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At least 271 records · Page 15
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Diverse ATPase Proteins in Mobilomes Constitute a Large Potential Sink for Prokaryotic Host ATP

Prokaryote mobilome genomes rely on host machineries for survival and replication. Given that mobile genetic elements (MGEs) derive their energy from host cells, we investigated the diversity of ATP-utilizing proteins in MGE genomes to determine whether they might be associated with proteins that could suppress related host proteins that consume energy. A comprehensive search of 353 huge phage genomes revealed that up to 9% of the proteins have ATPase domains. For example, ATPase proteins constitute ~3% of the genomes of Lak phages with ~550 kbp genomes that occur in the microbiomes of humans and other animals. Statistical analysis shows the number of ATPase proteins increases linearly with genome length, consistent with a large sink for host ATP during replication of megaphages. Using metagenomic data from diverse environments, we found 505 mobilome proteins with ATPase domains fused to diverse functional domains. Among these composite ATPase proteins, 61.6% have known functional domains that could contribute to host energy diversion during the mobilome infection cycle. As many have domains that are known to interact with nucleic acids and proteins, we infer that numerous ATPase proteins are used during replication and for protection from host immune systems. We found a set of uncharacterized ATPase proteins with nuclease and protease activities, displaying unique domain architectures that are energy intensive based on the presence of multiple ATPase domains. In many cases, these composite ATPase proteins genomically co-localize with small proteins in genomic contexts that are reminiscent of toxin-antitoxin systems and phage helicase-antibacterial helicase systems. Small proteins that function as inhibitors may be a common strategy for control of cellular processes, thus could inspire future biochemical experiments for the development of new nucleic acid and protein manipulation tools, with diverse biotechnological applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Impact of storage and extraction methods on peat soil microbiomes

Recovered microbial community structure is known to be influenced by sample storage conditions and nucleic acid extraction methods, and the impact varies by sample type. Peat soils store a large portion of soil carbon and their microbiomes mediate climate feedbacks. Here, we tested three storage conditions and five extraction protocols on peat soils from three physicochemically distinct habitats in Stordalen Mire, Sweden, revealing significant methodological impacts on microbial (here, meaning bacteria and archaea) community structure. Initial preservation method impacted alpha but not beta diversity, with in-field storage in LifeGuard buffer yielding roughly two-thirds the richness of in-field flash-freezing or transport from the field on ice (all samples were stored at −80 °C after return from the field). Nucleic acid extraction method impacted both alpha and beta diversity; one method (the PowerSoil Total RNA Isolation kit with DNA Elution Accessory kit) diverged from the others (PowerMax Soil DNA Isolation kit-High Humic Acid Protocol, and three variations of a modifiedPowerMax Soil DNA/RNA isolation kit), capturing more diverse microbial taxa, with divergent community structures. Although habitat and sample depth still consistently dominated community variation, method-based biases in microbiome recovery for these climatologically-relevant soils are significant, and underscore the importance of methodological consistency for accurate inter-study comparisons, long-term monitoring, and consistent ecological interpretations.

Science & Technology - Other Topics↗
DOE OSTI2024

The CRISPR effector Cam1 mediates membrane depolarization for phage defence

Prokaryotic type III CRISPR–Cas systems provide immunity against viruses and plasmids using CRISPR-associated Rossman fold (CARF) protein effectors. Recognition of transcripts of these invaders with sequences that are complementary to CRISPR RNA guides leads to the production of cyclic oligoadenylate second messengers, which bind CARF domains and trigger the activity of an effector domain. Whereas most effectors degrade host and invader nucleic acids, some are predicted to contain transmembrane helices without an enzymatic function. Whether and how these CARF–transmembrane helix fusion proteins facilitate the type III CRISPR–Cas immune response remains unknown. Here we investigate the role of cyclic oligoadenylate-activated membrane protein 1 (Cam1) during type III CRISPR immunity. Structural and biochemical analyses reveal that the CARF domains of a Cam1 dimer bind cyclic tetra-adenylate second messengers. In vivo, Cam1 localizes to the membrane, is predicted to form a tetrameric transmembrane pore, and provides defence against viral infection through the induction of membrane depolarization and growth arrest. These results reveal that CRISPR immunity does not always operate through the degradation of nucleic acids, but is instead mediated via a wider range of cellular responses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Six-Letter DNA Nanotechnology: Incorporation of Z-P Base Pairs into Self-Assembling 3D Crystals

Artificially expanded genetic information systems (AEGIS) were developed to expand the diversity and functionality of biological systems. Recent experiments have shown that these expanded DNA molecular systems are robust platforms for information storage and retrieval as well as useful for basic biotechnologies. In tandem, nucleic acid nanotechnology has seen the use of information-based “semantomorphic” encoding to drive the self-assembly of a vast array of supramolecular devices. To establish the effectiveness of AEGIS toward nanotechnological applications, we investigated the ability of a six-letter alphabet composed of A:T, G:C and synthetic Z:P (Z, 6-amino-3-(1'-β- D-2'-deoxy ribofuranosyl)-5-nitro-(1H)-pyridin-2-one; P, 2-amino-8-(1'- β-D-2'-deoxyribofuranosyl)-imidazo-[1,2a]-1,3,5-triazin-(8H)-4-one) base pairs to engage in 3D self-assembly. We found that crystals could be programmably assembled from AEGIS oligomers. We conclude that unnatural base pairs can be used for the topological self-assembly of crystals. We anticipate the expansion of AEGISbased nucleic acid nanotechnologies to enable the development of novel nanomaterials, high-fidelity signal cascades, and dynamic nanoscale devices.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Novel Z-DNA binding domains in giant viruses

Z-nucleic acid structures play vital roles in cellular processes and have implications in innate immunity due to their recognition by Zα domains containing proteins (Z-DNA/Z-RNA binding proteins, ZBPs). Although Zα domains have been identified in six proteins, including viral E3L, ORF112, and I73R, as well as, cellular ADAR1, ZBP1, and PKZ, their prevalence across living organisms remains largely unexplored. In this study, we introduce a computational approach to predict Zα domains, leading to the revelation of previously unidentified Zα domain-containing proteins in eukaryotic organisms, including non-metazoan species. Our findings encompass the discovery of new ZBPs in previously unexplored giant viruses, members of the Nucleocytoviricota phylum. Through experimental validation, we confirm the Zα functionality of select proteins, establishing their capability to induce the B-to-Z conversion. Additionally, we identify Zα-like domains within bacterial proteins. While these domains share certain features with Zα domains, they lack the ability to bind to Z-nucleic acids or facilitate the B-to-Z DNA conversion. Our findings significantly expand the ZBP family across a wide spectrum of organisms and raise intriguing questions about the evolutionary origins of Zα-containing proteins. Moreover, our study offers fresh perspectives on the functional significance of Zα domains in virus sensing and innate immunity and opens avenues for exploring hitherto undiscovered functions of ZBPs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Construction of Reconfigurable and Polymorphic DNA Origami Assemblies with Coiled‐Coil Patches and Patterns

Abstract DNA origami nanodevices achieve programmable structure and tunable mechanical and dynamic properties by leveraging the sequence‐specific interactions of nucleic acids. Previous advances have also established DNA origami as a useful building block to make well‐defined micron‐scale structures through hierarchical self‐assembly, but these efforts have largely leveraged the structural features of DNA origami. The tunable dynamic and mechanical properties also provide an opportunity to make assemblies with adaptive structures and properties. Here the integration of DNA origami hinge nanodevices and coiled‐coil peptides are reported into hybrid reconfigurable assemblies. With the same dynamic device and peptide interaction, it is made multiple higher‐order assemblies (i.e., polymorphic assembly) by organizing clusters of peptides into patches or arranging single peptides into patterns on the surfaces of DNA origami to control the relative orientation of devices. The coiled‐coil interactions are used to construct circular and linear assemblies whose structure and mechanical properties can be modulated with DNA‐based reconfiguration. Reconfiguration of linear assemblies leads to micron scale motions and ≈2.5‐10‐fold increase in bending stiffness. The results provide a foundation for stimulus‐responsive hybrid assemblies that can adapt their structure and properties in response to nucleic acid, peptide, protein, or other triggers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

DOE JGI Metagenome Workflow

The DOE Joint Genome Institute (JGI) Metagenome Workflow performs metagenome data processing, including assembly; structural, functional, and taxonomic annotation; and binning of metagenomic data sets that are subsequently included into the Integrated Microbial Genomes and Microbiomes (IMG/M) (I.-M. A. Chen, K. Chu, K. Palaniappan, A. Ratner, et al., Nucleic Acids Res, 49:D751–D763, 2021, https://doi.org/10.1093/nar/gkaa939) comparative analysis system and provided for download via the JGI data portal (https://genome.jgi.doe.gov/portal/). This workflow scales to run on thousands of metagenome samples per year, which can vary by the complexity of microbial communities and sequencing depth. Here, we describe the different tools, databases, and parameters used at different steps of the workflow to help with the interpretation of metagenome data available in IMG and to enable researchers to apply this workflow to their own data. We use 20 publicly available sediment metagenomes to illustrate the computing requirements for the different steps and highlight the typical results of data processing. The workflow modules for read filtering and metagenome assembly are available as a workflow description language (WDL) file (https://code.jgi.doe.gov/BFoster/jgi_meta_wdl). The workflow modules for annotation and binning are provided as a service to the user community at https://img.jgi.doe.gov/submit and require filling out the project and associated metadata descriptions in the Genomes OnLine Database (GOLD) (S. Mukherjee, D. Stamatis, J. Bertsch, G. Ovchinnikova, et al., Nucleic Acids Res, 49:D723–D733, 2021, https://doi.org/10.1093/nar/gkaa983).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

The promising role of proteomes and metabolomes in defining the single-cell landscapes of plants

The plant community has a strong track-record of RNA sequencing technology deployment, which combined with the recent advent of spatial platforms (e.g., 10x genomics), has resulted in an explosion of outstanding single cell and nuclei datasets that can be put in an in situ context within tissues (e.g., a cell atlas)1. In the genomics era, application of proteomics technologies in the plant sciences has always trailed behind that of RNA sequencing technologies, largely due to accessibility, ease-of-use and access to expertise along with depth of analysis benefits. On the other hand, the use of early analytical tools for characterizing small molecules (metabolites) from plant systems predates nucleic acid sequencing and proteomics analysis2, as the search for plant-based natural products has played a significant role in improving human health throughout history. However, the employment of proteomics and metabolomics assays for characterizing plant cell processes now remains significantly behind transcriptional approaches, even though both provide a direct functional readout of cell states and phenotypes.

Anderton, Christopher R. [BATTELLE (PACIFIC NW LAB↗
DOE OSTI2025

A novel regulator of the fungal phosphate starvation response revealed by transcriptional profiling and DNA affinity purification sequencing

Cells must accurately sense and respond to nutrients to compete for resources and establish growth. Phosphate is a critical nutrient source necessary for signaling, energy metabolism, and synthesis of nucleic acids, phospholipids, and cellular metabolites. During phosphate limitation, fungi import phosphate from the environment and liberate phosphate from phosphate-containing molecules in the cell. In the model filamentous fungus Neurospora crassa, the phosphate starvation response is regulated by the conserved transcription factor NUC-1. The activity of NUC-1 is repressed by a complex of the cyclin-dependent kinase MDK-1 and the cyclin PREG when phosphate is plentiful. When phosphate is limiting, NUC-1 repression by MDK-1/PREG is relieved by the cyclin-dependent kinase inhibitor NUC-2. We investigated the global response of N. crassa to phosphate starvation. During phosphate starvation, NUC-1 directly activated the expression of genes encoding phosphatases, nucleases, and a phosphate transporter and directly repressed genes associated with the ribosome. Additionally, NUC-1 indirectly activated the expression of an uncharacterized transcription factor, which we named nuc-3. NUC-3 directly repressed the expression of genes involved in phosphate acquisition and liberation after an extended period of phosphate starvation. Additionally, NUC-3 directly repressed the expression of the cyclin-dependent kinase inhibitor nuc-2. Thus, through the combination of NUC-3 direct repression of genes in the phosphate starvation response and nuc-2, an activator of the phosphate starvation response, NUC-3 serves to act as a brake on the phosphate starvation response after an extended period of phosphate starvation. This braking mechanism could reduce transcription, a phosphate-intensive process, under conditions of extended phosphate limitation.IMPORTANCEFungi have evolved regulatory networks to respond to available nutrients. Phosphate is often a limiting nutrient for fungi that is critical for many cellular functions, including nucleic acid and phospholipid biosynthesis, cell signaling, and energy metabolism. The fungal response to phosphate limitation is important in interactions with plants and animals. We investigated the global transcriptional response to phosphate starvation and the role of a major transcriptional regulator, NUC-1, in the model filamentous fungus Neurospora crassa. Our data show that NUC-1 is a bifunctional transcription factor that directly activates phosphate acquisition genes, while directly repressing genes associated with phosphate-intensive processes. NUC-1 indirectly regulates an uncharacterized transcription factor, which we named nuc-3. NUC-3 directly represses phosphate acquisition genes and nuc-2, an activator of the phosphate starvation response, during extended periods of phosphate starvation. Thus, NUC-3 acts as a brake on the phosphate starvation response to reduce phosphate-intensive activities, like transcriptional activation, when phosphate starvation persists.

DNA affinity purification sequencing↗
DOE OSTI2024

Triple tandem trimer immunogens for HIV-1 and influenza nucleic acid-based vaccines

Recombinant native-like HIV-1 envelope glycoprotein (Env) trimers are used in candidate vaccines aimed at inducing broadly neutralizing antibodies. While state-of-the-art SOSIP or single-chain Env designs can be expressed as native-like trimers, undesired monomers, dimers and malformed trimers that elicit non-neutralizing antibodies are also formed, implying that these designs could benefit from further modifications for gene-based vaccination approaches. Here, we describe the triple tandem trimer (TTT) design, in which three Env protomers are genetically linked in a single open reading frame and express as native-like trimers. Viral vectored Env TTT induced similar neutralization titers but with a higher proportion of trimer-specific responses. The TTT design was also applied to generate influenza hemagglutinin (HA) trimers without the need for trimerization domains. Additionally, we used TTT to generate well-folded chimeric Env and HA trimers that harbor protomers from three different strains. In summary, the TTT design is a useful platform for the design of HIV-1 Env and influenza HA immunogens for a multitude of vaccination strategies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Nucleic Acid-Based Detection Protease Activity

Proteases include clinically relevant markers for clotting disorders, certain cancers as well as toxins. Assays for protease activity often use designed peptides mimicking natural substrates and detection with colorometric and fluorescence-based detection that is difficult to multiplex without expensive and resource demanding instruments. This work demonstrates detection of proteolytic activity using PCR and sequencing-readable reporter molecules. The assay development focused on binding the constructed peptide-oligonucleotide chimera to immobilized streptavidin. Thrombin, an essential component of the clotting cascade, was used as a model system for testing peptide substrate recognition and release of a designed oligonucleotide for detection. Detection of protease activity was demonstrated in a concentration-dependent manner using MALDI-MS, RT-PCR and DNA sequencing.

Wunschel, David S [Pacific Northwest National Labo↗
NASA NTRS1985

Template-directed synthesis of novel, nucleic acid-like structures

In studying the origins of life, it is important to examine reactions of substrate mixtures that could plausibly have accumulated on the primitive earth. Nucleoside diphosphates would probably have been synthesized along with the standard nucleotides under prebiotic conditions. For these reasons, the template-directed reactions of activated derivatives of these diphosphates, alone or mixed with activated nucleotides, were investigated. An activated derivative of deoxyguanosine 3',5'-didphosphate condensed efficiently on a polycytidylate template to give oligonucleotide analogous in which each 3,5'ophosphodiester bond was replaced by a pyrophosphate linkage. Oligomers were formed even in the absence of a template, but much more slowly. Template-directed condensation occurred also with an analogous deoxadenosine derivative on polyuridylic acid and with an analogous acycloguanosine derivative on polycytidylic acid.

Schwartz, A. W.↗
NASA NTRS1984

An Investigation of the Adsorption Characteristics of 5'ATP and 5'AMP onto the Surface of Caso4 x 2H2O

A model has been proposed in which solid surfaces can act as a site for cataletic activity of condensation reactions for certain biomolecules. From this model, the adsorption characteristics of 5'ATP and 5'AMP onto the surface of CaSO4.2H2O was chosen for study. It has been proven that 5'ATP and 5'AMP do adsorb onto the surface of CaSO4. Studies were then made to determine the dependence of absorption versus time, concentration, ionic strength and pH. It was found that the adsorption of the nucleotides is highly pH dependent, primarily determined by the phosphate acid groups of the nucleic acid molecule. From this investigation, the data obtained is discussed in relation to the model for the prebiotic earth.

Calderon, J.↗
NASA NTRS1986

An investigation of the adsorption characteristics of 5 prime ATP and 5 prime AMP onto the surface of CaSO sub 4 x 2H sub 2 O

A model has been proposed (Lahev and Chans, 1982) in which solid surfaces can act as a site for catalytic activity of condensation reactions for certain biomolecules. From this model, the adsorption characteristics of 5'ATP and 5'AMP onto the surface of CaSO4 2H2O was chosen for study. It has been proven that 5'ATP and 5'AMP do adsorb onto the surface of CaSO4. Studies were then made to determine the dependence of adsorption versus time, concentration, ionic strength and pH. It was found that the adsorption of the nucleotides is highly pH dependent, primarily determined by the phosphate acid groups of the nucleic acid molecule. From this investigation, the data obtained are discussed in relation to the model for the prebiotic earth.

Calderon, J.↗
NASA NTRS1971

Experimental investigation on the origin of the genetic code.

A simple model of interacting complex systems of species is tested to assess the binding behavior of monomeric nucleic acid and protein components during evolution. Nine representative amino acids are immobilized by the formation of an amide linkage on a prepared chromatographic support. Selective binding of ribonucleoside 5-phosphates in these amino acids is achieved under standardized conditions, and a site-binding model is derived to characterize the binding. It is shown that the binding behavior of the reactants during nucleic acid-protein interactions depends on the nature of the base and the amino acid. The results of the study are assessed as useful for the interpretation of more complex nucleic acid-protein systems and of their role in the evolution of the cell.

Saxinger, C.↗
NASA NTRS1994

The sequence, and its evolutionary implications, of a Thermococcus celer protein associated with transcription

Through random search, a gene from Thermococcus celer has been identified and sequenced that appears to encode a transcription-associated protein (110 amino acid residues). The sequence has clear homology to approximately the last half of an open reading frame reported previously for Sulfolobus acidocaldarius [Langer, D. & Zillig, W. (1993) Nucleic Acids Res. 21, 2251]. The protein translations of these two archaeal genes in turn are homologs of a small subunit found in eukaryotic RNA polymerase I (A12.2) and the counterpart of this from RNA polymerase II (B12.6). Homology is also seen with the eukaryotic transcription factor TFIIS, but it involves only the terminal 45 amino acids of the archaeal proteins. Evolutionary implications of these homologies are discussed.

Non-NASA Center↗