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.

Gross Chromosomal Rearrangements in Kluyveromyces marxianus Revealed by Illumina and Oxford Nanopore Sequencing.

Kluyveromyces marxianus (K. marxianus) is an increasingly popular industrially relevant yeast. It is known to possess a highly efficient non-homologous end joining (NHEJ) pathway that promotes random integration of non-homologous DNA fragments into its genome. The nature of the integration events was traditionally analyzed by Southern blot hybridization. However, the precise DNA sequence at the insertion sites were not fully explored. We transformed a PCR product of the Saccharomyces cerevisiae URA3 gene (ScURA3) into an uracil auxotroph K. marxianus otherwise wildtype strain and picked 24 stable Ura+ transformants for sequencing analysis. We took advantage of rapid advances in DNA sequencing technologies and developed a method using a combination of Illumina MiSeq and Oxford Nanopore sequencing. This approach enables us to uncover the gross chromosomal rearrangements (GCRs) that are associated with the ScURA3 random integration. Moreover, it will shine a light on understanding DNA repair mechanisms in eukaryotes, which could potentially provide insights for cancer research.

Polar Effects of Transposon Insertion into a Minimal Bacterial Genome.

Global transposon mutagenesis is a valuable tool for identifying genes required for cell viability. Here we present a global analysis of the orientation of viable Tn5-Puror (Tn5-puromycin resistance) insertions into the near-minimal bacterial genome of JCVI-syn2.0. Sixteen of the 478 protein-coding genes show a noticeable asymmetry in the orientation of disrupting insertions of Tn5-Puror Ten of these are located in operons, upstream of essential or quasi-essential genes. Inserts transcribed in the same direction as the downstream gene are favored, permitting read-through transcription of the essential or quasi-essential gene. Some of these genes were classified as quasi-essential solely because of polar effects on the expression of downstream genes. Three genes showing asymmetry in Tn5-Puror insertion orientation prefer the orientation that avoids collisions between read-through transcription of Tn5-Puror and transcription of an adjacent gene. One gene (JCVISYN2_0132 [abbreviated here as "_0132"]) shows a strong preference for Tn5-Puror insertions transcribed upstream, away from the downstream nonessential gene _0133. This suggested that expression of _0133 due to read-through from Tn5-Puror is lethal when _0132 function is disrupted by transposon insertion. This led to the identification of genes _0133 and _0132 as a toxin-antitoxin pair. The three remaining genes show read-through transcription of Tn5-Puror directed downstream and away from sizable upstream intergenic regions (199 bp to 363 bp), for unknown reasons. In summary, polar effects of transposon insertion can, in a few cases, affect the classification of genes as essential, quasi-essential, or nonessential and sometimes can give clues to gene function.IMPORTANCE In studies of the minimal genetic requirements for life, we used global transposon mutagenesis to identify genes needed for a minimal bacterial genome. Transposon insertion can disrupt the function of a gene but can also have polar effects on the expression of adjacent genes. In the Tn5-Puror construct used in our studies, read-through transcription from Tn5-Puror can drive expression of downstream genes. This results in a preference for Tn5-Puror insertions transcribed toward a downstream essential or quasi-essential gene within the same operon. Such polar effects can have an impact on the classification of genes as essential, quasi-essential, or nonessential, but this has been observed in only a few cases. Also, polar effects of Tn5-Puror insertion can sometimes give clues to gene function.

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.

Direct transfer of whole genomes from bacteria to yeast.

Transfer of genomes into yeast facilitates genome engineering for genetically intractable organisms, but this process has been hampered by the need for cumbersome isolation of intact genomes before transfer. Here we demonstrate direct cell-to-cell transfer of bacterial genomes as large as 1.8 megabases (Mb) into yeast under conditions that promote cell fusion. Moreover, we discovered that removal of restriction endonucleases from donor bacteria resulted in the enhancement of genome transfer.

Designed surface residue substitutions in [NiFe] hydrogenase that improve electron transfer characteristics.

Photobiological hydrogen production is an attractive, carbon-neutral means to convert solar energy to hydrogen. We build on previous research improving the Alteromonas macleodii "Deep Ecotype" [NiFe] hydrogenase, and report progress towards creating an artificial electron transfer pathway to supply the hydrogenase with electrons necessary for hydrogen production. Ferredoxin is the first soluble electron transfer mediator to receive high-energy electrons from photosystem I, and bears an electron with sufficient potential to efficiently reduce protons. Thus, we engineered a hydrogenase-ferredoxin fusion that also contained several other modifications. In addition to the C-terminal ferredoxin fusion, we truncated the C-terminus of the hydrogenase small subunit, identified as the available terminus closer to the electron transfer region. We also neutralized an anionic patch surrounding the interface Fe-S cluster to improve transfer kinetics with the negatively charged ferredoxin. Initial screening showed the enzyme tolerated both truncation and charge neutralization on the small subunit ferredoxin-binding face. While the enzyme activity was relatively unchanged using the substrate methyl viologen, we observed a marked improvement from both the ferredoxin fusion and surface modification using only dithionite as an electron donor. Combining ferredoxin fusion and surface charge modification showed progressively improved activity in an in vitro assay with purified enzyme.

A broad survey reveals substitution tolerance of residues ligating FeS clusters in [NiFe] hydrogenase.

BACKGROUND: In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii "deep ecotype" as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported "G1" (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys. RESULTS: Among all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the "G1" enzyme. CONCLUSIONS: We have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii "deep ecotype", to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of active enzyme.

Sequence analysis of a complete 1.66 Mb Prochlorococcus marinus MED4 genome cloned in yeast.

Marine cyanobacteria of the genus Prochlorococcus represent numerically dominant photoautotrophs residing throughout the euphotic zones in the open oceans and are major contributors to the global carbon cycle. Prochlorococcus has remained a genetically intractable bacterium due to slow growth rates and low transformation efficiencies using standard techniques. Our recent successes in cloning and genetically engineering the AT-rich, 1.1 Mb Mycoplasma mycoides genome in yeast encouraged us to explore similar methods with Prochlorococcus. Prochlorococcus MED4 has an AT-rich genome, with a GC content of 30.8%, similar to that of Saccharomyces cerevisiae (38%), and contains abundant yeast replication origin consensus sites (ACS) evenly distributed around its 1.66 Mb genome. Unlike Mycoplasma cells, which use the UGA codon for tryptophane, Prochlorococcus uses the standard genetic code. Despite this, we observed no toxic effects of several partial and 15 whole Prochlorococcus MED4 genome clones in S. cerevisiae. Sequencing of a Prochlorococcus genome purified from yeast identified 14 single base pair missense mutations, one frameshift, one single base substitution to a stop codon and one dinucleotide transversion compared to the donor genomic DNA. We thus provide evidence of transformation, replication and maintenance of this 1.66 Mb intact bacterial genome in S. cerevisiae.

Chemical synthesis of the mouse mitochondrial genome.

We describe a one-step, isothermal assembly method for synthesizing DNA molecules from overlapping oligonucleotides. The method cycles between in vitro recombination and amplification until the desired length is reached. As a demonstration of its simplicity and robustness, we synthesized the entire 16.3-kilobase mouse mitochondrial genome from 600 overlapping 60-mers.

Enzymatic assembly of DNA molecules up to several hundred kilobases.

We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.

Cloning whole bacterial genomes in yeast.

Most microbes have not been cultured, and many of those that are cultivatable are difficult, dangerous or expensive to propagate or are genetically intractable. Routine cloning of large genome fractions or whole genomes from these organisms would significantly enhance their discovery and genetic and functional characterization. Here we report the cloning of whole bacterial genomes in the yeast Saccharomyces cerevisiae as single-DNA molecules. We cloned the genomes of Mycoplasma genitalium (0.6 Mb), M. pneumoniae (0.8 Mb) and M. mycoides subspecies capri (1.1 Mb) as yeast circular centromeric plasmids. These genomes appear to be stably maintained in a host that has efficient, well-established methods for DNA manipulation.

[NiFe] hydrogenase from Alteromonas macleodii with unusual stability in the presence of oxygen and high temperature.

Hydrogenases are enzymes involved in the bioproduction of hydrogen, a clean alternative energy source whose combustion generates water as the only end product. In this article we identified and characterized a [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii "deep ecotype" with unusual stability toward oxygen and high temperature. The A. macleodii hydrogenase (HynSL) can catalyze both H(2) evolution and H(2) uptake reactions. HynSL was expressed in A. macleodii under aerobic conditions and reached the maximum activity when the cells entered the late exponential phase. The higher level of hydrogenase activity was accompanied by a greater abundance of the HynSL protein in the late-log or stationary phase. The addition of nickel to the growth medium significantly enhanced the hydrogenase activity. Ni treatment affected the level of the protein, but not the mRNA, indicating that the effect of Ni was exerted at the posttranscriptional level. Hydrogenase activity was distributed ∼30% in the membrane fraction and ∼70% in the cytoplasmic fraction. Thus, HynSL appears to be loosely membrane-bound. Partially purified A. macleodii hydrogenase demonstrated extraordinary stability. It retained 84% of its activity after exposure to 80°C for 2 h. After exposure to air for 45 days at 4°C, it retained nearly 100% of its activity when assayed under anaerobic conditions. Its catalytic activity in the presence of O(2) was evaluated by the hydrogen-deuterium (H-D) exchange assay. In 1% O(2), 20.4% of its H-D exchange activity was retained. The great stability of HynSL makes it a potential candidate for biotechnological applications.

Single-step linker-based combinatorial assembly of promoter and gene cassettes for pathway engineering.

A linker-based approach for combinatorial assembly of promoter and gene cassettes into a biochemical pathway is developed. A synthetic library containing 144 combinations, with 3 promoters and 4 gene variants, was constructed for the ackA and pta genes of the acetate utilization pathway in E. coli. The in vitro isothermal assembled library was then introduced into E. coli mutant (acs-, pta-, ackA-) and selected for restoration of acetate utilization. 81% of the colonies screened contained the complete functional pathway. Thirty positive clones were analyzed and accounted for 10% of the 144 promoter-gene combinations.

One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome.

We previously reported assembly and cloning of the synthetic Mycoplasma genitalium JCVI-1.0 genome in the yeast Saccharomyces cerevisiae by recombination of six overlapping DNA fragments to produce a 592-kb circle. Here we extend this approach by demonstrating assembly of the synthetic genome from 25 overlapping fragments in a single step. The use of yeast recombination greatly simplifies the assembly of large DNA molecules from both synthetic and natural fragments.

The evolution of RecD outside of the RecBCD complex.

The common understanding of the function of RecD, as derived predominantly from studies in Escherichia coli, is that RecD is one of three enzymes in the RecBCD double-stranded break repair DNA recombination complex. However, comparative genomics has revealed that many organisms possess a recD gene even though the other members of the complex, recB and recC, are not present. Further, bioinformatic analyses have shown that there is substantial sequence dissimilarity between recD genes associated with recB and recC (recD1), and those that are not associated with recBC (recD2). Deinococcus radiodurans, known for its extraordinary DNA repair capability, is one such organism that does not possess either recB or recC, and yet does possess a recD gene. The recD of D. radiodurans was deleted and this mutant was shown to have a capacity to repair double-stranded DNA breaks equivalent to wild-type. The phylogenetic history of recD was studied using a dataset of 120 recD genes from 91 fully sequenced species. The analysis focused upon the role of gene duplication and functional genomic context in the evolution of recD2, which appears to have undergone numerous independent events resulting in duplicate recD2 genes. The role of RecD as part of the RecBCD complex appears to have a divergence from an earlier ancestral RecD function still preserved in many species including D. radiodurans.

Discovery of [NiFe] hydrogenase genes in metagenomic DNA: cloning and heterologous expression in Thiocapsa roseopersicina.

Using a metagenomics approach, we have cloned a piece of environmental DNA from the Sargasso Sea that encodes an [NiFe] hydrogenase showing 60% identity to the large subunit and 64% to the small subunit of a Thiocapsa roseopersicina O2-tolerant [NiFe] hydrogenase. The DNA sequence of the hydrogenase identified by the metagenomic approach was subsequently found to be 99% identical to the hyaA and hyaB genes of an Alteromonas macleodii hydrogenase, indicating that it belongs to the Alteromonas clade. We were able to express our new Alteromonas hydrogenase in T. roseopersicina. Expression was accomplished by coexpressing only two accessory genes, hyaD and hupH, without the need to express any of the hyp accessory genes (hypABCDEF). These results suggest that the native accessory proteins in T. roseopersicina could substitute for the Alteromonas counterparts that are absent in the host to facilitate the assembly of a functional Alteromonas hydrogenase. To further compare the complex assembly machineries of these two [NiFe] hydrogenases, we performed complementation experiments by introducing the new Alteromonas hyaD gene into the T. roseopersicina hynD mutant. Interestingly, Alteromonas endopeptidase HyaD could complement T. roseopersicina HynD to cleave endoproteolytically the C-terminal end of the T. roseopersicina HynL hydrogenase large subunit and activate the enzyme. This study refines our knowledge on the selectivity and pleiotropy of the elements of the [NiFe] hydrogenase assembly machineries. It also provides a model for functionally analyzing novel enzymes from environmental microbes in a culture-independent manner.

Kinetic Modeling of the Genetic Information Processes in a Minimal Cell.

JCVI-syn3A is a minimal bacterial cell with a 543 kbp genome consisting of 493 genes. For this slow growing minimal cell with a 105 min doubling time, we recently established the essential metabolism including the transport of required nutrients from the environment, the gene map, and genome-wide proteomics. Of the 452 protein-coding genes, 143 are assigned to metabolism and 212 are assigned to genetic information processing. Using genome-wide proteomics and experimentally measured kinetic parameters from the literature we present here kinetic models for the genetic information processes of DNA replication, replication initiation, transcription, and translation which are solved stochastically and averaged over 1,000 replicates/cells. The model predicts the time required for replication initiation and DNA replication to be 8 and 50 min on average respectively and the number of proteins and ribosomal components to be approximately doubled in a cell cycle. The model of genetic information processing when combined with the essential metabolic and cell growth networks will provide a powerful platform for studying the fundamental principles of life.

Direct Transfer of a Mycoplasma mycoides Genome to Yeast Is Enhanced by Removal of the Mycoides Glycerol Uptake Factor Gene glpF.

We previously discovered that intact bacterial chromosomes can be directly transferred to a yeast host cell where they can propagate as centromeric plasmids by fusing bacterial cells with S accharomyces cerevisiae spheroplasts. Inside the host any desired number of genetic changes can be introduced into the yeast centromeric plasmid to produce designer genomes that can be brought to life using a genome transplantation protocol. Earlier research demonstrated that the removal of restriction-systems from donor bacteria, such as Mycoplasma mycoides, Mycoplasma capricolum, or Haemophilus influenzae increased successful genome transfers. These findings suggested that other genetic factors might also impact the bacteria-to-yeast genome transfer process. In this study, we demonstrated that the removal of a particular genetic factor, the glycerol uptake facilitator protein gene glpF from M. mycoides, significantly increased direct genome transfer by up to 21-fold. Additionally, we showed that intact bacterial cells were endocytosed by yeast spheroplasts producing organelle-like structures within these yeast cells. These might lead to the possibility of creating novel synthetic organelles.

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.