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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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102 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

In silico analyses of diversity and dissemination of antimicrobial resistance genes and mobile genetics elements, for plasmids of enteric pathogens

The antimicrobial resistance (AMR) mobilome plays a key role in the dissemination of resistance genes encoded by mobile genetics elements (MGEs) including plasmids, transposons (Tns), and insertion sequences (ISs). These MGEs contribute to the dissemination of multidrug resistance (MDR) in enteric bacterial pathogens which have been considered as a global public health risk. To further understand the diversity and distribution of AMR genes and MGEs across different plasmid types, we utilized multiple sequence-based computational approaches to evaluate AMR-associated plasmid genetics. A collection of 1,309 complete plasmid sequences from Gammaproteobacterial species, including 100 plasmids from each of the following 14 incompatibility (Inc) types: A/C, BO, FIA, FIB, FIC, FIIA, HI1, HI2, I1, K, M, N, P except W, where only 9 sequences were available, was extracted from the National Center for Biotechnology Information (NCBI) GenBank database using BLAST tools. The extracted FASTA files were analyzed using the AMRFinderPlus web-based tools to detect antimicrobial, disinfectant, biocide, and heavy metal resistance genes and ISFinder to identify IS/Tn MGEs within the plasmid sequences. In silico prediction based on plasmid replicon types showed that the resistance genes were diverse among plasmids, yet multiple genes were widely distributed across the plasmids from enteric bacterial species. These findings provide insights into the diversity of resistance genes and that MGEs mediate potential transmission of these genes across multiple plasmid replicon types. This notion was supported by the observation that many IS/Tn MGEs and resistance genes known to be associated with them were common across multiple different plasmid types. Our results provide critical insights about how the diverse population of resistance genes that are carried by the different plasmid types can allow for the dissemination of AMR across enteric bacteria. The results also highlight the value of computational-based approaches and in silico analyses for the assessment of AMR and MGEs, which are important elements of molecular epidemiology and public health outcomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Large-scale genetic characterization of the model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough

Sulfate-reducing bacteria (SRB) are obligate anaerobes that can couple their growth to the reduction of sulfate. Despite the importance of SRB to global nutrient cycles and their damage to the petroleum industry, our molecular understanding of their physiology remains limited. To systematically provide new insights into SRB biology, we generated a randomly barcoded transposon mutant library in the model SRB Desulfovibrio vulgaris Hildenborough (DvH) and used this genome-wide resource to assay the importance of its genes under a range of metabolic and stress conditions. In addition to defining the essential gene set of DvH, we identified a conditional phenotype for 1,137 non-essential genes. Through examination of these conditional phenotypes, we were able to make a number of novel insights into our molecular understanding of DvH, including how this bacterium synthesizes vitamins. For example, we identified DVU0867 as an atypical L-aspartate decarboxylase required for the synthesis of pantothenic acid, provided the first experimental evidence that biotin synthesis in DvH occurs via a specialized acyl carrier protein and without methyl esters, and demonstrated that the uncharacterized dehydrogenase DVU0826:DVU0827 is necessary for the synthesis of pyridoxal phosphate. In addition, we used the mutant fitness data to identify genes involved in the assimilation of diverse nitrogen sources and gained insights into the mechanism of inhibition of chlorate and molybdate. Our large-scale fitness dataset and RB-TnSeq mutant library are community-wide resources that can be used to generate further testable hypotheses into the gene functions of this environmentally and industrially important group of bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Genome-wide identification of novel flagellar motility genes in Pseudomonas syringae pv. tomato DC3000

Pseudomonas syringaepv.tomatoDC3000 (PstDC3000) is a plant pathogenic bacterium that possesses complicated motility regulation pathways including a typical chemotaxis system. A significant portion of our understanding about the genes functioning inPstDC3000 motility is based on comparison to other bacteria. This leaves uncertainty about whether gene functions are conserved, especially since specific regulatory modules can have opposite functions in sets ofPseudomonas. In this study, we used a competitive selection to enrich for mutants with altered swimming motility and used random barcode transposon-site sequencing (RB-TnSeq) to identify genes with significant roles in swimming motility. Besides many of the known or predicted chemotaxis and motility genes, our method identified PSPTO_0406 (dipA), PSPTO_1042 (chrR) and PSPTO_4229 (hypothetical protein) as novel motility regulators. PSPTO_0406 is a homolog ofdipA, a known cyclic di-GMP degrading enzyme inP. aeruginosa. PSPTO_1042 is part of an extracytoplasmic sensing system that controls gene expression in response to reactive oxygen species, suggesting that PSPTO_1042 may function as part of a mechanism that enablesPstDC3000 to alter motility when encountering oxidative stressors. PSPTO_4229 encodes a protein containing an HD-related output domain (HDOD), but with no previously identified functions. We found that deletion and overexpression of PSPTO_4229 both reduce swimming motility, suggesting that its function is sensitive to expression level. We used the overexpression phenotype to screen for nonsense and missense mutants of PSPTO_4229 that no longer reduce swimming motility and found a pair of conserved arginine residues that are necessary for motility suppression. Together these results provide a global perspective on regulatory and structural genes controlling flagellar motility inPstDC3000.

Microbiology↗
DOE OSTI2026

Advances in genetic tools for metabolic engineering of non-conventional yeasts

Non-conventional yeasts are emerging as powerful alternatives to Saccharomyces cerevisiae for metabolic engineering, owing to their innate stress tolerance, broad substrate utilization, and distinctive metabolic capabilities. These attributes position them as promising chassis for producing biofuels, pharmaceuticals, and specialty chemicals. This review synthesizes recent advances in genetic toolkits for four such species—Pichia kudriavzevii (Issatchenkia orientalis), Starmerella bombicola, Debaryomyces hansenii, and Pachysolen tannophilus—highlighting progress across plasmid architectures (episomal and integrative), identification of autonomously replicating sequences and centromeric elements, and the development of safe-harbor genomic loci. We summarize promoter and terminator libraries enabling tunable expression, the expansion of auxotrophic and antifungal selection markers with recycling strategies, and the rapid adaptation of CRISPR-based systems (Cas9 and Cas12a) with optimized guide RNA expression, multiplex editing, and approaches that enhance homologous recombination (e.g., KU70/80 disruption). We also review landing-pad platforms for modular, repeated integrations and transposon-based tools (e.g., piggyBac) that facilitate multigene pathway assembly. Collectively, these innovations are accelerating design-build-test-learn cycles and enabling precise, scalable engineering of non-conventional yeasts. Remaining challenges—including limited species-specific episomal systems, variable transformation efficiencies, genome-stability concerns, and alternative codon usage—define clear priorities for future toolkit development. Together, these advances and open needs chart a path toward robust, sustainable biomanufacturing using diverse non-conventional yeast chassis.

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 OSTI2024

Altering translation allows E. coli to overcome chemically stabilized G-quadruplexes

Genomic DNA from each sample was prepared using the Wizard Genomic DNA Purification Kit (Promega) and after, DNA was quantified using the QuantiFluor ONE dsDNA System (Promega). Genomic DNA underwent shearing to ~200 bp fragments via sonication and the gDNA fragments were prepared for sequencing using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB). Bead-based size selection was used to select ~200 bp fragments and the fragments then underwent a splinkerette PCR using a Tn5-enriching forward primer and custom reverse primers for multiplexing. A final bead-based size selection was used to select for the correct length DNA. DNA was sequenced at the University of Michigan Advanced Genomics Core using Illumina sequencing with a custom read primer reading the last 10 nt of the transposon. PhiX174 DNA spike was added to the run to ensure sufficient sequence diversity on the flow cell. Then, a custom index read primer and standard Illumina primer were used to sequence the index reads and PhiX174, respectively.

Keck, James L.↗
DOE OSTI2023

ABF DFO with Technology Holding, Inc.

This Agile BioFoundry Directed Funding Opportunity project with Technology Holding and partners focuses on the development of both a strain of Pseudomonas putida KT2440 and a corresponding bioprocess to convert cellulosic sugars to beta-ketoadipic acid, which can be used in performance nylons and polyesters. Our approach follows the Design-Build-Test-Learn cycle wherein we have transferred learnings from muconic acid production in P. putida to develop a glucose and xylose-utilizing beta-ketoadipic acid production strain. This strain achieves 65 g/L of beta-ketoadipic acid at 0.7 g/L/hr and a C-mol yield of 0.40. We are currently on-boarding arabinose utilization as well. To identify non-intuitive strain modifications as well, we are deploying a beta-ketoadipic acid biosensor and building randomly barcoded transposon insertion sequencing (RB-TnSeq) libraries and gene over-expression libraries in beta-ketoadipic acid production strains. Moreover, we are using global metabolomics and other systems biology tools to identify off-target pathways. Lastly, we are scaling up beta-ketoadipic acid production to kg-scale production for Technology Holding to evaluate in performance polymers with their partners.

beta-ketoadipic acid↗
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 OSTI2021

A High-Throughput Method for Identifying Novel Genes That Influence Metabolic Pathways Reveals New Iron and Heme Regulation in Pseudomonas aeruginosa

The ability to simultaneously and more directly correlate genes with metabolite levels on a global level would provide novel information for many biological platforms yet has thus far been challenging. Here, we describe a method to help address this problem, which we dub “Met-Seq” ( met abolite-coupled Tn seq uencing).

59 BASIC BIOLOGICAL SCIENCES↗