that approach the minimal complement necessary for life and reflect multiple evolutionary instances of genomic reduction. Lateral gene transfer may play a critical role in shaping the mobile gene pool in these organisms, yet complex mobile elements have not been reported within this genus. We describe here a large ( approximately 23-kb) genetic element with unique features that is present in four copies in the Mycoplasma fermentans PG18 chromosome, accounting for approximately 8% of the genome. These novel elements, designated ICEF (integrative conjugal elements of M. fermentans), resemble conjugative, self-transmissible integrating elements (constins) in that circular, nonreplicative extrachromosomal forms occur in which the left and right termini of the integrated element are juxtaposed and separated by a coupling sequence derived from direct repeats flanking chromosomal copies of ICEF as a result of target site duplication. ICEF contain multiple similarly oriented open reading frames (ORFs), of which some have homology to products of known conjugation genes but others have no known counterparts. Surprisingly, unlike other constins, ICEF lack homologs of known integrases, transposases, or recombinases, suggesting that a novel enzyme may be employed for integration-excision. Skewed distribution and varied sites of chromosomal integration among M. fermentans isolates suggest a role for ICEF in promoting genomic and phenotypic variation in this species. Identification of homologs of terminal ICEF ORFs in two additional mycoplasma species indicates that ICEF is the prototype member of a family of ICE-related elements that may be widespread among pathogenic mycoplasmas infecting diverse vertebrate hosts.

Identification and functional mapping of the Mycoplasma fermentans P29 adhesin.

Initial adherence interactions between mycoplasmas and mammalian cells are important for host colonization and may contribute to subsequent pathogenic processes. Despite significant progress toward understanding the role of specialized, complex tip structures in the adherence of some mycoplasmas, particularly those that infect humans, less is known about adhesins through which other mycoplasmas of this host bind to diverse cell types, even though simpler surface components are likely to be involved. We show by flow cytometric analysis that a soluble recombinant fusion protein (FP29), representing the abundant P29 surface lipoprotein of Mycoplasma fermentans, binds human HeLa cells and inhibits M. fermentans binding to these cells, in both a quantitative and a saturable manner, whereas analogous fusion proteins representing other mycoplasma surface proteins did not. Constructs representing nested N- or C-terminal truncations of FP29 allowed initial mapping of this specific adherence function to a central region of the P29 sequence containing a 36-amino-acid disulfide loop. A derivative of FP29 containing a mutation converting one participating Cys to Ser, precluding intrachain disulfide bond formation, retained full activity. Together these results suggest that the direct interaction of M. fermentans with a ligand on the HeLa cell surface involves a limited segment of the P29 surface lipoprotein and requires neither the disulfide bond nor the contribution of adjacent portions of the protein. Earlier results indicating phase-variable display of monoclonal antibody surface epitopes on P29, now recognized to be outside this ligand binding region, raise the possibility that variation of mycoplasma surface architecture might alter the presentation of the binding region and the adherence phenotype. Preliminary results further indicated that FP29 could inhibit binding to HeLa cells by Mycoplasma hominis, a distinct human mycoplasma species displaying the phase-variable adhesin Vaa, but not that by Mycoplasma capricolum, an organism infecting caprine species. This result raises the additional, testable possibility that a common host cell ligand for two human mycoplasma species may be recognized through structurally dissimilar adhesins that undergo phase variation by two distinct mechanisms, governing protein expression (Vaa) or surface masking (P29).

Site-specific proteolysis of the MALP-404 lipoprotein determines the release of a soluble selective lipoprotein-associated motif-containing fragment and alteration of the surface phenotype of Mycoplasma fermentans.

The mature MALP-404 surface lipoprotein of Mycoplasma fermentans comprises a membrane-anchored N-terminal lipid-modified region responsible for macrophage activation (P. F. Mühlradt, M. Kiess, H. Meyer, R. Süssmuth, and G. Jung, J. Exp. Med. 185:1951-1958, 1997) and an external hydrophilic region that contains the selective lipoprotein-associated (SLA) motif defining a family of lipoproteins from diverse but selective prokaryotes, including mycoplasmas (M. J. Calcutt, M. F. Kim, A. B. Karpas, P. F. Mühlradt, and K. S. Wise, Infect. Immun. 67:760-771, 1999). This family generally corresponds to a computationally defined group of orthologs containing the basic membrane protein (BMP) domain. Two discrete lipid-modified forms of the abundant MALP product which vary dramatically in ratio among isolates of M. fermentans occur on the mycoplasma surface: (i) MALP-404, the full-length mature product, and (ii) MALP-2, the Toll-like receptor 2-mediated macrophage-activating lipopeptide containing the N-terminal 14 residues of the mature lipoprotein. The role of posttranslational processing in the biogenesis of MALP-2 from the prototype MALP-404 SLA-containing lipoprotein was investigated. Detergent phase fractionation of cell-bound products and N-terminal sequencing of a newly discovered released fragment (RF) demonstrated that MALP-404 was subject to site-specific proteolysis between residues 14 and 15 of the mature lipoprotein, resulting in the cell-bound MALP-2 and soluble RF products. This previously unknown mechanism of posttranslational processing among mycoplasmas suggests that specific cleavage of some surface proteins may confer efficient "secretion" of extracellular products by these organisms, with concurrent changes in the surface phenotype. This newly identified form of variation may have significant implications for host adaptation by mycoplasmas, as well as other pathogens expressing lipoproteins of the SLA (BMP) family.