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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 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
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

CRAGE-RB-PI-seq reveals transcriptional dynamics of plant-associated bacteria during root colonization

Plant roots release a wide array of metabolites into the rhizosphere, shaping microbial communities and their functions. While metagenomics has expanded our understanding of these communities, little is known about the physiology of their members in host environments. Transcriptome analysis via RNA sequencing is a common approach to learning more, but its use has been challenging because of low bacterial biomass and interference from plant RNA. To overcome this, we developed a randomly-barcoded promoter-library insertion sequencing (RB-PI-seq) combined with chassis-independent recombinase-assisted genome engineering (CRAGE). Using Pseudomonas simiae WCS417 as a model rhizobacterium, this method enabled targeted amplification of barcoded transcripts, bypassing plant RNA interference and allowing measurement of thousands of promoter activities during Arabidopsis root colonization. Our analysis revealed temporally resolved transcriptional regulation, including those associated with cell growth, chemotaxis, plant immune suppression, biofilm formation, and stress responses, reflecting the coordinated physiological adaptation to the root environment. Additionally, we discovered that transcriptional activation of xanthine dehydrogenase and a lysozyme inhibitor is crucial for evading plant immune systems. This framework is scalable to other bacterial species and provides new opportunities for understanding rhizobacterial gene regulation in native environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Barcoded overexpression screens in gut Bacteroidales identify genes with roles in carbon utilization and stress resistance

Abstract A mechanistic understanding of host-microbe interactions in the gut microbiome is hindered by poorly annotated bacterial genomes. While functional genomics can generate large gene-to-phenotype datasets to accelerate functional discovery, their applications to study gut anaerobes have been limited. For instance, most gain-of-function screens of gut-derived genes have been performed in Escherichia coli and assayed in a small number of conditions. To address these challenges, we develop Barcoded Overexpression BActerial shotgun library sequencing (Boba-seq). We demonstrate the power of this approach by assaying genes from diverse gut Bacteroidales overexpressed in Bacteroides thetaiotaomicron . From hundreds of experiments, we identify new functions and phenotypes for 29 genes important for carbohydrate metabolism or tolerance to antibiotics or bile salts. Highlights include the discovery of a d -glucosamine kinase, a raffinose transporter, and several routes that increase tolerance to ceftriaxone and bile salts through lipid biosynthesis. This approach can be readily applied to develop screens in other strains and additional phenotypic assays.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Avirulent Isolates of Penicillium chrysogenum to Control the Blue Mold of Apple Caused by P. expansum

Blue mold is an economically significant postharvest disease of pome fruit that is primarily caused by Penicillium expansum. To manage this disease and sustain product quality, novel decay intervention strategies are needed that also maintain long-term efficacy. Biocontrol organisms and natural products are promising tools for managing postharvest diseases. Here, two Penicillium chrysogenum isolates, 404 and 413, were investigated as potential biocontrol agents against P. expansum in apple. Notably, 404 and 413 were non-pathogenic in apple, yet they grew vigorously in vitro when compared to the highly aggressive P. expansum R19 and Pe21 isolates. Whole-genome sequencing and species-specific barcoding identified both strains as P. chrysogenum. Each P. chrysogenum strain was inoculated in apple with the subsequent co-inoculation of R19 or Pe21 simultaneously, 3, or 7 days after prior inoculation with 404 or 413. The co-inoculation of these isolates showed reduced decay incidence and severity, with the most significant reduction from the longer establishment of P. chrysogenum. In vitro growth showed no antagonism between species, further suggesting competitive niche colonization as the mode of action for decay reduction. Both P. chrysogenum isolates had incomplete patulin gene clusters but tolerated patulin treatment. Finally, hygromycin resistance was observed for both P. chrysogenum isolates, yet they are not multiresistant to apple postharvest fungicides. Overall, we demonstrate the translative potential of P. chrysogenum to serve as an effective biocontrol agent against blue mold decay in apples, pending practical optimization and formulation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Origin of replication discovery for environmentally isolated Pantoea strain enables expression of heterologous proteins, pathways and products

Leveraging predicted origin sequences from a previously characterized groundwater plasmidome, we constructed a barcoded plasmid library to screen for previously unknown origins. Testing this library against a panel of representative bacterial strains led to the identification of 3 previously unknown origins that replicate in gram-negative bacteria not previously associated with these origin sequences. Experimental validation confirmed that a plasmid bearing origin 6911 as the sole origin could replicate with a copy number of 9 (±2) in Pantoea sp. MT58, a fast growing and metal tolerant, environmentally important bacterium. Plasmids based on this new origin were used to express the reporter protein GFP, and non-native metabolite pathways for the natural product indigoidine and the terpenoid compound isoprenol. Functional previously unknown origins of replication in such non-model organisms can expand the toolkit for genetic manipulations of both model and less-studied bacteria.

molecular biology↗
DOE OSTI2022

High throughput single cell multiomics platform [Abstract]

In this collaborative project, PNNL and Scienion will co-develop an integrated microfluidic technology to co-measure the transcriptome and proteome in single cells. The technology will enable us to efficiently separate proteins from mRNA transcripts between two microchips, barcode the molecules, and measure them separately with next-generation sequencing and mass spectrometry, respectively. Multicellular organisms contain diverse cell types and tremendous cell-to-cell heterogeneity that dictates a multitude of biological functions in both physiological and pathological environments. Even in the case of microbes, these genetically identical organisms can randomly differentiate into many subpopulations that assume different roles for the survival of the community. Bulk-scale measurements are insufficient to resolve such complexities. The development and applications of high throughput single-cell omics technologies have transformed our understanding of cellular heterogeneities and their differential responses to internal signaling events or external stimulations. Despite these advances, most current single-cell omic technologies provide information on only one type of biomolecule. Perse, such measurements provide incomplete information because the cell phenotype is determined by multiple layers of biomolecules and the interplay between genome, epigenome, transcriptome, and proteome. For example, mRNA abundance in one cell can not be precisely referred to the corresponding DNA and protein in other cells because of the potential subtle difference in genotype (e.g., somatic mutation or copy number variation) or phenotype (external microenvironment and cell-cell interactions). As such, parallel measurement of multiple biomolecules in the same single cells can offer unique advantages compared with measuring them separately in different single cells. Scienion is a world-leading biotech company focusing on precision liquid handling and its application in single-cell whole-genome sequencing and RNA sequencing. PNNL is the leading institution in ultrasensitive mass spectrometry, microfluidics, and untargeted single-cell proteomics (scProteomics). This collaboration will facilitate a unique fusion between scTranscriptomics capability at Scienion and scProteomics capabilities at PNNL to, for the first time, perform both untargeted transcriptomics and proteomics from the same single cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

FUSARIUM-ID v.3.0: An Updated, Downloadable Resource for Fusarium Species Identification

Species within Fusarium are of global agricultural, medical, and food/feed safety concern and have been extensively characterized. However, accurate identification of species is challenging and usually requires DNA sequence data. FUSARIUM-ID ( http://isolate.fusariumdb.org/blast.php ) is a publicly available database designed to support the identification of Fusarium species using sequences of multiple phylogenetically informative loci, especially the highly informative ~680-bp 5' portion of the translation elongation factor 1-alpha (TEF1) gene that has been adopted as the primary barcoding locus in the genus. However, FUSARIUM-ID v.1.0 and 2.0 had several limitations, including inconsistent metadata annotation for the archived sequences and poor representation of some species complexes and marker loci. Here, we present FUSARIUM-ID v.3.0, which provides the following improvements: (i) additional and updated annotation of metadata for isolates associated with each sequence, (ii) expanded taxon representation in the TEF1 sequence database, (iii) availability of the sequence database as a downloadable file to enable local BLAST queries, and (iv) a tutorial file for users to perform local BLAST searches using either freely available software, such as SequenceServer, BLAST+ executable in the command line, and Galaxy, or the proprietary Geneious software. FUSARIUM-ID will be updated on a regular basis by archiving sequences of TEF1 and other loci from newly identified species and greater in-depth sampling of currently recognized species.

Plant Sciences↗
DOE OSTI2021

Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering

Abstract Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Combined Imaging and RNA-Seq on a Microfluidic Platform for Viral Infection Studies

The goal of this work was to pioneer a novel, low-overhead protocol for simultaneously assaying cell-surface markers and intracellular gene expression in a single mammalian cell. The purpose of developing such a method is to be able to understand the mechanisms by which pathogens engage with individual mammalian cells, depending on their cell surface proteins, and how both host and pathogen gene expression changes are reflective of these mechanisms. The knowledge gained from such analyses of single cells will ultimately lead to more robust pathogen detection and countermeasures. Our method was aimed at streamlining both the upstream cell sample preparation using microfluidic methods, as well as the actual library making protocol. Specifically, we wanted to implement a random hexamer-based reverse transcription of all RNA within a single cell (as opposed to oligo dT-based which would only capture polyadenylated transcripts), and then use a CRISPR-based method called scDash to deplete ribosomal DNAs (since ribosomal RNAs make up the majority of the RNA in a mammalian cell). After significant troubleshooting, we demonstrate that we are able to prepare cDNA from RNA using the random hexamer primer, and perform the rDNA depletion. We also show that we can visualize individually stained cells, setting up the pipeline for connecting surface markers to RNA-sequencing profiles. Finally, we test a number of devices for various parts of the pipeline, including bead generation, optical barcoding and cell dispensing, and demonstrate that while some of these have potential, more work is needed to optimize this part of the pipeline.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Single-nuclei transcriptome analysis of channel catfish spleen provides insight into the immunome of an aquaculture-relevant species

The catfish industry is the largest sector of U.S. aquaculture production. Given its role in food production, the catfish immune response to industry-relevant pathogens has been extensively studied and has provided crucial information on innate and adaptive immune function during disease progression. To further examine the channel catfish immune system, we performed single-cell RNA sequencing on nuclei isolated from whole spleens, a major lymphoid organ in teleost fish. Libraries were prepared using the 10X Genomics Chromium X with the Next GEM Single Cell 3’ reagents and sequenced on an Illumina sequencer. Each demultiplexed sample was aligned to the Coco_2.0 channel catfish reference assembly, filtered, and counted to generate feature-barcode matrices. From whole spleen samples, outputs were analyzed both individually and as an integrated dataset. The three splenic transcriptome libraries generated an average of 278,717,872 reads from a mean 8,157 cells. The integrated data included 19,613 cells, counts for 20,121 genes, with a median 665 genes/cell. Cluster analysis of all cells identified 17 clusters which were classified as erythroid, hematopoietic stem cells, B cells, T cells, myeloid cells, and endothelial cells. Subcluster analysis was carried out on the immune cell populations. Here, distinct subclusters such as immature B cells, mature B cells, plasma cells, γδ T cells, dendritic cells, and macrophages were further identified. Differential gene expression analyses allowed for the identification of the most highly expressed genes for each cluster and subcluster. This dataset is a rich cellular gene expression resource for investigation of the channel catfish and teleost splenic immunome.

Science & Technology - Other Topics↗
DOE OSTI2023

Automated methods for scalable, parallelized enzymatic biopolymer synthesis and modification using microfluidic devices

Methods for the automated template-free synthesis of user-defined sequence controlled biopolymers using microfluidic devices are described. The methods facilitate simultaneous synthesis of up to thousands of uniquely addressed biopolymers from the controlled movement and combination of regents as fluid droplets using microfluidic and EWOD-based systems. In some forms, biopolymers including nucleic acids, peptides, carbohydrates, and lipids are synthesized from step-wise assembly of building blocks based on a user-defined sequence of droplet movements. In some forms, the methods synthesize uniquely addressed nucleic acids of up to 1,000 nucleotides in length. Methods for adding, removing and changing barcodes on biopolymers are also provided. Biopolymers synthesized according to the methods, and libraries and databases thereof are also described. Modified biopolymers, including chemically modified nucleotides and biopolymers conjugated to other molecules are described.

Banal, James↗
DOE OSTI2022

A high-throughput skim-sequencing approach for genotyping, dosage estimation and identifying translocations

The development of next-generation sequencing (NGS) enabled a shift from array-based genotyping to directly sequencing genomic libraries for high-throughput genotyping. Even though whole-genome sequencing was initially too costly for routine analysis in large populations such as breeding or genetic studies, continued advancements in genome sequencing and bioinformatics have provided the opportunity to capitalize on whole-genome information. As new sequencing platforms can routinely provide high-quality sequencing data for sufficient genome coverage to genotype various breeding populations, a limitation comes in the time and cost of library construction when multiplexing a large number of samples. Here we describe a high-throughput whole-genome skim-sequencing (skim-seq) approach that can be utilized for a broad range of genotyping and genomic characterization. Using optimized low-volume Illumina Nextera chemistry, we developed a skim-seq method and combined up to 960 samples in one multiplex library using dual index barcoding. With the dual-index barcoding, the number of samples for multiplexing can be adjusted depending on the amount of data required, and could be extended to 3,072 samples or more. Panels of doubled haploid wheat lines ( Triticum aestivum , CDC Stanley x CDC Landmark), wheat-barley ( T . aestivum x Hordeum vulgare ) and wheat-wheatgrass ( Triticum durum x Thinopyrum intermedium ) introgression lines as well as known monosomic wheat stocks were genotyped using the skim-seq approach. Bioinformatics pipelines were developed for various applications where sequencing coverage ranged from 1 × down to 0.01 × per sample. Using reference genomes, we detected chromosome dosage, identified aneuploidy, and karyotyped introgression lines from the skim-seq data. Leveraging the recent advancements in genome sequencing, skim-seq provides an effective and low-cost tool for routine genotyping and genetic analysis, which can track and identify introgressions and genomic regions of interest in genetics research and applied breeding programs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Methods for generating barcoded combinatorial libraries

Provided herein are methods and composition for trackable genetic variant libraries. Further provided herein are methods and compositions for recursive engineering. Further provided herein are methods and compositions for multiplex engineering. Further provided herein are methods and compositions for enriching for editing and trackable engineered sequences and cells using nucleic acid-guided nucleases.

Gill, Ryan T.↗
DOE OSTI2020

Distinctiveness of genes contributing to growth of Pseudomonas syringae in diverse host plant species

A variety of traits are necessary for bacterial colonization of the interior of plant hosts, including well-studied virulence effectors as well as other phenotypes contributing to bacterial growth and survival within the apoplast. High-throughput methods such as transposon sequencing (TnSeq) are powerful tools to identify such genes in bacterial pathogens. However, there is little information as to the distinctiveness of traits required for bacterial colonization of different hosts. Here, we utilize randomly barcoded TnSeq (RB-TnSeq) to identify the genes that contribute to the ability of Pseudomonas syringae strain B728a to grow within common bean (Phaseolus vulgaris), lima bean (Phaseolus lunatus), and pepper (Capsicum annuum); species representing two different plant families. The magnitude of contribution of most genes to apoplastic fitness in each of the plant hosts was similar. However, 50 genes significantly differed in their fitness contributions to growth within these species. These genes encoded proteins in various functional categories including polysaccharide synthesis and transport, amino acid metabolism and transport, cofactor metabolism, and phytotoxin synthesis and transport. Six genes that encoded unannotated, hypothetical proteins also contributed differentially to growth in these hosts. The genetic repertoire of a relatively promiscuous pathogen such as P. syringae may thus be shaped, at least in part, by the conditional contribution of some fitness determinants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Elucidation of a bacterial pathway for catabolism of the β–β-linked dilignol pinoresinol

Monolignol-derived dimers containing β–β linkages are synthesized by vascular plants and can be released during lignin depolymerization. In this work, we isolated a bacterium, Novosphingobium rhizosphaerae LY, that grows with the β–β lignan (+)-pinoresinol as a sole growth substrate. Sequence analysis suggested that this strain encodes a broad range of pathways for assimilation of aromatic monomers as well as one enzyme implicated in pinoresinol catabolism but lacks other known pathways for aromatic dimer catabolism. We constructed a genome-wide barcoded transposon library and identified genes required for pinoresinol catabolism. Using feeding studies, compound isolation, targeted synthesis, and analysis of purified enzymes, we elucidated the biochemical intermediates and reaction pathway involved in pinoresinol catabolism. We demonstrated that the first enzymatic reaction is the reductive cleavage of a furan ring in (±)-pinoresinol with retention of configuration to yield lariciresinol. We additionally confirmed that the final pathway enzyme, PinU, is related to lignostilbene dioxygenases and oxidatively cleaves a diguaiacylbutadiene intermediate to yield vanillin and coniferaldehyde. Finally, based on the enzyme characterization, we demonstrated that the strain can grow with a second β–β lignan, (–)-syringaresinol, as a sole growth substrate. In combination, these results demonstrate a new biocatalytic route for transforming a widely occurring group of plant phenylpropanoid natural products.

Novosphingobium↗
DOE OSTI2024

Measurements of soil protist richness and community composition are influenced by primer pair, annealing temperature, and bioinformatics choices

ABSTRACT Protists are a diverse and understudied group of microbial eukaryotic organisms especially in terrestrial environments. Advances in molecular methods are increasing our understanding of the distribution and functions of these creatures; however, there is a vast array of choices researchers make including barcoding genes, primer pairs, PCR settings, and bioinformatic options that can impact the outcome of protist community surveys. Here, we tested four commonly used primer pairs targeting the V4 and V9 regions of the 18S rRNA gene using different PCR annealing temperatures and processed the sequences with different bioinformatic parameters in 10 diverse soils to evaluate how primer pair, amplification parameters, and bioinformatic choices influence the composition and richness of protist and non-protist taxa using Illumina sequencing. Our results showed that annealing temperature influenced sequencing depth and protist taxon richness for most primer pairs, and that merging forward and reverse sequencing reads for the V4 primer pairs dramatically reduced the number of sequences and taxon richness of protists. The data sets of primers that targeted the same 18S rRNA gene region (e.g., V4 or V9) had similar protist community compositions; however, data sets from primers targeting the V4 18S rRNA gene region detected a greater number of protist taxa compared to those prepared with primers targeting the V9 18S rRNA region. There was limited overlap of protist taxa between data sets targeting the two different gene regions (80/549 taxa). Together, we show that laboratory and bioinformatic choices can substantially affect the results and conclusions about protist diversity and community composition using metabarcoding. IMPORTANCE Ecosystem functioning is driven by the activity and interactions of the microbial community, in both aquatic and terrestrial environments. Protists are a group of highly diverse, mostly unicellular microbes whose identity and roles in terrestrial ecosystem ecology have been largely ignored until recently. This study highlights the importance of choices researchers make, such as primer pair, on the results and conclusions about protist diversity and community composition in soils. In order to better understand the roles protist taxa play in terrestrial ecosystems, biases in methodological and analytical choices should be understood and acknowledged.

Biotechnology & Applied Microbiology↗
DOE OSTI2024

Potential for functional divergence in ectomycorrhizal fungal communities across a precipitation gradient

Abstract Functional traits influence the assembly of microbial communities, but identifying these traits in the environment has remained challenging. We studied ectomycorrhizal fungal (EMF) communities inhabiting Populus trichocarpa roots distributed across a precipitation gradient in the Pacific Northwest, USA. We profiled these communities using taxonomic (meta-barcoding) and functional (metagenomic) approaches. We hypothesized that genes involved in fungal drought-stress tolerance and fungal mediated plant water uptake would be most abundant in drier soils. We were unable to detect support for this hypothesis; instead, the abundance of genes involved in melanin synthesis, hydrophobins, aquaporins, trehalose-synthases, and other gene families exhibited no significant shifts across the gradient. Finally, we studied variation in sequence homology for certain genes, finding that fungal communities in dry soils are composed of distinct aquaporin and hydrophobin gene sequences. Altogether, our results suggest that while EMF communities exhibit significant compositional shifts across this gradient, coupled functional turnover, at least as inferred using community metagenomics is limited. Accordingly, the consequences of these distinct EMF communities on plant water uptake remain critically unknown, and future studies targeting the expression of genes involved in drought stress tolerance are required.

59 BASIC BIOLOGICAL SCIENCES↗