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At least 991 records · Page 55
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Tailoring poplar lignin without yield penalty by combining a null and haploinsufficient CINNAMOYL-CoA REDUCTASE2 allele

Lignin causes lignocellulosic biomass recalcitrance to enzymatic hydrolysis. Engineered low-lignin plants have reduced recalcitrance but often exhibit yield penalties, offsetting their gains in fermentable sugar yield. Here, CRISPR/Cas9-generated CCR2(–/*) line 12 poplars have one knockout CCR2 allele while the other contains a 3-bp deletion, resulting in a 114I115A-to-114T conversion in the corresponding protein. Despite having 10% less lignin, CCR2(–/*) line 12 grows normally. On a plant basis, the saccharification efficiency of CCR2(–/*) line 12 is increased by 25–41%, depending on the pretreatment. Analysis of monoallelic CCR2 knockout lines shows that the reduced lignin amount in CCR2(–/*) line 12 is due to the combination of a null and the specific haploinsufficient CCR2 allele. Analysis of another CCR2(–/*) line shows that depending on the specific CCR2 amino-acid change, lignin amount and growth can be affected to different extents. Furthermore, our findings open up new possibilities for stably fine-tuning residual gene function in planta.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Development of genetic tools for heterologous protein expression in a pentose‐utilizing environmental isolate of Pseudomonas putida

Abstract Pseudomonas putida has emerged as a promising host for the conversion of biomass‐derived sugars and aromatic intermediates into commercially relevant biofuels and bioproducts. Most of the strain development studies previously published have focused on P. putida KT2440, which has been engineered to produce a variety of non‐native bioproducts. However, P. putida is not capable of metabolizing pentose sugars, which can constitute up to 25% of biomass hydrolysates. Related P. putida isolates that metabolize a larger fraction of biomass‐derived carbon may be attractive as complementary hosts to P. putida KT2440. Here we describe genetic tool development for P. putida M2, a soil isolate that can metabolize pentose sugars. The functionality of five inducible promoter systems and 12 ribosome binding sites was assessed to regulate gene expression. The utility of these expression systems was confirmed by the production of indigoidine from C6 and C5 sugars. Chromosomal integration and expression of non‐native genes was achieved by using chassis‐independent recombinase‐assisted genome engineering (CRAGE) for single‐step gene integration of biosynthetic pathways directly into the genome of P. putida M2. These genetic tools provide a foundation to develop hosts complementary to P. putida KT2440 and expand the ability of this versatile microbial group to convert biomass to bioproducts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Data for Characterization of the Ghd8 Flowering Time Gene in a Mini-Core Collection of Miscanthus sinensis

The optimal flowering time for bioenergy crop miscanthus is essential for environmental adaptability and biomass accumulation. However, little is known about how genes controlling flowering in other grasses contribute to flowering regulation in miscanthus. Here, we report on the sequence characterization and gene expression of Miscanthus sinensisGhd8 , a transcription factor encoding a HAP3/NF-YB DNA-binding domain, which has been identified as a major quantitative trait locus in rice, with pleiotropic effects on grain yield, heading date and plant height. In M. sinensis , we identified two homoeologous loci, MsiGhd8A located on chromosome 13 and MsiGhd8B on chromosome 7, with one on each of this paleo-allotetraploid species’ subgenomes. A total of 46 alleles and 28 predicted protein sequence types were identified in 12 wild-collected accessions. Several variants of MsiGhd8 showed a geographic and latitudinal distribution. Quantitative real-time PCR revealed that MsiGhd8 expressed under both long days and short days, and MsiGhd8B showed a significantly higher expression than MsiGhd8A . The comparison between flowering time and gene expression indicated that MsiGhd8B affected flowering time in response to day length for some accessions. This study provides insight into the conserved function of Ghd8 in the Poaceae, and is an important initial step in elucidating the flowering regulatory network of Miscanthus.

Feedstock Production↗
NASA NTRS2016

Developing Personalized Sensorimotor Adaptability Countermeasures for Spaceflight

Astronauts experience sensorimotor disturbances during their initial exposure to microgravity and during the re-adaptation phase following a return to an Earth-gravitational environment. Interestingly, astronauts who return from spaceflight show substantial differences in their abilities to readapt to a gravitational environment. The ability to predict the manner and degree to which individual astronauts would be affected would improve the effectiveness of countermeasure training programs designed to enhance sensorimotor adaptability. In this paper we will be presenting results from our ground-based study that show how behavioral, brain imaging and genomic data may be used to predict individual differences in sensorimotor adaptability to novel sensorimotor environments. This approach will allow us to better design and implement sensorimotor adaptability training countermeasures against decrements in post-mission adaptive capability that are customized for each crewmember's sensory biases, adaptive capacity, brain structure, functional capacities, and genetic predispositions. The ability to customize adaptability training will allow more efficient use of crew time during training and will optimize training prescriptions for astronauts to ensure expected outcomes.

Mulavara, A. P.↗
DOE OSTI2021

Molecular principles of Piwi-mediated cotranscriptional silencing through the dimeric SFiNX complex

Nuclear Argonaute proteins, guided by their bound small RNAs to nascent target transcripts, mediate cotranscriptional silencing of transposons and repetitive genomic loci through heterochromatin formation. The molecular mechanisms involved in this process are incompletely understood. Here, we show that the SFiNX complex, a silencing mediator downstream from nuclear Piwi-piRNA complexes in Drosophila, facilitates cotranscriptional silencing as a homodimer. The dynein light chain protein Cut up/LC8 mediates SFiNX dimerization, and its function can be bypassed by a heterologous dimerization domain, arguing for a constitutive SFiNX dimer. Dimeric, but not monomeric SFiNX, is capable of forming molecular condensates in a nucleic acid-stimulated manner. Mutations that prevent SFiNX dimerization result in loss of condensate formation in vitro and the inability of Piwi to initiate heterochromatin formation and silence transposons in vivo. We propose that multivalent SFiNX-nucleic acid interactions are critical for heterochromatin establishment at piRNA target loci in a cotranscriptional manner.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Hyporheic zone, river, and groundwater metagenome resolved genomes and rpS3 genes in East River Watershed, Colorado USA Summer 2020, 2021

Here we present metagenome assembled genomes (MAGs) for the bacterial and archaeal communities from water filter collected across 8 locations along the East River Watershed, CO, and 1 nearby groundwater well. The purpose was to look for connectivity and similarities across the network and to see the impact of the groundwater. As a part of Lawrence Berkeley National Laboratory (LBNL) Watershed Science Focus Area (SFA), we assessed community composition and strain similarities between the sites and we also compared it to previous metagenomic studies within the watershed looking at floodplain (Matheus Carnevali et al. 2021) and hillslope (Lavy et al. 2019) microbiomes. Here we present metagenome assembled genomes (MAGs) for the bacterial and archaeal communities from filters across 8 locations during August 2020 and July 2021. This resulted in 32 samples. The groundwater sample was sequenced at UC Berkley's QB3. The other 31 samples were sequenced at University of Maryland. Metagenomes were assembled using four autobinners and the best bins were selected using dasTool. The genomes were dereplicated at 95% with dRep and the subset of winning genomes were manually curated based on visual inspection of taxonomic profile, GC content, coverage, and a set of 51 bacterial single copy genes (BSCG), and 38 archaeal signal copy genes (ASCG). The dataset includes a zip file of 311 genomes (HZ_River_SW_MAGS_Dereplicated_95.zip). The dataset additionally includes a zipped file of ribosomal protein small subunit 3 (rpS3) proteins from the hyporheic zone and river data (rpS3_Proteins_HZ_River.zip), a metadata file used to register associated samples with IGSNs (International Generic Sample Numbers) (samples.csv), a location metadata file (locations.csv). This work was supported by the Watershed Function Science Focus Area at Lawrence Berkeley National Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-05CH11231.

DNA↗
DOE OSTI2026

Impact of Selenium and Vitamin E Deficiency on Zika Virus Pathogenesis and Immune Response in Mice

Micronutrient status is recognized to influence host susceptibility to viral infections, yet its impact on Zika virus (ZIKV) pathogenesis remains incompletely understood. We investigated the effects of dietary selenium and combined selenium plus vitamin E deficiency on ZIKV infection outcomes in a type I interferon α/β receptor knockout (Ifnar1 −/− ) murine model. Mice maintained on deficient diets exhibited significantly lower neutralizing antibody titers and reduced levels of key antiviral cytokines (IFN-γ, TNF-α, IFN-α, IFN-β, IL-12p70, CCL5) compared to controls. Correspondingly, higher viral RNA loads were detected in the brains of double-deficient mice, which also experienced greater weight loss and increased mortality. Deep sequencing revealed no major differences in overall viral genome diversity across diet groups; however, specific mutations, including V330L and D67E in the E gene, and V360I in the NS3 gene, were enriched or detected in nutritionally deficient animals. These findings suggest that antioxidant micronutrient deficiency impairs both humoral and cellular immune responses to ZIKV, potentially facilitating enhanced neuroinvasion. While the functional consequences of the identified mutations warrant further investigation, our results underscore the importance of adequate micronutrient intake for optimal antiviral defense. Further studies are needed to clarify the epidemiological significance of these observations.

Biological and medical sciences↗
DOE OSTI2021

Global overview and major challenges of host prediction methods for uncultivated phages

Bacterial communities play critical roles across all of Earth’s biomes, affecting human health and global ecosystem functioning. They do so under strong constraints exerted by viruses, i.e., bacteriophages or “phages”. Phages can reshape bacterial communities’ structure, influence long-term evolution of bacterial populations, and alter host cell metabolism during infection. Metagenomics approaches, i.e., shotgun sequencing of environmental DNA or RNA, recently enabled large-scale exploration of phage genomic diversity, yielding several millions of phage genomes now to be further analyzed and characterized. One major challenge however is the lack of direct host information for these phages. Several methods and tools have been proposed to bioinformatically predict the potential host(s) of uncultivated phages based only on genome sequence information. Here we review these different approaches and highlight their distinct strengths and limitations. We also outline complementary experimental assays which are being proposed to validate and refine these bioinformatic predictions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A call for caution in the biological interpretation of viral auxiliary metabolic genes

Virus-encoded auxiliary metabolic genes (AMGs) are non-essential genes that increase viral fitness by maintaining or manipulating host metabolism during infection. AMGs are intriguing from an evolutionary perspective, as most viral genomes are highly compact and have limited coding capacity for accessory genes. Advances in viral (meta)genomics have expanded the detection of putative AMGs from viruses in diverse environments. However, this has also led to many instances of misannotation due to the limitations of annotation tools, resulting in misinterpretations about the roles of some viral genes. Here, we highlight studies that support claims about AMGs with more than just function predictions for guidance on best practices. We then propose the adoption of an expanded, inclusive view of all genes auxiliary to core viral functions with the term ‘auxiliary viral genes’ (AVGs), alongside an associated eco-evolutionary framework for considering the types of analyses that can better support claims made about AVGs.

Environmental microbiology↗
DOE OSTI2021

The F-box protein gene exo-1 is a target for reverse engineering enzyme hypersecretion in filamentous fungi

Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein–encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain ?cre-1, while glucose repression was still mostly functional in ?exo-1. Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in ?exo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in ?exo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.

Gabriel, Raphael↗
DOE OSTI2025

Revisiting synthetic lethality of Gcn5-related N-acetyltransferase (GNAT) family mutations in Haloferax volcanii

ABSTRACT Lysine acetylation is a post-translational modification that occurs in all domains of life, highlighting its evolutionary significance. Previous genome comparison identified three Gcn5-related N-acetyltransferase (GNAT) family members as lysine acetyltransferase homologs (Pat1, Pat2, and Elp3) and two deacetylase homologs (Sir2 and HdaI) in the halophilic archaeonHaloferax volcanii, withelp3andpat2proposed as a synthetic lethal gene pair. Here, we advance these findings by performing single and double mutagenesis ofelp3with thepat1andpat2lysine acetyltransferase gene homologs. Genome sequencing and PCR screens of these strains reveal successful generation of Δelp3,Δpat1Δelp3, and Δpat2Δelp3mutant strains. Although these mutant strains exhibited a reduced growth rate compared to the parent, they remained viable. Overall, this study provides genetic evidence thatelp3andpat2, while impacting cell growth, are not a synthetic lethal gene pair as previously reported. IMPORTANCE Here, we reveal by whole-genome sequencing that the GNAT family gene homologselp3andpat2can be deleted in the sameHaloferax volcaniistrain. Beyond the targeted deletions, minimal differences between the parent and Δelp3Δpat2mutant were observed, suggesting that suppressor mutations are not responsible for our ability to generate this double mutant strain. Elp3 and Pat2, thus, may not share as close a functional relationship as implied by earlier study. Our finding is significant as Elp3 is thought to function in acetylation in tRNA modification, while Pat2 likely functions in the lysine acetylation of proteins.

Microbiology↗
DOE OSTI2024

The Exometabolome of Xylella fastidiosa in Contact with Paraburkholderia phytofirmans Supernatant Reveals Changes in Nicotinamide, Amino Acids, Biotin, and Plant Hormones

Microbial competition within plant tissues affects invading pathogens’ fitness. Metabolomics is a great tool for studying their biochemical interactions by identifying accumulated metabolites. Xylella fastidiosa, a Gram-negative bacterium causing Pierce’s disease (PD) in grapevines, secretes various virulence factors including cell wall-degrading enzymes, adhesion proteins, and quorum-sensing molecules. These factors, along with outer membrane vesicles, contribute to its pathogenicity. Previous studies demonstrated that co-inoculating X. fastidiosa with the Paraburkholderia phytofirmans strain PsJN suppressed PD symptoms. Here, we further investigated the interaction between the phytopathogen and the endophyte by analyzing the exometabolome of wild-type X. fastidiosa and a diffusible signaling factor (DSF) mutant lacking quorum sensing, cultivated with 20% P. phytofirmans spent media. Liquid chromatography–mass spectrometry (LC-MS) and the Method for Metabolite Annotation and Gene Integration (MAGI) were used to detect and map metabolites to genomes, revealing a total of 121 metabolites, of which 25 were further investigated. These metabolites potentially relate to host adaptation, virulence, and pathogenicity. Notably, this study presents the first comprehensive profile of X. fastidiosa in the presence of a P. phytofirmans spent media. The results highlight that P. phytofirmans and the absence of functional quorum sensing affect the ratios of glutamine to glutamate (Gln:Glu) in X. fastidiosa. Additionally, two compounds with plant metabolism and growth properties, 2-aminoisobutyric acid and gibberellic acid, were downregulated when X. fastidiosa interacted with P. phytofirmans. These findings suggest that P. phytofirmans-mediated disease suppression involves modulation of the exometabolome of X. fastidiosa, impacting plant immunity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Genomic discovery of an evolutionarily programmed modality for small-molecule targeting of an intractable protein surface

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of “undruggability” for an intracellular target. Structural studies reveal extensive protein–WDB002 and protein–protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise “undruggable” targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Tapping the treasure trove of atypical phages

With advancements in genomics technologies, a vast diversity of ‘atypical’ phages, that is, with single-stranded DNA or RNA genomes, are being uncovered from different ecosystems. Though these efforts have revealed the existence and prevalence of these nonmodel phages, computational approaches often fail to associate these phages with their specific bacterial host(s), while the lack of methods to isolate these phages has limited our ability to characterize infectivity pathways and new gene function. In this review, we call for the development of generalizable experimental methods to better capture this understudied viral diversity via isolation and study them through gene-level characterization and engineering. Establishing a diverse set of new ‘atypical’ phage model systems has the potential to provide many new biotechnologies, including potential uses of these atypical phages in halting the spread of antibiotic resistance and engineering of microbial communities for beneficial outcomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Advanced genomics identifies growth effectors for proteotoxic ER stress recovery in Arabidopsis thaliana

Abstract Adverse environmental and pathophysiological situations can overwhelm the biosynthetic capacity of the endoplasmic reticulum (ER), igniting a potentially lethal condition known as ER stress. ER stress hampers growth and triggers a conserved cytoprotective signaling cascade, the unfolded protein response (UPR) for ER homeostasis. As ER stress subsides, growth is resumed. Despite the pivotal role of the UPR in growth restoration, the underlying mechanisms for growth resumption are yet unknown. To discover these, we undertook a genomics approach in the model plant species Arabidopsis thaliana and mined the gene reprogramming roles of the UPR modulators, basic leucine zipper28 (bZIP28) and bZIP60, in ER stress resolution. Through a network modeling and experimental validation, we identified key genes downstream of the UPR bZIP-transcription factors (bZIP-TFs), and demonstrated their functional roles. Our analyses have set up a critical pipeline for functional gene discovery in ER stress resolution with broad applicability across multicellular eukaryotes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Coexpressed subunits of dual genetic origin define a conserved supercomplex mediating essential protein import into chloroplasts

Significance Chloroplasts are of vital importance in photosynthetic eukaryotic organisms. Like mitochondria, they contain their own genomes. Nevertheless, most chloroplast proteins are encoded by nuclear genes, translated in the cytosol, and must cross the chloroplast envelope membranes to reach their proper destinations inside the organelle. Despite its fundamental role, our knowledge of the machinery catalyzing chloroplast protein import is incomplete and controversial. Here, we address the evolutionary conservation, composition, and function of the chloroplast protein import machinery using the green alga Chlamydomonas reinhardtii as a model system. Our findings help clarify the current debate regarding the composition of the chloroplast protein import machinery, provide evidence for cross-compartmental coordination of its biogenesis, and open promising avenues for its structural characterization.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

High-throughput identification of novel heat tolerance genes via genome-wide pooled mutant screens in the model green alga Chlamydomonas reinhardtii

Different high temperatures adversely affect crop and algal yields with various responses in photosynthetic cells. The list of genes required for thermotolerance remains elusive. Additionally, it is unclear how carbon source availability affects heat responses in plants and algae. Here, we utilized the insertional, indexed, genome-saturating mutant library of the unicellular, eukaryotic green alga Chlamydomonas reinhardtii to perform genome-wide, quantitative, pooled screens under moderate (35°C) or acute (40°C) high temperatures with or without organic carbon sources. We identified heat-sensitive mutants based on quantitative growth rates and identified putative heat tolerance genes (HTGs). By triangulating HTGs with heat-induced transcripts or proteins in wildtype cultures and MapMan functional annotations, we presented a high/medium-confidence list of 933 Chlamydomonas genes with putative roles in heat tolerance. Triangulated HTGs include those with known thermotolerance roles and novel genes with little or no functional annotation. About 50% of these high-confidence HTGs in Chlamydomonas have orthologs in green lineage organisms, including crop species. Arabidopsis thaliana mutants deficient in the ortholog of a high-confidence Chlamydomonas HTG were also heat sensitive. This work expands our knowledge of heat responses in photosynthetic cells and provides engineering targets to improve thermotolerance in algae and crops.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Virulence factors and antimicrobial resistance profiles of Campylobacter isolates recovered from consecutively reused broiler litter

ABSTRACT Campylobacterinfections are a leading cause of bacterial diarrhea in humans globally. Infections are due to consumption of contaminated food products and are highly associated with chicken meat, with chickens being an important reservoir forCampylobacter. Here, we characterized the genetic diversity ofCampylobacter jejuni(C. jejuni) andCampylobacter coli(C. coli) detected in broiler chicken litter over three consecutive flocks and determined their antimicrobial resistance (ARM) and virulence factor (VF) profiles.Campylobacterwas detected in 9.38% (27/288) of litter samples collected. Antimicrobial susceptibility testing and whole genome sequencing were performed onC. jejuni(n= 39) andC. coli(n= 5) isolates.Campylobactervirulence factors differed within and across broiler houses but were explained by the broiler flock cohort raised on litter,Campylobacterspecies andCampylobactermultilocus sequence type (MLST). Virulence factors involved in the ability to invade and colonize host tissues and evade host defenses were present inC. jejuniisolates (ST-464) from flock cohorts 1 and 2 but absent inC. jejuniisolates (ST-48) from flock cohort 3.C. jejuniisolates from house three harbored a significantly higher proportion of virulence genes with functions related to glycosylation and immune evasion thanC. jejuniisolates from houses 1 and 2 (P< 0.01). AllC. jejuniisolates were susceptible to all antibiotics tested whileC. coli(n= 4) were resistant to tetracycline and harbored the tetracycline resistant ribosomal protection protein (TetO). Our results suggest that house environment and broiler management practices imposed selective pressures on virulence factors and antimicrobial resistance genes ofCampylobacter. IMPORTANCE Campylobacteris a leading cause of foodborne illness in the United States due to consumption of contaminated or mishandled food products, often associated with chicken meat.Campylobacteris common in the microbiota of avian and mammalian gut; however, acquisition of antimicrobial resistance genes (ARGs) and virulence factors (VFs) may result in strains that pose significant threat to public health. Although there are studies investigating the genetic diversity ofCampylobacterstrains isolated from post-harvest chicken samples, there are limited data on the genome characteristics of isolates recovered from preharvest broiler production. Here, we show thatCampylobacter jejuniandCampylobacter colidiffer in their carriage of antimicrobial resistance and virulence factors may also differ in their ability to persist in litter during consecutive grow-out of broiler flocks. We found that presence/absence of virulence factors needed for evasion of host defense mechanisms and gut colonization played an integral role in differentiatingCampylobacterstrains.

Microbiology↗