Search NASA⌕ Search

SEARCH · Search NASA

Results for “gene transfer”

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.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 253 records · Page 14
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

A t FUT4 and A t FUT6 Are Arabinofuranose-Specific Fucosyltransferases

The bulk of plant biomass is comprised of plant cell walls, which are complex polymeric networks, composed of diverse polysaccharides, proteins, polyphenolics, and hydroxyproline-rich glycoproteins (HRGPs). Glycosyltransferases (GTs) work together to synthesize the saccharide components of the plant cell wall. The Arabidopsis thaliana fucosyltransferases (FUTs), At FUT4, and At FUT6, are members of the plant-specific GT family 37 (GT37). At FUT4 and At FUT6 transfer fucose (Fuc) onto arabinose (Ara) residues of arabinogalactan (AG) proteins (AGPs) and have been postulated to be non-redundant AGP-specific FUTs. At FUT4 and At FUT6 were recombinantly expressed in mammalian HEK293 cells and purified for biochemical analysis. We report an updated understanding on the specificities of At FUT4 and At FUT6 that are involved in the synthesis of wall localized AGPs. Our findings suggest that they are selective enzymes that can utilize various arabinogalactan (AG)-like and non-AG-like oligosaccharide acceptors, and only require a free, terminal arabinofuranose. We also report with GUS promoter-reporter gene studies that AtFUT4 and AtFUT6 gene expression is sub-localized in different parts of developing A. thaliana roots.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Endothelium-specific endothelin-1 expression promotes pro-inflammatory macrophage activation by regulating miR-33/NR4A axis

The hallmark of atherogenesis is characterized as endothelial dysfunction and subsequent macrophage activation. Although our previous study has demonstrated that endothelin-1 (ET-1) plays an important role in atherogenesis, the underlying mechanism remains deeply investigation. Enhanced atherosclerotic plaques were observed in endothelium-specific ET-1 overexpression ApoE{sup −/-} mice (eET-1/ApoE{sup −/-}) concomitant with increased secretion of pro-inflammatory adhesion molecules and cytokines. The conditional media used for culturing human umbilical vein endothelial cells (HUVECs) with AdET-1 infection and subjected to OX-LDL stimulation, was collected and utilized for bone marrow-derived macrophages (BMDMs) culturing. RT-PCR analysis showed increased genes expression related to classical M1 macrophages but decreased alternative activated M2 macrophages genes expression in macrophage culturing with the conditional media. Furthermore, consistent regulations of macrophage polarization were observed using isolated exosomes from the conditional media. More importantly, we noticed that miR-33 was enriched in the exosomes derived by HUVECs with AdET-1 infection, while bioinformatics analysis further indicated that miR-33 directly targeted NR4A and miR-33/NR4A axis was required for the effect of endothelial-specific ET-1 overexpression on pro-inflammatory macrophage activation. By contrast, such effects could be reversed by ET-1 knockdown. Taken together, our study indicated that the exosomes derived by HUVECs with AdET-1 infection can transfer miR-33 to macrophages and subsequently promote pro-inflammatory macrophage activation by directly targeting to NR4A. These evidences clearly revealed that miR-33/NR4A axis was the important mechanism underlying the effect of ET-1 on macrophage activation and indicated that ET-1 may act as a promising target for atherosclerosis management.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Identification of a pathway for electron uptake in Shewanella oneidensis

Extracellular electron transfer (EET) could enable electron uptake into microbial metabolism for the synthesis of complex, energy dense organic molecules from CO 2 and renewable electricity. Theoretically EET could do this with an efficiency comparable to H 2 -oxidation but without the need for a volatile intermediate and the problems it causes for scale up. However, significant gaps remain in understanding the mechanism and genetics of electron uptake. For example, studies of electron uptake in electroactive microbes have shown a role for the Mtr EET complex in the electroactive microbe Shewanella oneidensis MR-1, though there is substantial variation in the magnitude of effect deletion of these genes has depending on the terminal electron acceptor used. This speaks to the potential for previously uncharacterized and/or differentially utilized genes involved in electron uptake. To address this, we screened gene disruption mutants for 3667 genes, representing ≈99% of all nonessential genes, from the S. oneidensis whole genome knockout collection using a redox dye oxidation assay. Confirmation of electron uptake using electrochemical testing allowed us to identify five genes from S. oneidensis that are indispensable for electron uptake from a cathode. Knockout of each gene eliminates extracellular electron uptake, yet in four of the five cases produces no significant defect in electron donation to an anode. This result highlights both distinct electron uptake components and an electronic connection between aerobic and anaerobic electron transport chains that allow electrons from the reversible EET machinery to be coupled to different respiratory processes in S. oneidensis. Homologs to these genes across many different genera suggesting that electron uptake by EET coupled to respiration could be widespread. These gene discoveries provide a foundation for: studying this phenotype in exotic metal-oxidizing microbes, genetic optimization of electron uptake in S. oneidensis; and genetically engineering electron uptake into a highly tractable host like E. coli to complement recent advances in synthetic CO2 fixation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A method for achieving complete microbial genomes and improving bins from metagenomics data

Metagenomics facilitates the study of the genetic information from uncultured microbes and complex microbial communities. Assembling complete genomes from metagenomics data is difficult because most samples have high organismal complexity and strain diversity. Some studies have attempted to extract complete bacterial, archaeal, and viral genomes and often focus on species with circular genomes so they can help confirm completeness with circularity. However, less than 100 circularized bacterial and archaeal genomes have been assembled and published from metagenomics data despite the thousands of datasets that are available. Circularized genomes are important for (1) building a reference collection as scaffolds for future assemblies, (2) providing complete gene content of a genome, (3) confirming little or no contamination of a genome, (4) studying the genomic context and synteny of genes, and (5) linking protein coding genes to ribosomal RNA genes to aid metabolic inference in 16S rRNA gene sequencing studies. We developed a semi-automated method called Jorg to help circularize small bacterial, archaeal, and viral genomes using iterative assembly, binning, and read mapping. In addition, this method exposes potential misassemblies from k-mer based assemblies. We chose species of the Candidate Phyla Radiation (CPR) to focus our initial efforts because they have small genomes and are only known to have one ribosomal RNA operon. In addition to 34 circular CPR genomes, we present one circular Margulisbacteria genome, one circular Chloroflexi genome, and two circular megaphage genomes from 19 public and published datasets. We demonstrate findings that would likely be difficult without circularizing genomes, including that ribosomal genes are likely not operonic in the majority of CPR, and that some CPR harbor diverged forms of RNase P RNA. Code and a tutorial for this method is available at https://github.com/lmlui/Jorg and is available on the DOE Systems Biology KnowledgeBase as a beta app.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Metagenomic Insight into Environmentally Challenged Methane-Fed Microbial Communities

In this study, we aimed to investigate, through high-resolution metagenomics and metatranscriptomics, the composition and the trajectories of microbial communities originating from a natural sample, fed exclusively with methane, over 14 weeks of laboratory incubation. This study builds on our prior data, suggesting that multiple functional guilds feed on methane, likely through guild-to-guild carbon transfer, and potentially through intraguild and intraspecies interactions. We observed that, under two simulated dioxygen partial pressures—low versus high—community trajectories were different, with considerable variability among the replicates. In all microcosms, four major functional guilds were prominently present, representing Methylococcaceae (the true methanotrophs), Methylophilaceae (the nonmethanotrophic methylotrophs), Burkholderiales, and Bacteroidetes. Additional functional guilds were detected in multiple samples, such as members of Opitutae, as well as the predatory species, suggesting additional complexity for methane-oxidizing communities. Metatranscriptomic analysis suggested simultaneous expression of the two alternative types of methanol dehydrogenases in both Methylococcaceae and Methylophilaceae, while high expression of the oxidative/nitrosative stress response genes suggested competition for dioxygen among the community members. The transcriptomic analysis further suggested that Burkholderiales likely feed on acetate that is produced by Methylococcaceae under hypoxic conditions, while Bacteroidetes likely feed on biopolymers produced by both Methylococcaceae and Methylophilaceae.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Measurement of Volatile Compounds for Real-Time Analysis of Soil Microbial Metabolic Response to Simulated Snowmelt

Snowmelt dynamics are a significant determinant of microbial metabolism in soil and regulate global biogeochemical cycles of carbon and nutrients by creating seasonal variations in soil redox and nutrient pools. With an increasing concern that climate change accelerates both snowmelt timing and rate, obtaining an accurate characterization of microbial response to snowmelt is important for understanding biogeochemical cycles intertwined with soil. However, observing microbial metabolism and its dynamics non-destructively remains a major challenge for systems such as soil. Microbial volatile compounds (mVCs) emitted from soil represent information-dense signatures and when assayed non-destructively using state-of-the-art instrumentation such as Proton Transfer Reaction-Time of Flight-Mass Spectrometry (PTR-TOF-MS) provide time resolved insights into the metabolism of active microbiomes. In this study, we used PTR-TOF-MS to investigate the metabolic trajectory of microbiomes from a subalpine forest soil, and their response to a simulated wet-up event akin to snowmelt. Using an information theory approach based on the partitioning of mutual information, we identified mVC metabolite pairs with robust interactions, including those that were non-linear and with time lags. The biological context for these mVC interactions was evaluated by projecting the connections onto the Kyoto Encyclopedia of Genes and Genomes (KEGG) network of known metabolic pathways. Simulated snowmelt resulted in a rapid increase in the production of trimethylamine (TMA) suggesting that anaerobic degradation of quaternary amine osmo/cryoprotectants, such as glycine betaine, may be important contributors to this resource pulse. Unique and synergistic connections between intermediates of methylotrophic pathways such as dimethylamine, formaldehyde and methanol were observed upon wet-up and indicate that the initial pulse of TMA was likely transformed into these intermediates by methylotrophs. Increases in ammonia oxidation signatures (transformation of hydroxylamine to nitrite) were observed in parallel, and while the relative role of nitrifiers or methylotrophs cannot be confirmed, the inferred connection to TMA oxidation suggests either a direct or indirect coupling between these processes. Overall, it appears that such mVC time-series from PTR-TOF-MS combined with causal inference represents an attractive approach to non-destructively observe soil microbial metabolism and its response to environmental perturbation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Establishment of FXS-A9 panel with a single human X chromosome from fragile X syndrome-associated individual

Highlights: • We established mouse A9 cells with human X chromosome from FXS carrier and patient. • FXS-associated CGG repeats were stably maintained in established rodent cells. • Epigenetic status of CGG repeat region were maintained in established rodent cells. • Human FMR1 expression in these cells was according to each CGG repeat length. Fragile X syndrome (FXS) is the most common inheritable form of intellectual disability. FMR1, the gene responsible for FXS, is located on human chromosome Xq27.3 and contains a stretch of CGG trinucleotide repeats in its 5′ untranslated region. FXS is caused by CGG repeats that expand beyond 200, resulting in FMR1 silencing via promoter hypermethylation. The molecular mechanism underlying CGG repeat expansion, a fundamental cause of FXS, remains poorly understood, partly due to a lack of experimental systems. Accumulated evidence indicates that the large chromosomal region flanking a CGG repeat is critical for repeat dynamics. In the present study, we isolated and introduced whole human X chromosomes from healthy, FXS premutation carriers, or FXS patients who carried disease condition-associated CGG repeat lengths, into mouse A9 cells via microcell-mediated chromosome transfer. The CGG repeat length-associated methylation status and human FMR1 expression in these monochromosomal hybrid cells mimicked those in humans. Thus, this set of A9 cells containing CGG repeats from three different origins (FXS-A9 panel) may provide a valuable resource for investigating a series of genetic and epigenetic CGG repeat dynamics during FXS pathogenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Light Energy Transduction in Green Sulfur Bacteria

Green sulfur bacteria (GSB) are exquisitely adapted for growth at extraordinarily low light intensities. They are important primary producers of biomass in many anoxic environments, and they contribute significantly to the biogeochemical cycling of carbon, nitrogen, and sulfur on Earth. Green bacteria more generally share the property of using chlorosomes for light-harvesting, and these unusual organelles have many unique features. These include the presence of a monolayer lipid-protein envelope, self-assembling bacteriochlorophylls (BChl) in which pigment-pigment interactions predominate, and the presence of redox components ([2Fe-2S] ferredoxins and quinones) that play a role in regulating excitation energy transfer to the type-1 homodimeric reaction centers. The reaction centers of GSB are related to Photosystem I of cyanobacteria and higher plants but also exhibit several unique structural and functional features. The long-term objectives of this research program are to understand the structure, functions, and biogenesis of the chlorosomes, reaction centers, and electron transport chains that carry out the photochemical transduction of light energy into chemical energy in the model green sulfur bacterium, Chlorobaculum (formerly Chlorobium) tepidum. Over a period of 26 years, we characterized chlorosomes in detail. We identified the proteins in the chlorosome envelopes of diverse organisms, identified the nearest neighbors of those proteins, and characterized the chlorosomes of mutant strains lacking one to five of these proteins. After the genome sequence of Cba. tepidum became available, we identified all genes encoding enzymes for BChl a , BChl c/d/e/ƒ , and Chl a biosynthesis Cba. tepidum . We additionally identified all genes encoding enzymes for carotenoid biosynthesis. By constructing a bchQRU mutant strain that produces [Et, Me]-BChl d , together with collaborators who are specialists in solid-state NMR and cryo-electron microscopy, we solved the structure of the BChls in chlorosomes. Using similar methods and chlorosomes containing [Et, Me]-BChl c , we then showed that alternative structures were possible using the same syn-anti BChl dimer. The structures were refined by introducing spectroscopic data from single-chlorosome measurements. Over the course of this project, we sequenced the genomes of approximately twenty GSB strains, which provided important information for comparative analyses. Through analyses of metagenomic data from Mushroom and Octopus Springs in Yellowstone National Park, we identified a novel chlorophototroph belonging to the phylum Acidobacteriota. We successfully isolated an axenic culture of this organism. We characterized the photosynthetic apparatus of this bacterium, named Chloracidobacterium thermophilum, in significant detail, in particular its type-1, homodimeric reaction centers. Surprisingly, these reaction centers contain three types of Chls, BChl a , Chl a , and Zn-BChl a' . We showed that a dimer of Zn-BChl a' is the primary donor and Chl a the primary acceptor of electrons in this reaction center by using advanced spectroscopic techniques. We isolated eight additional strains of Chloracidobacterium spp. from Mushroom Spring and Rupite hot springs in Bulgaria. Comparative genomes showed that these represent three species, Cab. thermophilum , Cab. aggregatum , and Cab. validum . By applying comparative genomics, genetic, biochemical biophysical and physiological approaches to study green bacteria, we produced a wealth of new information about the remarkable light-harvesting and energy transduction capabilities of these poorly characterized microorganisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Mixed waste contamination selects for a mobile genetic element population enriched in multiple heavy metal resistance genes

Abstract Mobile genetic elements (MGEs) like plasmids, viruses, and transposable elements can provide fitness benefits to their hosts for survival in the presence of environmental stressors. Heavy metal resistance genes (HMRGs) are frequently observed on MGEs, suggesting that MGEs may be an important driver of adaptive evolution in environments contaminated with heavy metals. Here, we report the meta-mobilome of the heavy metal-contaminated regions of the Oak Ridge Reservation subsurface. This meta-mobilome was compared with one derived from samples collected from unimpacted regions of the Oak Ridge Reservation subsurface. We assembled 1615 unique circularized DNA elements that we propose to be MGEs. The circular elements from the highly contaminated subsurface were enriched in HMRG clusters relative to those from the nearby unimpacted regions. Additionally, we found that these HMRGs were associated with Gamma and Betaproteobacteria hosts in the contaminated subsurface and potentially facilitate the persistence and dominance of these taxa in this region. Finally, the HMRGs were associated with conjugative elements, suggesting their potential for future lateral transfer. We demonstrate how our understanding of MGE ecology, evolution, and function can be enhanced through the genomic context provided by completed MGE assemblies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

HSQC spectra of lignin isolated from poplar stems

Here we present a curated dataset of two-dimensional heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectra of lignin isolated from stems of genetically engineered poplar through auxin signaling gene modification. The plants were grown in greenhouse with temperatures between 21 and 23 °C. Plants were harvested and the aboveground stems were cut off an approximately five-inch-long segment from the bottom end of the plant stem, debarked and air-dried for three weeks. The dried stem samples were Wiley milled (mesh size 20), Soxhlet-extracted with toluene/ethanol for 24 h to remove extractives. The extracted biomass was ball-milled in a Retsch PM100 planetary ball mill using a porcelain jar with ceramic balls at 600 rpm for 2 h (in 5 min on and 5 min off cycles to avoid excessive sample heating). The ball-milled materials were then subjected to enzymatic hydrolysis for 48 h followed by centrifugation and washing with deionized water. The solid residue was freeze-dried to recover the lignin. The dry stem lignin samples were dissolved in deuterated dimethyl sulfoxide (d6) and transferred into a 5 mm tube. 13C–1H HSQC experiments were performed in a Bruker Avance III HD 500 MHz NMR spectrometer operating at a frequency of 125.12 MHz for the 13C nucleus using a standard Bruker pulse sequence on a Prodigy platform cryoprobe. The NMR spectra were acquired under the following acquisition conditions: 230 ppm spectral width in F1 (13C) dimension with 256 data points and 12 ppm spectral width in F2 (1H) dimension with 2048 data points, a 90° pulse, a one bond C–H coupling constant of 145 Hz, a 1.0 s pulse delay, and 64 scans. Spectra were processed using the Bruker TopSpin 3.6 software. Additional meta data is embedded in the raw spectra figures.

HSQC, lignin, poplar, stems, CBI↗
DOE OSTI2022

Immunogenicity and therapeutic targeting of a public neoantigen derived from mutated PIK3CA

Public neoantigens (NeoAgs) represent an elite class of shared cancer-specific epitopes derived from recurrently mutated driver genes. Here we describe a high-throughput platform combining single-cell transcriptomic and T cell receptor (TCR) sequencing to establish whether mutant PIK3CA, among the most frequently genomically altered driver oncogenes, generates an immunogenic public NeoAg. Using this strategy, we developed a panel of TCRs that recognize an endogenously processed neopeptide encompassing a common PIK3CA hotspot mutation restricted by the prevalent human leukocyte antigen (HLA)-A*03:01 allele. Mechanistically, immunogenicity to this public NeoAg arises from enhanced neopeptide/HLA complex stability caused by a preferred HLA anchor substitution. Structural studies indicated that the HLA-bound neopeptide presents a comparatively ‘featureless’ surface dominated by the peptide’s backbone. To bind this epitope with high specificity and affinity, we discovered that a lead TCR clinical candidate engages the neopeptide through an extended interface facilitated by an unusually long CDR3β loop. In patients with diverse malignancies, we observed NeoAg clonal conservation and spontaneous immunogenicity to the neoepitope. Finally, adoptive transfer of TCR-engineered T cells led to tumor regression in vivo in mice bearing PIK3CA-mutant tumors but not wild-type PIK3CA tumors. Together, these findings establish the immunogenicity and therapeutic potential of a mutant PIK3CA-derived public NeoAg.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Insights into the physiological and genomic characterization of three bacterial isolates from a highly alkaline, terrestrial serpentinizing system

The terrestrial serpentinite-hosted ecosystem known as “The Cedars” is home to a diverse microbial community persisting under highly alkaline (pH ~ 12) and reducing (Eh < -550 mV) conditions. This extreme environment presents particular difficulties for microbial life, and efforts to isolate microorganisms from The Cedars over the past decade have remained challenging. Herein, we report the initial physiological assessment and/or full genomic characterization of three isolates: Paenibacillus sp. Cedars (‘Paeni-Cedars’), Alishewanella sp. BS5-314 (‘Ali-BS5-314’), and Anaerobacillus sp. CMMVII (‘Anaero-CMMVII’). Paeni-Cedars is a Gram-positive, rod-shaped, mesophilic facultative anaerobe that grows between pH 7–10 (minimum pH tested was 7), temperatures 20–40°C, and 0–3% NaCl concentration. The addition of 10–20 mM CaCl 2 enhanced growth, and iron reduction was observed in the following order, 2-line ferrihydrite > magnetite > serpentinite ~ chromite ~ hematite. Genome analysis identified genes for flavin-mediated iron reduction and synthesis of a bacillibactin-like, catechol-type siderophore. Ali-BS5-314 is a Gram-negative, rod-shaped, mesophilic, facultative anaerobic alkaliphile that grows between pH 10–12 and temperatures 10–40°C, with limited growth observed 1–5% NaCl. Nitrate is used as a terminal electron acceptor under anaerobic conditions, which was corroborated by genome analysis. The Ali-BS5-314 genome also includes genes for benzoate-like compound metabolism. Anaero-CMMVII remained difficult to cultivate for physiological studies; however, growth was observed between pH 9–12, with the addition of 0.01–1% yeast extract. Anaero-CMMVII is a probable oxygen-tolerant anaerobic alkaliphile with hydrogenotrophic respiration coupled with nitrate reduction, as determined by genome analysis. Based on single-copy genes, ANI, AAI and dDDH analyses, Paeni-Cedars and Ali-BS5-314 are related to other species (P. glucanolyticus and A. aestuarii, respectively), and Anaero-CMMVII represents a new species. The characterization of these three isolates demonstrate the range of ecophysiological adaptations and metabolisms present in serpentinite-hosted ecosystems, including mineral reduction, alkaliphily, and siderophore production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Modulating metal-centered dimerization of a lanthanide chaperone protein for separation of light lanthanides

Elucidating details of biology’s selective uptake and trafficking of rare earth elements, particularly the lanthanides, has the potential to inspire sustainable biomolecular separations of these essential metals for myriad modern technologies. Here, we biochemically and structurally characterize Methylobacterium (Methylorubrum) extorquens LanD, a periplasmic protein from a bacterial gene cluster for lanthanide uptake. This protein provides only four ligands at its surface-exposed lanthanide-binding site, allowing for metal-centered protein dimerization that favors the largest lanthanide, La III . However, the monomer prefers Nd III and Sm III , which are disfavored lanthanides for cellular utilization. Structure-guided mutagenesis of a metal-ligand and an outer-sphere residue weakens metal binding to the LanD monomer and enhances dimerization for Pr III and Nd III by 100-fold. Selective dimerization enriches high-value Pr III and Nd III relative to low-value La III and Ce III in an all-aqueous process, achieving higher separation factors than lanmodulins and comparable or better separation factors than common industrial extractants. Finally, we show that LanD interacts with lanmodulin (LanM), a previously characterized periplasmic protein that shares LanD’s preference for Nd III and Sm III . Our results suggest that LanD’s unusual metal-binding site transfers less-desirable lanthanides to LanM to siphon them away from the pathway for cytosolic import. The properties of LanD show how relatively weak chelators can achieve high selectivity, and they form the basis for the design of protein dimers for separation of adjacent lanthanide pairs and other metal ions.

lanthanide biochemistry↗
DOE OSTI2022

Propagation of viral genomes by replicating ammonia-oxidising archaea during soil nitrification

Ammonia-oxidising archaea (AOA) are a ubiquitous component of microbial communities and dominate the first stage of nitrification in some soils. While we are beginning to understand soil virus dynamics, we have no knowledge of the composition or activity of those infecting nitrifiers or their potential to influence processes. This study aimed to characterise viruses having infected autotrophic AOA in two nitrifying soils of contrasting pH by following transfer of assimilated CO 2 -derived 13 C from host to virus via DNA stable-isotope probing and metagenomic analysis. Incorporation of 13 C into low GC mol% AOA and virus genomes increased DNA buoyant density in CsCl gradients but resulted in co-migration with dominant non-enriched high GC mol% genomes, reducing sequencing depth and contig assembly. We therefore developed a hybrid approach where AOA and virus genomes were assembled from low buoyant density DNA with subsequent mapping of 13 C isotopically enriched high buoyant density DNA reads to identify activity of AOA. Metagenome-assembled genomes were different between the two soils and represented a broad diversity of active populations. Sixty-four AOA-infecting viral operational taxonomic units (vOTUs) were identified with no clear relatedness to previously characterised prokaryote viruses. These vOTUs were also distinct between soils, with 42% enriched in 13 C derived from hosts. The majority were predicted as capable of lysogeny and auxiliary metabolic genes included an AOA-specific multicopper oxidase suggesting infection may augment copper uptake essential for central metabolic functioning. These findings indicate virus infection of AOA may be a frequent process during nitrification with potential to influence host physiology and activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Different chemical scaffolds bind to L-phe site in Mycobacterium tuberculosis Phe-tRNA synthetase

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mt), is one of the deadliest infectious diseases. The rise of multidrug-resistant strains represents a major public health threat, requiring new therapeutic options. Bacterial aminoacyl-tRNA synthetases (aaRS) have been shown to be highly promising drug targets, including for TB treatment. These enzymes play an essential role in translating the DNA gene code into protein sequence by attaching specific amino acid to their cognate tRNAs. They have multiple binding sites that can be targeted for inhibitor discovery: amino acid binding pocket, ATP binding pocket, tRNA binding site and an editing domain. Recently we reported several high-resolution structures of M. tuberculosis phenylalanyl-tRNA synthetase (MtPheRS) complexed with tRNA Phe and either L-Phe or a nonhydrolyzable phenylalanine adenylate analog. Here, in this study, using Nucleic Magnetic Resonance (NMR) and Surface Plasmon Resonance (SPR) we identified fragments that bind to MtPheRS and we determined crystal structures of their complexes with MtPheRS/tRNA Phe . All the binders interact with the L-Phe amino acid binding site. The analysis of interactions of the new compounds combined with adenylate analog structure provides insights for the rational design of antituberculosis drugs. The 3 ' arm of the tRNA Phe in all the structures was disordered with exception of one complex with D-735 compound. In this structure the 3' CCA end of the acceptor stem is observed in the editing domain of MtPheRS providing insights regarding the post-transfer editing activity of class II aaRS.

Gade, Priyanka [Univ. of Chicago, IL (United State↗
DOE OSTI2024

Control of hydrogen concentrations by microbial sulfate reduction in two contrasting anoxic coastal sediments

Introduction Molecular hydrogen is produced by the fermentation of organic matter and consumed by organisms including hydrogenotrophic methanogens and sulfate reducers in anoxic marine sediment. The thermodynamic feasibility of these metabolisms depends strongly on organic matter reactivity and hydrogen concentrations; low organic matter reactivity and high hydrogen concentrations can inhibit fermentation so when organic matter is poor, fermenters might form syntrophies with methanogens and/or sulfate reducers who alleviate thermodynamic stress by keeping hydrogen concentrations low and tightly controlled. However, it is unclear how these metabolisms effect porewater hydrogen concentrations in natural marine sediments of different organic matter reactivities. Methods We measured aqueous concentrations of hydrogen, sulfate, methane, dissolved inorganic carbon, and sulfide with high-depth-resolution and 16S rRNA gene assays in sediment cores with low carbon reactivity in White Oak River (WOR) estuary, North Carolina, and those with high carbon reactivity in Cape Lookout Bight (CLB), North Carolina. We calculated the Gibbs energies of sulfate reduction and hydrogenotrophic methanogenesis. Results Hydrogen concentrations were significantly higher in the sulfate reduction zone at CLB than WOR (mean: 0.716 vs. 0.437 nM H 2 ) with highly contrasting hydrogen profiles. At WOR, hydrogen was extremely low and invariant (range: 0.41–0.52 nM H 2 ) in the upper 15 cm. Deeper than 15 cm, hydrogen became more variable (range: 0.312–2.56 nM H 2 ) and increased until methane production began at ~30 cm. At CLB, hydrogen was highly variable in the upper 15 cm (range: 0.08–2.18 nM H 2 ). Ratios of inorganic carbon production to sulfate consumption show AOM drives sulfate reduction in WOR while degradation of organics drive sulfate reduction in CLB. Discussion We conclude more reactive organic matter increases hydrogen concentrations and their variability in anoxic marine sediments. In our AOM-dominated site, WOR, sulfate reducers have tight control on hydrogen via consortia with fermenters which leads to the lower observed variance due to interspecies hydrogen transfer. After sulfate depletion, hydrogen accumulates and becomes variable, supporting methanogenesis. This suggests that CLB’s more reactive organic matter allows fermentation to occur without tight metabolic coupling of fermenters to sulfate reducers, resulting in high and variable porewater hydrogen concentrations that prevent AOM from occurring through reverse hydrogenotrophic methanogenesis.

Microbiology↗
DOE OSTI2022

Establishing Butyribacterium methylotrophicum as a Platform Organism for the Production of Biocommodities from Liquid C 1 Metabolites

Using the Wood-Ljungdahl pathway, acetogens can nonphotosynthetically fix gaseous C 1 molecules, preventing them from entering the atmosphere. Many acetogens can also grow on liquid C 1 compounds such as formate and methanol, which avoid the storage and mass transfer issues associated with gaseous C 1 compounds. Substrate redox state also plays an important role in acetogen metabolism and can modulate products formed by these organisms. Butyribacterium methylotrophicum is an acetogen known for its ability to synthesize longer-chained molecules such as butyrate and butanol, which have significantly higher values than acetate or ethanol, from one-carbon (C 1 ) compounds. We explored B. methylotrophicum’s C 1 metabolism by varying substrates, substrate concentrations, and substrate feeding strategies to improve four-carbon product titers. Our results showed that formate utilization by B. methylotrophicum favored acetate production and methanol utilization favored butyrate production. Cofeeding of both substrates produced a high butyrate titer of 4 g/liter when methanol was supplied in excess to formate. Testing of formate feeding strategies, in the presence of methanol, led to further increases in the butyrate to acetate ratio. Mixotrophic growth of liquid and gaseous C 1 substrates expanded the B. methylotrophicum product profile, as ethanol, butanol, and lactate were produced under these conditions. We also showed that B. methylotrophicum is capable of producing caproate, a six-carbon product, presumably through chain elongation cycles of the reverse β-oxidation pathway. Furthermore, we demonstrated butanol production via heterologous gene expression. Finally, our results indicate that both selection of appropriate substrates and genetic engineering play important roles in determining titers of desired products.

09 BIOMASS FUELS↗
DOE OSTI2020

Factors Defining the Intercalation Electrochemistry of CaFe 2 O 4 -Type Manganese Oxides

Oxides with the CaFe 2 O 4 -type structure have been predicted as being suitable hosts for reactions of intercalation of light cations such as Li and Mg because of their favorable cationic diffusion. Although Li has been shown to intercalate into the Mn 2 O 4 variant, the key structure property correlations determining function are not fully ascertained. This basic information is needed before attempting the intercalation of divalent cations, which face comparably higher migration barriers. For this purpose, here the electrode function of CaFe 2 O 4 -type Li 0.8 Mn 2 O 4 was compared for materials made by a direct high-pressure route or through cation exchange from NaMn 2 O 4 . X-ray diffraction and absorption spectroscopy revealed that, despite having largely the same bulk structure, the presence of surface defects blocked Li + transfer in Li 0.8 Mn 2 O 4 made at high pressure. These defects were not present in the cation-exchanged material, which resulted in highly reversible Li intercalation with very fast kinetics in micrometric crystals. Delithiated electrodes from the cation-exchange synthesis were subsequently reduced in an ionic liquid electrolyte containing Mg 2+ . The process induced topotactic changes in the bulk, strongly suggesting the existence of intercalation, but it is accompanied by severe reactivity with the electrolyte that impedes reversibility. This study uncovers that defects affect the fundamentals of cation intercalation in this novel class of materials. The ability of the cation-exchanged material to conduct fast reactions with Li is consistent with calculated activation energy barriers and creates promise for their use as Mg hosts, provided that novel electrolytes enhanced stability at high potential can be realized.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗