Publications by authors named "Kim S Wise"

The bacterial strain JCVI-syn3.0 stands as the first example of a living organism with a minimized synthetic genome, derived from the genome and chemically synthesized . Here, we report the experimental evolution of a syn3.

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Analysis of the genes retained in the minimized 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.

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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 bacterium-based CFE system using lysates of the genome-minimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative (Mcap).

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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.

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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.

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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, , 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.

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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.

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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.

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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.

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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.

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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.

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Article Synopsis
  • Mycoplasma mycoides subsp. mycoides small colony biotype (SC) is a serious animal pathogen responsible for contagious bovine pleuropneumonia.
  • The complete genome sequences of the pathogenic strain M. mycoides subsp. mycoides SC Gladysdale and a related pathogen, Mycoplasma leachii PG50(T), have been reported.
  • These studies enhance our understanding of the genetic makeup of these harmful bacteria within the same phylogenetic group affecting cattle.
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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.

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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).

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Article Synopsis
  • - The study of Mollicutes (mycoplasmas) shows that surface variation among different species is caused by random expression of various lipoprotein genes, crucial for their adaptability and survival as wall-less organisms.
  • - Research focused on two specific mycoplasma species revealed six novel lipoprotein genes (vmcA to vmcF) that contribute to a unique surface architecture, with two distinct gene locations influencing their expression patterns.
  • - These findings suggest that the variable lipoprotein genes not only highlight differences in mycoplasma pathogens' adaptability but also present potential targets for diagnostics in veterinary medicine.
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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.

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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.

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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.

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