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At least 541 records · Page 30
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Phyllosticta paracitricarpa is synonymous with the EU quarantine fungus P. citricarpa based on phylogenomic analyses

Phyllosticta citricarpa is an important citrus-pathogen and a quarantine organism in the European Union. Its recently described relative, P. paracitricarpa, is very closely related and not listed as a quarantine organism. P. paracitricarpa is very difficult to distinguish from P. citricarpa, since its morphological features overlap and the barcoding gene sequences that were originally used to delimit them as distinct species have a low number of species-specific polymorphisms that have subsequently been shown to overlap between the two clades. Therefore, we performed extensive genomic analyses to determine whether the genetic variation between P. citricarpa and P. paracitricarpa strains should be considered to represent infraspecific variation within P. citricarpa, or whether it is indicative of distinct species. Using a phylogenomic analysis with 3,000 single copy ortholog genes and whole-genome comparisons, we determined that the variation between P. citricarpa and P. paracitricarpa can be considered as infraspecies variation within P. citricarpa. We also determined the level of variation in mitochondrial assemblies of several Phyllosticta species and concluded there are only minimal differences between the assemblies of P. citricarpa and P. paracitricarpa. Thus, using several orthogonal approaches, we here demonstrate that variation within the nuclear and mitochondrial genomes of other Phyllosticta species is larger than variation between genomes obtained from P. citricarpa and P. paracitricarpa strains. Thus, P. citricarpa and P. paracitricarpa should be considered as conspecific.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Exceptional subgenome stability and functional divergence in the allotetraploid Ethiopian cereal teff

Teff (Eragrostis tef) is a cornerstone of food security in the Horn of Africa, where it is prized for stress resilience, grain nutrition, and market value. Here, we report a chromosome-scale assembly of allotetraploid teff (variety Dabbi) and patterns of subgenome dynamics. The teff genome contains two complete sets of homoeologous chromosomes, with most genes maintaining as syntenic gene pairs. TE analysis allows us to estimate that the teff polyploidy event occurred ~1.1 million years ago (mya) and that the two subgenomes diverged ~5.0 mya. Despite this divergence, we detect no large-scale structural rearrangements, homoeologous exchanges, or biased gene loss, in contrast to many other allopolyploids. The two teff subgenomes have partitioned their ancestral functions based on divergent expression across a diverse expression atlas. Together, these genomic resources will be useful for accelerating breeding of this underutilized grain crop and for fundamental insights into polyploid genome evolution.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

Multiple Wheat Genomes Reveal Novel Gli-2 Sublocus Location and Variation of Celiac Disease Epitopes in Duplicated α-Gliadin Genes

The seed protein α-gliadin is a major component of wheat flour and causes gluten-related diseases. However, due to the complexity of this multigene family with a genome structure composed of dozens of copies derived from tandem and genome duplications, little was known about the variation between accessions, and thus little effort has been made to explicitly target α-gliadin for bread wheat breeding. Here, we analyzed genomic variation in α-gliadins across 11 recently published chromosome-scale assemblies of hexaploid wheat, with validation using long-read data. We unexpectedly found that the Gli-B2 locus is not a single contiguous locus but is composed of two subloci, suggesting the possibility of recombination between the two during breeding. We confirmed that the number of immunogenic epitopes among 11 accessions varied. The D subgenome of a European spelt line also contained epitopes, in agreement with its hybridization history. Evolutionary analysis identified amino acid sites under diversifying selection, suggesting their functional importance. The analysis opens the way for improved grain quality and safety through wheat breeding.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Clostridium autoethanogenum isopropanol production via native plasmid pCA replicon

Clostridium autoethanogenum is a model gas-fermenting acetogen for commercial ethanol production. It is also a platform organism being developed for the carbon-negative production of acetone and isopropanol by gas fermentation. We have assembled a 5.5 kb pCA plasmid for type strain DSM10061 (JA1-1) using three genome sequence datasets. pCA is predicted to encode seven open-reading frames and estimated to be a low-copy number plasmid present at approximately 12 copies per chromosome. RNA-seq analyses indicate that pCA genes are transcribed at low levels and two proteins, CAETHG_05090 (putative replication protein) and CAETHG_05115 (hypothetical, a possible Mob protein), were detected at low levels during batch gas fermentations. Thiolase (thlA), CoA-transferase (ctfAB), and acetoacetate decarboxylase (adc) genes were introduced into a vector for isopropanol production in C. autoethanogenum using the native plasmid origin of replication. The availability of the pCA sequence will facilitate studies into its physiological role and could form the basis for genetic tool optimization.

09 BIOMASS FUELS↗
DOE OSTI2023

Ex vivo to in vivo model of malignant peripheral nerve sheath tumors for precision oncology

Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas that often develop in patients with neurofibromatosis type 1 (NF1). To address the critical need for novel therapeutics in MPNST, we aimed to establish an ex vivo 3D platform that accurately captured the genomic diversity of MPNST and could be utilized in a medium-throughput manner for drug screening studies to be validated in vivo using patient-derived xenografts (PDX). Genomic analysis was performed on all PDX-tumor pairs. Selected PDX were harvested for assembly into 3D microtissues. Based on prior work in our labs, we evaluated drugs (trabectedin, olaparib, and mirdametinib) ex vivo and in vivo. For 3D microtissue studies, cell viability was the endpoint as assessed by Zeiss Axio Observer. For PDX drug studies, tumor volume was measured twice weekly. Bulk RNA sequencing was performed to identify pathways enriched in cells. We developed 13 NF1-associated MPNST-PDX and identified mutations or structural abnormalities in NF1 (100%), SUZ12 (85%), EED (15%), TP53 (15%), CDKN2A (85%), and chromosome 8 gain (77%). We successfully assembled PDX into 3D microtissues, categorized as robust (>90% viability at 48 h), good (>50%), or unusable (<50%). We evaluated drug response to “robust” or “good” microtissues, namely MN-2, JH-2-002, JH-2-079-c, and WU-225. Drug response ex vivo predicted drug response in vivo, and enhanced drug effects were observed in select models. These data support the successful establishment of a novel 3D platform for drug discovery and MPNST biology exploration in a system representative of the human condition.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Probing photosynthesis by altering chloroplast proteins (Final Technical Report)

The goal of this project was to explore the effect of altering the carbon fixing enzyme Rubisco in the model C3 plant tobacco on photosynthesis and plant growth and development. We introduced the gene sequences encoding Rubisco from a Limonium species and from a red alga into tobacco and found that the enzyme did not assemble properly, undoubtedly requiring additional assembly factors from the source organism. We then developed a method to make single amino-acid changes into the Rubisco subunit that is encoded by the chloroplast genome. We used the method to investigate the role of particular amino acid residues in kinetic properties of Rubisco. Finally, we improved a bacterial expression system that has allowed assembly of active tobacco Rubisco in E. coli. We used the improved system to investigate the kinetic properties of Rubisco enzymes that contain only one type of Rubisco small subunit along with the large subunit.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Telomere-to-telomere assemblies of chromosome 10 reveal complex adaptive variation of 3-ketoacyl-CoA-synthases in Populus trichocarpa likely driven by Helitrons

The model woody plant Populus trichocarpa displays an atypical alkene-diverse wax cuticle likely driven by copy number variation (CNV) of 3-ketoacyl-CoA synthases ( KCS ), which has been difficult to confirm with short-read assemblies. Long-read sequencing enables the development of telomere-to-telomere resources to detect cryptic variation, including CNVs, which are currently missed. Integrating this information can improve genomic prediction for breeding and provide insights into the evolutionary basis of important traits. Our analysis of 78 long-read haplotypes from chromosome 10 identified more than twice as many KCS genes as previously reported, and numerous intragenic non-synonymous substitutions. Random Forest predictive models highlighted the importance of Potri.010G079500 in producing very long chain alkenes; however, its absence did not predict previously reported alkene-deficient phenotypes. Instead, alkene levels are best predicted by the combinations of KCS copies. Additionally, amino acid substitutions clustered around ligand and donor binding pockets, suggesting they contribute to differing wax cuticle composition. Finally, each KCS gene and copy was linked to a Helitron transposon. A phylogenetic analysis suggests Helitrons are the evolutionary mechanism for generating KCS tandem arrays. Long-read generated telomere-to-telomere assemblies of P. trichocarpa chromosome 10 revealed large-effect loci critical to genetic studies that are unattainable from short-reads. This new resource produced novel insights into genome structure and function, and a novel mechanism for generating tandem gene duplication. Our results highlight that, given current challenges in annotation and assembly, detailed and focused long-read sequences are key to interpreting complex genomic regions that contain tandem copy number variants.

09 BIOMASS FUELS↗
DOE OSTI2023

Genome features of a novel hydrocarbonoclastic Chryseobacterium oranimense strain and its comparison to bacterial oil-degraders and to other C. oranimense strains

For the first time, we report the whole genome sequence of a hydrocarbonoclastic Chryseobacterium oranimense strain isolated from Trinidad and Tobago (COTT) and its genes involved in the biotransformation of hydrocarbons and xenobiotics through functional annotation. The assembly consisted of 11 contigs with 2,794 predicted protein-coding genes which included a diverse group of gene families involved in aliphatic and polycyclic hydrocarbon degradation. Comparative genomic analyses with 18 crude-oil degrading bacteria in addition to two C. oranimense strains not associated with oil were carried out. The data revealed important differences in terms of annotated genes involved in the hydrocarbon degradation process that may explain the molecular mechanisms of hydrocarbon and xenobiotic biotransformation. Notably, many gene families were expanded to explain COTT’s competitive ability to manage habitat-specific stressors. Gene-based evidence of the metabolic potential of COTT supports the application of indigenous microbes for the remediation of polluted terrestrial environments and provides a genomic resource for improving our understanding of how to optimize these characteristics for more effective bioremediation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Testing at scale during the COVID-19 pandemic

Assembly and publication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome in January 2020 enabled the immediate development of tests to detect the new virus. This began the largest global testing programme in history, in which hundreds of millions of individuals have been tested to date. The unprecedented scale of testing has driven innovation in the strategies, technologies and concepts that govern testing in public health. In this Review, we describe the changing role of testing during the COVID-19 pandemic, including the use of genomic surveillance to track SARS-CoV-2 transmission around the world, the use of contact tracing to contain disease outbreaks and testing for the presence of the virus circulating in the environment. Despite these efforts, widespread community transmission has become entrenched in many countries and has required the testing of populations to identify and isolate infected individuals, many of whom are asymptomatic. The diagnostic and epidemiological principles that underpin such population-scale testing are also considered, as are the high-throughput and point-of-care technologies that make testing feasible on a massive scale.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Genome Sequence of the Chestnut Blight Fungus Cryphonectria parasitica EP155: A Fundamental Resource for an Archetypical Invasive Plant Pathogen

Cryphonectria parasitica is the causal agent of chestnut blight, a fungal disease that almost entirely eliminated mature American chestnut from North America over a 50-year period. Here, we formally report the genome of C. parasitica EP155 using a Sanger shotgun sequencing approach. After finishing and integration with simple-sequence repeat markers, the assembly was 43.8 Mb in 26 scaffolds (L 50 = 5; N 50 = 4.0Mb). Eight chromosomes are predicted: five scaffolds have two telomeres and six scaffolds have one telomere sequence. In total, 11,609 gene models were predicted, of which 85% show similarities to other proteins. Finally, this genome resource has already increased the utility of a fundamental plant pathogen experimental system through new understanding of the fungal vegetative incompatibility system, with significant implications for enhancing mycovirus-based biological control.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Giant Starship Elements Mobilize Accessory Genes in Fungal Genomes

Accessory genes are variably present among members of a species and are a reservoir of adaptive functions. In bacteria, differences in gene distributions among individuals largely result from mobile elements that acquire and disperse accessory genes as cargo. In contrast, the impact of cargo-carrying elements on eukaryotic evolution remains largely unknown. Here, we show that variation in genome content within multiple fungal species is facilitated by Starships, a newly discovered group of massive mobile elements that are 110 kb long on average, share conserved components, and carry diverse arrays of accessory genes. We identified hundreds of Starship-like regions across every major class of filamentous Ascomycetes, including 28 distinct Starships that range from 27 to 393 kb and last shared a common ancestor ca. 400 Ma. Using new long-read assemblies of the plant pathogen Macrophomina phaseolina, we characterize four additional Starships whose activities contribute to standing variation in genome structure and content. One of these elements, Voyager, inserts into 5S rDNA and contains a candidate virulence factor whose increasing copy number has contrasting associations with pathogenic and saprophytic growth, suggesting Voyager’s activity underlies an ecological trade-off. We propose that Starships are eukaryotic analogs of bacterial integrative and conjugative elements based on parallels between their conserved components and may therefore represent the first dedicated agents of active gene transfer in eukaryotes. Our results suggest that Starships have shaped the content and structure of fungal genomes for millions of years and reveal a new concerted route for evolution throughout an entire eukaryotic phylum.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Data for "RT-EZ: A Golden Gate Assembly Toolkit for Streamlined Genetic Engineering of Rhodotorula toruloides"

For economic and sustainable biomanufacturing, the oleaginous yeast Rhodotorula toruloides has emerged as a promising platform for producing biofuels, pharmaceuticals, and other valuable chemicals. However, genetic manipulation of R. toruloides has been limited by its high GC content and the lack of a replicating plasmid, necessitating gene integration into the genome of the yeast. To address these challenges, we developed the RT-EZ ( R. toruloides Efficient Zipper) toolkit, a versatile tool based on Golden Gate assembly, designed to streamline R. toruloides engineering with improved efficiency and flexibility. The RT-EZ toolkit simplifies vector construction by incorporating new features such as bidirectional promoters and 2A peptides, color-based screening using RFP, and sequences optimized for both Agrobacterium tumefaciens-mediated transformation (ATMT) and easy linearization, enabling straightforward selection and transformation. Notably, the RT-EZ kit can be used to construct an expression cassette with four different genes in one assembly reaction, significantly improving vector construction speed and efficiency. The utility of the RT-EZ toolkit was demonstrated through the successful synthesis of arachidonic acid in R. toruloides by coexpressing fatty acid elongases and desaturases. This result underscores the potential of the RT-EZ toolkit to advance synthetic biology in R. toruloides , providing a streamlined method for addressing genetic engineering challenges in the yeast.

gene editing↗
DOE OSTI2025

RT-EZ: A Golden Gate Assembly Toolkit for Streamlined Genetic Engineering of Rhodotorula toruloides

For economic and sustainable biomanufacturing, the oleaginous yeast Rhodotorula toruloides has emerged as a promising platform for producing biofuels, pharmaceuticals, and other valuable chemicals. However, genetic manipulation of R. toruloides has been limited by its high GC content and the lack of a replicating plasmid, necessitating gene integration into the genome of the yeast. To address these challenges, we developed the RT-EZ (R. toruloides Efficient Zipper) toolkit, a versatile tool based on Golden Gate assembly, designed to streamline R. toruloides engineering with improved efficiency and flexibility. The RT-EZ toolkit simplifies vector construction by incorporating new features such as bidirectional promoters and 2A peptides, color-based screening using RFP, and sequences optimized for both Agrobacterium tumefaciens-mediated transformation (ATMT) and easy linearization, enabling straightforward selection and transformation. Notably, the RT-EZ kit can be used to construct an expression cassette with four different genes in one assembly reaction, significantly improving vector construction speed and efficiency. The utility of the RT-EZ toolkit was demonstrated through the successful synthesis of arachidonic acid in R. toruloides by coexpressing fatty acid elongases and desaturases. Furthermore, this result underscores the potential of the RT-EZ toolkit to advance synthetic biology in R. toruloides, providing a streamlined method for addressing genetic engineering challenges in the yeast.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Mechanism Across Scales: A Holistic Modeling Framework Integrating Laboratory and Field Studies for Microbial Ecology

Over the last century, leaps in technology for imaging, sampling, detection, high-throughput sequencing, and -omics analyses have revolutionized microbial ecology to enable rapid acquisition of extensive datasets for microbial communities across the ever-increasing temporal and spatial scales. The present challenge is capitalizing on our enhanced abilities of observation and integrating diverse data types from different scales, resolutions, and disciplines to reach a causal and mechanistic understanding of how microbial communities transform and respond to perturbations in the environment. This type of causal and mechanistic understanding will make predictions of microbial community behavior more robust and actionable in addressing microbially mediated global problems. To discern drivers of microbial community assembly and function, we recognize the need for a conceptual, quantitative framework that connects measurements of genomic potential, the environment, and ecological and physical forces to rates of microbial growth at specific locations. We describe the Framework for Integrated, Conceptual, and Systematic Microbial Ecology (FICSME), an experimental design framework for conducting process-focused microbial ecology studies that incorporates biological, chemical, and physical drivers of a microbial system into a conceptual model. Through iterative cycles that advance our understanding of the coupling across scales and processes, we can reliably predict how perturbations to microbial systems impact ecosystem-scale processes or vice versa. We describe an approach and potential applications for using the FICSME to elucidate the mechanisms of globally important ecological and physical processes, toward attaining the goal of predicting the structure and function of microbial communities in chemically complex natural environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Elemental profiling and genomewide association studies reveal genomic variants modulating ionomic composition in Populus trichocarpa leaves

Samples were collected from a population of 1,089 black cottonwood genotypes (P. trichocarpa) assembled from native stands to encompass the central portion of the natural range of the species, stretching from 38.8° to 54.3° N Q13 latitude from California, USA, to British Columbia, Canada. Establishment of the common garden, growth conditions, and site maintenance have been described by Muchero et al (2015). In this study, leaf samples for ionomic profiling were collected from 4-year-old trees, during the growing season, in July 2012, from a field located in Clatskanie, Oregon, USA (46°6′11″N 123°12′13″W). The field site was located in a protected alluvial floodplain containing a uniform Wauna-Locoda silt loam soil area characterized by an acidic pH, in Columbia County, Oregon. A subset of 584 out of the 1,089 P. trichocarpa genotypes were represented in this sampling. These genotypes were randomly selected to represent the geographical distribution of the population. A single fully mature (LPI 7-9) leaf on the south side of the tree exposed to full sunlight conditions was removed from the tree within a 6-hour window centering on solar noon and immediately frozen under dry ice before processing. Leaf samples of 584 P. trichocarpa genotypes were finely ground to 40 mm particle size using a mortar and pestle, and ionomic composition was analyzed using ICP-MS. In total, 20 elements were profiled, including aluminum (Al27), arsenic (As75), boron (B11), cadmium (Cd111), calcium (Ca43), cobalt (Co), copper (Cu), iron (Fe57), magnesium (Mg25), manganese (Mn55), molybdenum (Mo), nickel (Ni60), phosphorus (P31), potassium (K39), rubidium (Rb85), selenium (Se82), sodium (Na23), strontium (Sr88), sulfur (S34), and zinc (Zn66), following a protocol established by Ziegler et al. (2013). For each sample, 75mg of powder was digested overnight in 2.5 mL HNO3 containing 20 parts per billion (ppb) indium as an internal standard, following the protocol described in Ziegler et al. (2013). Following a dilution, concentration of the 20 elements was measured using an Elan 6000 DRC-e mass spectrometer (Perkin-Elmer SCIEX) connected to a PFA microflow nebulizer (Elemental Scientific) and Apex HF desolvator (Elemental Scientific). One measurement per sample per genotype was done. For subsequent analyses, the quantifications were converted to total element concentration.

CBI ionomics, GWAS, plasma-mass spectrometry, neut↗
DOE OSTI2021

Integrating genomics and metabolomics for scalable non-ribosomal peptide discovery

Non-Ribosomal Peptides (NRPs) represent a biomedically important class of natural products that include a multitude of antibiotics and other clinically used drugs. NRPs are not directly encoded in the genome but are instead produced by metabolic pathways encoded by biosynthetic gene clusters (BGCs). Since the existing genome mining tools predict many putative NRPs synthesized by a given BGC, it remains unclear which of these putative NRPs are correct and how to identify post-assembly modifications of amino acids in these NRPs in a blind mode, without knowing which modifications exist in the sample. To address this challenge, here we report NRPminer, a modification-tolerant tool for NRP discovery from large (meta)genomic and mass spectrometry datasets. We show that NRPminer is able to identify many NRPs from different environments, including four previously unreported NRP families from soil-associated microbes and NRPs from human microbiota. Furthermore, in this work we demonstrate the anti-parasitic activities and the structure of two of these NRP families using direct bioactivity screening and nuclear magnetic resonance spectrometry, illustrating the power of NRPminer for discovering bioactive NRPs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

SpacerExtractor v0.9

The SpacerExtractor tool is meant to robustly identify and extract CRISPR spacers from metagenome short reads. Working from a database of known CRISPR repeats, SpacerExtractor quickly scans short reads for the corresponding repeat sequences, extract the potential spacer between two repeats, apply several quality control, denoising, and clustering steps, and provides a full non-redundant complement of spacers for each detected repeat. Because of the high variability observed at CRISPR loci, this read mining approach typically recovers a much larger diversity of spacers than can be found in assembled contigs. SpacerExtractor also includes commands to run CRISPR-Cas Typer on a new set of genomes or MAGs, and add newly predicted repeats to the repeat database.

Bushnell, Brian↗
DOE OSTI2020

Functional and genetic markers of niche partitioning among enigmatic members of the human oral microbiome

Abstract Introduction Microbial residents of the human oral cavity have long been a major focus of microbiology due to their influence on host health and intriguing patterns of site specificity amidst the lack of dispersal limitation. However, the determinants of niche partitioning in this habitat are yet to be fully understood, especially among taxa that belong to recently discovered branches of microbial life. Results Here, we assemble metagenomes from tongue and dental plaque samples from multiple individuals and reconstruct 790 non-redundant genomes, 43 of which resolve to TM7, a member of the Candidate Phyla Radiation, forming six monophyletic clades that distinctly associate with either plaque or tongue. Both pangenomic and phylogenomic analyses group tongue-specific clades with other host-associated TM7 genomes. In contrast, plaque-specific TM7 group with environmental TM7 genomes. Besides offering deeper insights into the ecology, evolution, and mobilome of cryptic members of the oral microbiome, our study reveals an intriguing resemblance between dental plaque and non-host environments indicated by the TM7 evolution, suggesting that plaque may have served as a stepping stone for environmental microbes to adapt to host environments for some clades of microbes. Additionally, we report that prophages are widespread among oral-associated TM7, while absent from environmental TM7, suggesting that prophages may have played a role in adaptation of TM7 to the host environment. Conclusions Our data illuminate niche partitioning of enigmatic members of the oral cavity, including TM7, SR1, and GN02, and provide genomes for poorly characterized yet prevalent members of this biome, such as uncultivated Flavobacteriaceae.

60 APPLIED LIFE SCIENCES↗