Results for “genomic analysis” — Page 34

DOE OSTI2024

Mechanism-guided engineering of a minimal biological particle for genome editing

The widespread application of genome editing to treat and cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped delivery vehicles (EDVs) are engineered virally derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication proteins in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here, we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of ~80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs and demonstrated increased editing in cell lines and therapeutically relevant primary human T cells. These results show that virally derived particles can be streamlined to create efficacious genome editing delivery vehicles with simpler production and manufacturing.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

The flying spider-monkey tree fern genome provides insights into fern evolution and arborescence

To date, little is known about the evolution of fern genomes, with only two small genomes published from the heterosporous Salviniales. Here we assembled the genome of Alsophila spinulosa, known as the flying spider-monkey tree fern, onto 69 pseudochromosomes. The remarkable preservation of synteny, despite resulting from an ancient whole-genome duplication over 100 million years ago, is unprecedented in plants and probably speaks to the uniqueness of tree ferns. Our detailed investigations into stem anatomy and lignin biosynthesis shed new light on the evolution of stem formation in tree ferns. We identified a phenolic compound, alsophilin, that is abundant in xylem, and we provided the molecular basis for its biosynthesis. Finally, analysis of demographic history revealed two genetic bottlenecks, resulting in rapid demographic declines of A. spinulosa. The A. spinulosa genome fills a crucial gap in the plant genomic landscape and helps elucidate many unique aspects of tree fern biology.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Integrated Proteomic and Glycoproteomic Characterization of Human High-Grade Serous Ovarian Carcinoma

Many gene products exhibit great structural heterogeneity because of an array of modifications. These modifications are not directly encoded in the genomic template but often affect the functionality of proteins. Protein glycosylation plays a vital role in proper protein functions. However, the analysis of glycoproteins has been challenging compared with other protein modifications, such as phosphorylation. Here, we perform an integrated proteomic and glycoproteomic analysis of 83 prospectively collected high-grade serous ovarian carcinoma (HGSC) and 23 non-tumor tissues. Integration of the expression data from global proteomics and glycoproteomics reveals tumor-specific glycosylation, uncovers different glycosylation associated with three tumor clusters, and identifies glycosylation enzymes that were correlated with the altered glycosylation. In addition to providing a valuable resource, these results provide insights into the potentialroles of glycosylation in the pathogenesis of HGSC, with the possibility of distinguishing pathological outcomes of ovarian tumors from non-tumors, as well as classifying tumor clusters.

Hu, Yingwei↗

DOE OSTI2020

Autotrophic and mixotrophic metabolism of an anammox bacterium revealed by in vivo 13 C and 2 H metabolic network mapping

Anaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and use of different carbon and energy substrates beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus ‘Kuenenia stuttgartiensis’ using time-series 13 C and 2 H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis. Our findings confirm predicted metabolic pathways used for CO 2 fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis , and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of the oxidative branch of an incomplete tricarboxylic acid cycle for alpha-ketoglutarate biosynthesis, despite the genome not having an annotated citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood–Ljungdahl pathway instead of oxidizing it completely to CO 2 followed by reassimilation. In contrast, our data suggest that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor’s side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2020

What Is in Umbilicaria pustulata? A Metagenomic Approach to Reconstruct the Holo-Genome of a Lichen

Lichens are valuable models in symbiosis research and promising sources of biosynthetic genes for biotechnological applications. Most lichenized fungi grow slowly, resist aposymbiotic cultivation, and are poor candidates for experimentation. Obtaining contiguous, high-quality genomes for such symbiotic communities is technically challenging. Here, we present the first assembly of a lichen holo-genome from metagenomic whole-genome shotgun data comprising both PacBio long reads and Illumina short reads. The nuclear genomes of the two primary components of the lichen symbiosis—the fungus Umbilicaria pustulata (33 Mb) and the green alga Trebouxia sp. (53 Mb)—were assembled at contiguities comparable to single-species assemblies. The analysis of the read coverage pattern revealed a relative abundance of fungal to algal nuclei of ~20:1. Gap-free, circular sequences for all organellar genomes were obtained. The bacterial community is dominated by Acidobacteriaceae and encompasses strains closely related to bacteria isolated from other lichens. Gene set analyses showed no evidence of horizontal gene transfer from algae or bacteria into the fungal genome. Our data suggest a lineage-specific loss of a putative gibberellin-20-oxidase in the fungus, a gene fusion in the fungal mitochondrion, and a relocation of an algal chloroplast gene to the algal nucleus. Major technical obstacles during reconstruction of the holo-genome were coverage differences among individual genomes surpassing three orders of magnitude. Moreover, we show that GC-rich inverted repeats paired with nonrandom sequencing error in PacBio data can result in missing gene predictions. This likely poses a general problem for genome assemblies based on long reads.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2021

Mapping the Path Forward to Next Generation Algal Technologies: Workshop on Understanding the Rules of Life and Complexity in Algal Systems

Algal technology offers enormous potential as a source of renewable biomass and bioproducts for fuels, biochemicals, nutrients, and numerous other applications. While tremendous opportunities exist, significant challenges are also present in harnessing and utilizing this valuable and potentially sustainable bioproduction platform. Funded by a joint partnership between the Bioenergy Technologies Office (BETO) at the Department of Energy (DOE) and the National Science Foundation (NSF), the recent virtual workshop, "Understanding the Rules of Life: Complexity in Algal Systems Workshop," was organized to bring together thought leaders from academia, national laboratories and other government agencies, and industry to identify key challenges and propose research strategies to overcome these barriers. Two parallel sessions were undertaken: 1) algae strain and toolkit development and 2) algal ecosystem and microbiome dynamics, in order to advance algae engineering and cultivation efforts. In each session, attendees discussed the most pressing challenges from the perspective of model systems and omics analysis. Themes such as genetic engineering toolboxes, translation from the laboratory to outdoor cultivation, functional genomics, metabolomics, algal ecology, consortial design, microbiome dynamics, mathematical models, and data analytics emerged from the discussion. Overall, this workshop has served to elucidate the key hurdles and many opportunities present as we continue to advance algal science and technology from the laboratory and into the commercial realm.

09 BIOMASS FUELS↗

DOE OSTI2023

Impaired neuron differentiation in GBA-associated Parkinson’s disease is linked to cell cycle defects in organoids

The mechanisms underlying Parkinson’s disease (PD) etiology are only partially understood despite intensive research conducted in the field. Recent evidence suggests that early neurodevelopmental defects might play a role in cellular susceptibility to neurodegeneration. To study the early developmental contribution of GBA mutations in PD we used patient-derived iPSCs carrying a heterozygous N370S mutation in the GBA gene. Patient-specific midbrain organoids displayed GBA-PD relevant phenotypes such as reduction of GCase activity, autophagy impairment, and mitochondrial dysfunction. Genome-scale metabolic (GEM) modeling predicted changes in lipid metabolism which were validated with lipidomics analysis, showing significant differences in the lipidome of GBA-PD. In addition, patient-specific midbrain organoids exhibited a decrease in the number and complexity of dopaminergic neurons. This was accompanied by an increase in the neural progenitor population showing signs of oxidative stress-induced damage and premature cellular senescence. These results provide insights into how GBA mutations may lead to neurodevelopmental defects thereby predisposing to PD pathology.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Draft Genome Sequence of Pedococcus sp. Strain 5OH_020, Isolated from California Grassland Soil

The draft genome sequence of the soil bacterium Pedococcus sp. strain 5OH_020, isolated on a natural cobalamin analog, comprises 4.4 Mbp, with 4,108 protein-coding genes. Its genome encodes cobalamin-dependent enzymes, including methionine synthase and class II ribonucleotide reductase. Taxonomic analysis suggests that it is a novel species within the genus Pedococcus .

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Disruption of histone acetylation homeostasis reveals multilayered chromatin regulation for transcriptional resiliency

Background Epigenetic modifications, nucleosome occupancy, and three-dimensional chromatin architecture collectively create a multi-layered, highly interactive regulatory system for controlling genomic functionality. Dysregulation of epigenetic processes leads to a plethora of abnormalities including disease states. Therapies focused on epigenetic modulation can alter gene expression to correct dysfunction, though the perpetuation of these states and the relationships among chromatin regulatory layers is not well understood. Results Here, we investigated global and local chromatin structural and functional responses after acute histone deacetylase inhibitor treatment (suberoylanilide hydroxamic acid) in lung cancer cells across time. Treatment substantially increased global histone acetylation resulting in a pervasive but not distinctive signature. The spread of acetylation did not significantly impact global chromatin accessibility, and nucleosome remodeling largely occurred at finer scales in functionally relevant genomic regions. Indeed, both H 3 K 4 trimethylation, a mark of active transcription, and gene expression changes were altered in a controlled locus-specific manner, suggesting aberrant acetylation indirectly leads to balanced and bidirectional gene expression profiles from tighter regulation of other chromatin features. HDACi treatment induced (13%) genomic rearrangement in chromatin compartmentalization and moderate weakening of topologically associating domains. Conclusions Continuous wavelet analysis of these features demonstrates that scale-dependent, locus-specific factors influence the relationship between chromatin architecture and functional output, suggesting that regulation of transcription and nucleosome remodeling is not entirely (nor linearly) dependent upon large scale compartment exchange. Structural and functional responses are most pronounced early after treatment with partial persistence of differential local chromatin features and expression later in time; this highlights the plasticity of chromatin regulation, which may have implications for the efficacy of epigenetic treatments. These results demonstrate the effectiveness of multi-layered regulation of transcription: in resilient systems, disruption of one chromatin feature does not distort the regulation of other features in supporting a transcriptional program that allows for survival.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Novel viruses of the family Partitiviridae discovered in Saccharomyces cerevisiae

It has been 49 years since the last discovery of a new virus family in the model yeast Saccharomyces cerevisiae . A large-scale screen to determine the diversity of double-stranded RNA (dsRNA) viruses in S . cerevisiae has identified multiple novel viruses from the family Partitiviridae that have been previously shown to infect plants, fungi, protozoans, and insects. Most S . cerevisiae partitiviruses (ScPVs) are associated with strains of yeasts isolated from coffee and cacao beans. The presence of partitiviruses was confirmed by sequencing the viral dsRNAs and purifying and visualizing isometric, non-enveloped viral particles. ScPVs have a typical bipartite genome encoding an RNA-dependent RNA polymerase (RdRP) and a coat protein (CP). Phylogenetic analysis of ScPVs identified three species of ScPV, which are most closely related to viruses of the genus Cryspovirus from the mammalian pathogenic protozoan Cryptosporidium parvum . Molecular modeling of the ScPV RdRP revealed a conserved tertiary structure and catalytic site organization when compared to the RdRPs of the Picornaviridae . The ScPV CP is the smallest so far identified in the Partitiviridae and has structural homology with the CP of other partitiviruses but likely lacks a protrusion domain that is a conspicuous feature of other partitivirus particles. ScPVs were stably maintained during laboratory growth and were successfully transferred to haploid progeny after sporulation, which provides future opportunities to study partitivirus-host interactions using the powerful genetic tools available for the model organism S . cerevisiae .

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

SRX13165144: yHMH446_Pichia_sp_nov_plate33_SPADES

A novel budding yeast species was isolated from a soil sample collected in the United States of America. Phylogenetic analyses of multiple loci and phylogenomic analyses conclusively placed the species within the genus Pichia. Strain yHMH446 falls within a clade that includes Pichia norvegensis, Pichia pseudocactophila, Candida inconspicua, and Pichia cactophila. Whole genome sequence data were analyzed for the presence of genes known to be important for carbon and nitrogen metabolism, and the phenotypic data from the novel species were compared to all Pichia species with publicly available genomes. Across the genus, including the novel species candidate, we found that the inability to use many carbon and nitrogen sources correlated with the absence of metabolic genes. Based on these results, Pichia galeolata sp. nov. is proposed to accommodate yHMH446(T) (=NRRL Y-64187 = CBS 16864). This study shows how integrated taxogenomic analysis can add mechanistic insight to species descriptions.

genome sequence↗

DOE OSTI2022

Pichia sp. yHMH446, whole genome shotgun sequencing project

A novel budding yeast species was isolated from a soil sample collected in the United States of America. Phylogenetic analyses of multiple loci and phylogenomic analyses conclusively placed the species within the genus Pichia. Strain yHMH446 falls within a clade that includes Pichia norvegensis, Pichia pseudocactophila, Candida inconspicua, and Pichia cactophila. Whole genome sequence data were analyzed for the presence of genes known to be important for carbon and nitrogen metabolism, and the phenotypic data from the novel species were compared to all Pichia species with publicly available genomes. Across the genus, including the novel species candidate, we found that the inability to use many carbon and nitrogen sources correlated with the absence of metabolic genes. Based on these results, Pichia galeolata sp. nov. is proposed to accommodate yHMH446(T) (=NRRL Y-64187 = CBS 16864). This study shows how integrated taxogenomic analysis can add mechanistic insight to species descriptions.

genome sequence↗

DOE OSTI2021

Pichia sp. strain yHMH446 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene, complete sequence; and internal transcribed spacer 2, partial sequence

A novel budding yeast species was isolated from a soil sample collected in the United States of America. Phylogenetic analyses of multiple loci and phylogenomic analyses conclusively placed the species within the genus Pichia. Strain yHMH446 falls within a clade that includes Pichia norvegensis, Pichia pseudocactophila, Candida inconspicua, and Pichia cactophila. Whole genome sequence data were analyzed for the presence of genes known to be important for carbon and nitrogen metabolism, and the phenotypic data from the novel species were compared to all Pichia species with publicly available genomes. Across the genus, including the novel species candidate, we found that the inability to use many carbon and nitrogen sources correlated with the absence of metabolic genes. Based on these results, Pichia galeolata sp. nov. is proposed to accommodate yHMH446(T) (=NRRL Y-64187 = CBS 16864). This study shows how integrated taxogenomic analysis can add mechanistic insight to species descriptions.

genome sequence↗

DOE OSTI2021

Pichia sp. strain yHMH446 internal transcribed spacer 2 and large subunit ribosomal RNA gene, partial sequence

A novel budding yeast species was isolated from a soil sample collected in the United States of America. Phylogenetic analyses of multiple loci and phylogenomic analyses conclusively placed the species within the genus Pichia. Strain yHMH446 falls within a clade that includes Pichia norvegensis, Pichia pseudocactophila, Candida inconspicua, and Pichia cactophila. Whole genome sequence data were analyzed for the presence of genes known to be important for carbon and nitrogen metabolism, and the phenotypic data from the novel species were compared to all Pichia species with publicly available genomes. Across the genus, including the novel species candidate, we found that the inability to use many carbon and nitrogen sources correlated with the absence of metabolic genes. Based on these results, Pichia galeolata sp. nov. is proposed to accommodate yHMH446(T) (=NRRL Y-64187 = CBS 16864). This study shows how integrated taxogenomic analysis can add mechanistic insight to species descriptions.

genome sequence↗

DOE OSTI2022

Pichia sp. yHMH446 translation elongation factor 1-alpha gene, partial cds

A novel budding yeast species was isolated from a soil sample collected in the United States of America. Phylogenetic analyses of multiple loci and phylogenomic analyses conclusively placed the species within the genus Pichia. Strain yHMH446 falls within a clade that includes Pichia norvegensis, Pichia pseudocactophila, Candida inconspicua, and Pichia cactophila. Whole genome sequence data were analyzed for the presence of genes known to be important for carbon and nitrogen metabolism, and the phenotypic data from the novel species were compared to all Pichia species with publicly available genomes. Across the genus, including the novel species candidate, we found that the inability to use many carbon and nitrogen sources correlated with the absence of metabolic genes. Based on these results, Pichia galeolata sp. nov. is proposed to accommodate yHMH446(T) (=NRRL Y-64187 = CBS 16864). This study shows how integrated taxogenomic analysis can add mechanistic insight to species descriptions.

genome sequence↗

DOE OSTI2024

Complete genomes of Mucilaginibacter sabulilitoris SNA2 and Mucilaginibacter sp. cycad4: microbes with the potential for plant growth promotion

Mucilaginibacter species have been isolated from various environments, often in association with plants. Here, we report the complete genomes of Mucilaginibacter sabulilitoris SNA2 and Mucilaginibacter sp. cycad4. The former is the first available for that species, and based on 16S sequence analysis, the latter strain is likely a new species.

Mucilaginibacter↗

DOE OSTI2023

A High-Quality Genome-Scale Model for Rhodococcus opacus Metabolism

Rhodococcus opacus is a bacterium that has a high tolerance to aromatic compounds and can produce significant amounts of triacylglycerol (TAG). Here, we present iGR1773, the first genome-scale model (GSM) of R. opacus PD630 metabolism based on its genomic sequence and associated data. The model includes 1773 genes, 3025 reactions, and 1956 metabolites, was developed in a reproducible manner using CarveMe, and was evaluated through Metabolic Model tests (MEMOTE). We combine the model with two Constraint-Based Reconstruction and Analysis (COBRA) methods that use transcriptomics data to predict growth rates and fluxes: E-Flux2 and SPOT (Simplified Pearson Correlation with Transcriptomic data). Growth rates are best predicted by E-Flux2. Flux profiles are more accurately predicted by E-Flux2 than flux balance analysis (FBA) and parsimonious FBA (pFBA), when compared to 44 central carbon fluxes measured by 13C-Metabolic Flux Analysis (13C-MFA). Under glucose-fed conditions, E-Flux2 presents an R2 value of 0.54, while predictions based on pFBA had an inferior R2 of 0.28. We attribute this improved performance to the extra activity information provided by the transcriptomics data. For phenol-fed metabolism, in which the substrate first enters the TCA cycle, E-Flux2’s flux predictions display a high R2 of 0.96 while pFBA showed an R2 of 0.93. We also show that glucose metabolism and phenol metabolism function with similar relative ATP maintenance costs. These findings demonstrate that iGR1773 can help the metabolic engineering community predict aromatic substrate utilization patterns and perform computational strain design.

Roell, Garrett W.↗

DOE OSTI2024

Phylometagenomics of cycad coralloid roots reveals shared symbiotic signals

Cycads are known to host symbiotic cyanobacteria, includingNostocalesspecies, as well as other sympatric bacterial taxa within their specialized coralloid roots. Yet, it is unknown if these bacteria share a phylogenetic origin and/or common genomic functions that allow them to engage in facultative symbiosis with cycad roots. To address this, we obtained metagenomic sequences from 39 coralloid roots sampled from diverse cycad species and origins in Australia and Mexico. Culture-independent shotgun metagenomic sequencing was used to validate sub-community co-cultures as an efficient approach for functional and taxonomic analysis. Our metanalysis shows a host-independent microbiome core consisting of seven bacterial orders with high species diversity within the identified taxa. Moreover, we recovered 43 cyanobacterial metagenome-assembled genomes, and in addition toNostocspp., symbiotic cyanobacteria of the genusAulosirawere identified for the first time. Using this robust dataset, we used phylometagenomic analysis to reveal three monophyletic cyanobiont clades, two host-generalist and one cycad-specific that includesAulosiraspp. Although the symbiotic clades have independently arisen, they are enriched in certain functional genes, such as those related to secondary metabolism. Furthermore, the taxonomic composition of associated sympatric bacterial taxa remained constant. Our research quadruples the number of cycad cyanobiont genomes and provides a robust framework to decipher cyanobacterial symbioses, with the potential of improving our understanding of symbiotic communities. This study lays a solid foundation to harness cyanobionts for agriculture and bioprospection, and assist in conservation of critically endangered cycads.

Genetics & Heredity↗