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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 469 records · Page 26
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Unveiling the Arsenal of Apple Bitter Rot Fungi: Comparative Genomics Identifies Candidate Effectors, CAZymes, and Biosynthetic Gene Clusters in Colletotrichum Species

The bitter rot of apple is caused by Colletotrichum spp. and is a serious pre-harvest disease that can manifest in postharvest losses on harvested fruit. In this study, we obtained genome sequences from four different species, C. chrysophilum, C. noveboracense, C. nupharicola, and C. fioriniae, that infect apple and cause diseases on other fruits, vegetables, and flowers. Our genomic data were obtained from isolates/species that have not yet been sequenced and represent geographic-specific regions. Genome sequencing allowed for the construction of phylogenetic trees, which corroborated the overall concordance observed in prior MLST studies. Bioinformatic pipelines were used to discover CAZyme, effector, and secondary metabolic (SM) gene clusters in all nine Colletotrichum isolates. We found redundancy and a high level of similarity across species regarding CAZyme classes and predicted cytoplastic and apoplastic effectors. SM gene clusters displayed the most diversity in type and the most common cluster was one that encodes genes involved in the production of alternapyrone. Our study provides a solid platform to identify targets for functional studies that underpin pathogenicity, virulence, and/or quiescence that can be targeted for the development of new control strategies. With these new genomics resources, exploration via omics-based technologies using these isolates will help ascertain the biological underpinnings of their widespread success and observed geographic dominance in specific areas throughout the country.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Natural Product Gene Clusters in the Filamentous Nostocales Cyanobacterium HT-58-2

Cyanobacteria are known as rich repositories of natural products. One cyanobacterial-microbial consortium (isolate HT-58-2) is known to produce two fundamentally new classes of natural products: the tetrapyrrole pigments tolyporphins A–R, and the diterpenoid compounds tolypodiol, 6-deoxytolypodiol, and 11-hydroxytolypodiol. The genome (7.85 Mbp) of the Nostocales cyanobacterium HT-58-2 was annotated previously for tetrapyrrole biosynthesis genes, which led to the identification of a putative biosynthetic gene cluster (BGC) for tolyporphins. Here, bioinformatics tools have been employed to annotate the genome more broadly in an effort to identify pathways for the biosynthesis of tolypodiols as well as other natural products. A putative BGC (15 genes) for tolypodiols has been identified. Four BGCs have been identified for the biosynthesis of other natural products. Two BGCs related to nitrogen fixation may be relevant, given the association of nitrogen stress with production of tolyporphins. The results point to the rich biosynthetic capacity of the HT-58-2 cyanobacterium beyond the production of tolyporphins and tolypodiols.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Integration of Complete Plasmids Containing Bont Genes into Chromosomes of Clostridium parabotulinum , Clostridium sporogenes , and Clostridium argentinense

At least 40 toxin subtypes of botulinum neurotoxins (BoNTs), a heterogenous group of bacterial proteins, are produced by seven different clostridial species. A key factor that drives the diversity of neurotoxigenic clostridia is the association of bont gene clusters with various genomic locations including plasmids, phages and the chromosome. Analysis of Clostridium sporogenes BoNT/B1 strain CDC 1632, C. argentinense BoNT/G strain CDC 2741, and Clostridium parabotulinum BoNT/B1 strain DFPST0006 genomes revealed bont gene clusters within plasmid-like sequences within the chromosome or nested in large contigs, with no evidence of extrachromosomal elements. A nucleotide sequence (255,474 bp) identified in CDC 1632 shared 99.5% identity (88% coverage) with bont/B1-containing plasmid pNPD7 of C. sporogenes CDC 67071; CDC 2741 contig AYSO01000020 (1.1 MB) contained a ~140 kb region which shared 99.99% identity (100% coverage) with plasmid pRSJ17_1 of C. argentinense BoNT/G strain 89G; and DFPST0006 contig JACBDK0100002 (573 kb) contained a region that shared 100% identity (99%) coverage with the bont/B1-containing plasmid pCLD of C. parabotulinum Okra. This is the first report of full-length plasmid DNA-carrying complete neurotoxin gene clusters integrated in three distinct neurotoxigenic species: C. parabotulinum, C. sporogenes and C. argentinense.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Full-Length ASFV B646L Gene Sequencing by Nanopore Offers a Simple and Rapid Approach for Identifying ASFV Genotypes

African swine fever (ASF) is an acute, highly hemorrhagic viral disease in domestic pigs and wild boars. The disease is caused by African swine fever virus, a double stranded DNA virus of the Asfarviridae family. ASF can be classified into 25 different genotypes, based on a 478 bp fragment corresponding to the C-terminal sequence of the B646L gene, which is highly conserved among strains and encodes the major capsid protein p72. The C-terminal end of p72 has been used as a PCR target for quick diagnosis of ASF, and its characterization remains the first approach for epidemiological tracking and identification of the origin of ASF in outbreak investigations. Recently, a new classification of ASF, based on the complete sequence of p72, reduced the 25 genotypes into only six genotypes; therefore, it is necessary to have the capability to sequence the full-length B646L gene (p72) in a rapid manner for quick genotype characterization. Here, we evaluate the use of an amplicon approach targeting the whole B646L gene, coupled with nanopore sequencing in a multiplex format using Flongle flow cells, as an easy, low cost, and rapid method for the characterization and genotyping of ASF in real-time.

Virology↗
DOE OSTI2024

Assessment of the Effect of Deleting the African Swine Fever Virus Gene R298L on Virus Replication and Virulence of the Georgia2010 Isolate

African swine fever (ASF) is a lethal disease of domestic pigs that is currently challenging swine production in large areas of Eurasia. The causative agent, ASF virus (ASFV), is a large, double-stranded and structurally complex virus. The ASFV genome encodes for more than 160 proteins; however, the functions of most of these proteins are still in the process of being characterized. The ASF gene R298L, which has previously been characterized as able to encode a functional serine protein kinase, is expressed late in the virus infection cycle and may be part of the virus particle. There is no description of the importance of the R298L gene in basic virus functions such as replication or virulence in the natural host. Based on its evolution, it is proposed that there are four different phenotypes of R298L of ASFV in nature, which may have potential implications for R298L functionality. We report here that a recombinant virus lacking the R298L gene in the Georgia 2010 isolate, ASFV-G-∆R298L, does not exhibit significant changes in its replication in primary cultures of swine macrophages. In addition, when experimentally inoculated in pigs, ASFV-G-∆R298L induced a fatal form of the disease similar to that caused by the parental virulent ASFV-G. Therefore, deletion of R298L does not significantly affect virus replication and virulence in domestic pigs of the ASFV Georgia 2010 isolate.

Virology↗
DOE OSTI2021

Random sub-diffusion and capture of genes by the nuclear pore reduces dynamics and coordinates inter-chromosomal movement

Hundreds of genes interact with the yeast nuclear pore complex (NPC), localizing at the nuclear periphery and clustering with co-regulated genes. Dynamic tracking of peripheral genes shows that they cycle on and off the NPC and that interaction with the NPC slows their sub-diffusive movement. Furthermore, NPC-dependent inter-chromosomal clustering leads to coordinated movement of pairs of loci separated by hundreds of nanometers. We developed fractional Brownian motion simulations for chromosomal loci in the nucleoplasm and interacting with NPCs. These simulations predict the rate and nature of random sub-diffusion during repositioning from nucleoplasm to periphery and match measurements from two different experimental models, arguing that recruitment to the nuclear periphery is due to random sub-diffusion and transient capture by NPCs. Finally, the simulations do not lead to inter-chromosomal clustering or coordinated movement, suggesting that interaction with the NPC is necessary, but not sufficient, to cause clustering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Expanding standards in viromics: in silico evaluation of dsDNA viral genome identification, classification, and auxiliary metabolic gene curation

Viruses influence global patterns of microbial diversity and nutrient cycles. Though viral metagenomics (viromics), specifically targeting dsDNA viruses, has been critical for revealing viral roles across diverse ecosystems, its analyses differ in many ways from those used for microbes. To date, viromics benchmarking has covered read pre-processing, assembly, relative abundance, read mapping thresholds and diversity estimation, but other steps would benefit from benchmarking and standardization. Here we use in silico-generated datasets and an extensive literature survey to evaluate and highlight how dataset composition (i.e., viromes vs bulk metagenomes) and assembly fragmentation impact (i) viral contig identification tool, (ii) virus taxonomic classification, and (iii) identification and curation of auxiliary metabolic genes (AMGs). The in silico benchmarking of five commonly used virus identification tools show that gene-content-based tools consistently performed well for long (≥3 kbp) contigs, while k -mer- and blast-based tools were uniquely able to detect viruses from short (≤3 kbp) contigs. Notably, however, the performance increase of k -mer- and blast-based tools for short contigs was obtained at the cost of increased false positives (sometimes up to ~5% for virome and ~75% bulk samples), particularly when eukaryotic or mobile genetic element sequences were included in the test datasets. Furthermore, for viral classification, variously sized genome fragments were assessed using gene-sharing network analytics to quantify drop-offs in taxonomic assignments, which revealed correct assignations ranging from ~95% (whole genomes) down to ~80% (3 kbp sized genome fragments). A similar trend was also observed for other viral classification tools such as VPF-class, ViPTree and VIRIDIC, suggesting that caution is warranted when classifying short genome fragments and not full genomes. Finally, we highlight how fragmented assemblies can lead to erroneous identification of AMGs and outline a best-practices workflow to curate candidate AMGs in viral genomes assembled from metagenomes. Together, these benchmarking experiments and annotation guidelines should aid researchers seeking to best detect, classify, and characterize the myriad viruses ‘hidden’ in diverse sequence datasets.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Manipulating the circadian clock to increase gene expression

A method of increasing gene expression by manipulating the circadian clock is described that includes transforming a photosynthetic organism to include an expression control sequence that modulates the expression of a clock gene to increase expression of a target gene. Photosynthetic organism having a modified circadian cycle reflecting this method are also described.

Johnson, Charles Hirschie↗
DOE OSTI2021

Application of the Metabolic Modeling Pipeline in KBase to Categorize Reactions, Predict Essential Genes, and Predict Pathways in an Isolate Genome

The DOE Systems Biology Knowledgebase (KBase) platform offers a range of powerful tools for the reconstruction, refinement, and analysis of genome-scale metabolic models built from microbial isolate genomes. In this chapter, we describe and demonstrate these tools in action with an analysis of isoprene production in the Bacillus subtilis DSM genome. Two different methods are applied to build initial metabolic models for the DSM genome, then the models are gapfilled in three different growth conditions. Next, flux balance analysis (FBA) and flux variability analysis (FVA) techniques are applied to both study the growth of these models in minimal media and classify reactions within each model based on essentiality and functionality. The models are applied with the FBA method to predict essential genes, which are then compared to an updated list of essential genes obtained for B. subtilis 168, a very similar strain to the DSM isolate. The models are also applied to simulate Biolog growth conditions, and these results are compared with Biolog data collected for B. subtilis 168. Finally, the DSM metabolic models are applied to explore the pathways and genes responsible for producing isoprene in this strain. These studies demonstrate the accuracy and utility of models generated from the KBase pipelines, as well as exploring the tools available for analyzing these models.

Allen, Benjamin↗
DOE OSTI2022

Method of regulating gene expression in a cell

The disclosure provides methods of making a tetracycline inducible expression system in a cell. The methods include providing the cell with a first nucleic acid sequence comprising a first promoter operably linked to a tetracycline repressor gene coding sequence, providing the cell with a second nucleic acid sequence comprising a second promoter operably linked to a coding sequence of a gene of interest wherein the second promoter is modified to include one or more tetracycline repressor protein binding sites, and determining the expression of the gene of interest in the presence or absence of tetracycline. The disclosure further provides nucleic acid sequences, vectors and cells including the tetracycline inducible modified promoter.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Promoter useful for high expression of a heterologous gene of interest in Aspergillus niger

The present invention provides for an Aspergillus niger host cell comprising a gene of interest operatively linked to an ecm33 promoter of an ascomycete fungi, wherein the gene of interest is heterologous to the ecm33 promoter and/or to Aspergillus niger. In some embodiments, the gene of interest is a glycoside hydrolase enzyme. In some embodiments, the glycoside hydrolase enzyme is a glucosidase.

Gladden, John M.↗
DOE OSTI2022

Centromere/kinetochore protein genes for cancer diagnosis, prognosis and treatment selection

The invention described in the application relates to a panel of gene expression markers for patient with a tumor. The invention thus provides methods and compositions, e.g., kits, for evaluating gene expression levels of the markers and methods of using such gene expression levels to evaluate the likelihood of disease progression or response to chemotherapy or radiation therapy. Such information can be used in determining prognosis and treatment options for cancer patients.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Metabolite, annotation, and gene integration system and method

Disclosed herein are systems and methods for associating metabolites with genes. In some embodiments, after potential metabolites are identified based on spectroscopy data of the content of an organism, possible reactions capable of producing the potential metabolites are determined. The possible reactions are compared to gene sequences in a database, and an association score for the likelihood that a gene sequence is related to the potential metabolites is calculated.

Erbilgin, Onur↗
DOE OSTI2022

Dead but Not Forgotten: How Extracellular DNA, Moisture, and Space Modulate the Horizontal Transfer of Extracellular Antibiotic Resistance Genes in Soil

Antibiotic-resistant bacteria and the spread of antibiotic resistance genes (ARGs) pose a serious risk to human and veterinary health. While many studies focus on the movement of live antibiotic-resistant bacteria to the environment, it is unclear whether extracellular ARGs (eARGs) from dead cells can transfer to live bacteria to facilitate the evolution of antibiotic resistance in nature. Here, we use eARGs from dead, antibiotic-resistant Pseudomonas stutzeri cells to track the movement of eARGs to live P. stutzeri cells via natural transformation, a mechanism of horizontal gene transfer involving the genomic integration of eARGs. In sterile, antibiotic-free agricultural soil, we manipulated the eARG concentration, soil moisture, and proximity to eARGs. We found that transformation occurred in soils inoculated with just 0.25 μg of eDNA g –1 soil, indicating that even low concentrations of soil eDNA can facilitate transformation (previous estimates suggested ~2 to 40 μg eDNA g –1 soil). When eDNA was increased to 5 μg g –1 soil, there was a 5-fold increase in the number of antibiotic-resistant P. stutzeri cells. We found that eARGs were transformed under soil moistures typical of terrestrial systems (5 to 30% gravimetric water content) but inhibited at very high soil moistures (>30%). Altogether, this work demonstrates that dead bacteria and their eARGs are an overlooked path to antibiotic resistance. More generally, the spread of eARGs in antibiotic-free soil suggests that transformation allows genetic variants to establish in the absence of antibiotic selection and that the soil environment plays a critical role in regulating transformation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Developing multi-gene CRISPRa/i programs to accelerate DBTL cycles in ABF hosts engineered for chemical production

This project developed and implemented a modular CRISPR activation and interference (CRISPRa/i) platform to accelerate strain optimization and pathway development for industrially relevant microbial hosts. By integrating multiplexed transcriptional perturbation tools with data-driven Design–Build–Test–Learn (DBTL) workflows, the team achieved reductions in cycle time and enhanced production of industrial aromatics, particularly 4-aminocinnamic acid (4-ACA), in Pseudomonas putida. Key accomplishments included: ● Development of a robust, tunable CRISPRa/i system in P. putida that enabled efficient multi-target gene regulation via guide RNA (gRNA) programs ● Completion of two full DBTL cycles, guided by machine learning (ML) models trained on transcriptomic and performance data, reducing engineering time by over 30% ● Optimization of multi-gene regulatory programs to balance expression of host and pathway modules, improve 4-ACA titers, and resolve metabolic bottlenecks ● Demonstration of system portability through a limited proof-of-concept extension in Acinetobacter baylyi, underscoring the generalizability of the approach ● Evaluation of strain performance on lignocellulosic biomass-derived substrates, demonstrating the feasibility of converting renewable carbon into aromatic building blocks These results illustrate the feasibility of applying ML-guided CRISPRa/i perturbation strategies to accelerate strain development in complex microbial systems. The resulting tools and datasets contribute to DOE objectives by improving platform predictability, reducing development costs, and enabling broader access to sustainable, economically viable bioproduction technologies.

09 BIOMASS FUELS↗
DOE OSTI2022

Towards replacement of animal tests with in vitro assays: a gene expression biomarker predicts in vitro and in vivo estrogen receptor activity

High-throughput transcriptomics (HTTr) has the potential to support efforts to reduce or replace some animal tests. In past studies, we described a computational approach utilizing a gene expression biomarker consisting of 46 genes to predict estrogen receptor (ER) activity after chemical exposure in ER-positive human breast cancer cells including the MCF-7 cell line. We hypothesized that the biomarker model could identify ER activities of chemicals examined by Endocrine Disruptor Screening Program (EDSP) Tier 1 screening assays in which transcript profiles of the same chemicals were examined in MCF-7 cells. For the 62 chemicals examined including 5 chemicals examined in this study using RNA-Seq, the ER biomarker model accuracy was 1) 97% for in vitro reference chemicals, 2) 76–85% for guideline uterotrophic assays, and 3) 87–88% for guideline and nonguideline uterotrophic assays. For the same chemicals, these accuracies were similar or slightly better than those of the ToxCast ER model based on 18 in vitro assays. The performance of the ER biomarker model indicates that HTTr interpreted using the ER biomarker correctly identifies active and inactive ER reference chemicals. Finally, as part of the HTTr screening program the approach could rapidly identify chemicals with potential ER bioactivities for additional screening and testing.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Tying the knot in the tetrahydrofolate (THF) riboswitch: A molecular basis for gene regulation

Effective gene regulation by the tetrahydrofolate riboswitch depends not only on ligand affinity but also on the kinetics of ligand association, which involves two cooperative binding sites. Here, we have determined a 1.9-Å resolution crystal structure of the ligand-free THF riboswitch aptamer. The pseudoknot binding site ‘unwinds’ in the absence of ligand, whereby the adjacent helical domains (P1, P2, and P3) become disjointed, resulting in rotation and misalignment of the gene-regulatory P1 helix with respect to P3. In contrast, the second binding site at the three-way junction, which is the first to fold, is structurally conserved between apo and holo forms. This suggests a kinetic role for this site, in which binding of the first ligand molecule to the stably folded three-way junction promotes formation of the regulatory pseudoknot site and subsequent binding of the second molecule. As such, these findings provide a molecular basis for both conformational switching and kinetic control.

59 BASIC BIOLOGICAL SCIENCES↗