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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 613 records · Page 34
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation

Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2'-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2'-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2'-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon–anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. In conclusion, our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Selection of stable reference genes for RT-qPCR in Rhodococcus opacus PD630

Rhodococcus opacus PD630 is a gram-positive bacterium with promising attributes for the conversion of lignin into valuable fuels and chemicals. To develop an organism as a cellular factory, it is necessary to have a deep understanding of its metabolism and any heterologous pathways being expressed. For the purpose of quantifying gene transcription, reverse transcription quantitative PCR (RT-qPCR) is the gold standard due to its sensitivity and reproducibility. However, RT-qPCR requires the use of reference genes whose expression is stable across distinct growth or treatment conditions to normalize the results. Unfortunately, no in-depth analysis of stable reference genes has been conducted in Rhodococcus, inhibiting the utilization of RT-qPCR in R. opacus. In this work, ten candidate reference genes, chosen based on previously collected RNA sequencing data or literature, were examined under four distinct growth conditions using three mathematical programs (BestKeeper, Normfinder, and geNorm). Based on this analysis, the minimum number of reference genes required was found to be two, and two separate pairs of references genes were identified as optimal normalization factors for when ribosomal RNA is either present or depleted. This work represents the first validation of reference genes for Rhodococcus, providing a valuable starting point for future research.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

The 27 kDa Trypanosoma brucei Pentatricopeptide Repeat Protein is a G-tract Specific RNA Binding Protein

Pentatricopeptide repeat (PPR) proteins, a helical repeat family of organellar RNA binding proteins, play essential roles in post-transcriptional RNA processing. In Trypanosoma brucei, an expanded family of PPR proteins localize to the parasite’s single mitochondrion, where they are believed to perform important roles in both RNA processing and translation. We studied the RNA binding specificity of the simplest T. brucei PPR protein (KRIPP11) using electrophoretic mobility shift assays, fluorescence anisotropy, circular dichroism spectroscopy, and in vitro selection. We found KRIPP11 to be an RNA binding protein with specificity for sequences of four or more consecutive guanosine residues (G-tracts). Such G-tracts are dramatically enriched in T. brucei mitochondrial transcripts that are destined for extensive uridine insertion/deletion editing but are not present in mRNAs following editing. We further found that the quadruplex oligoguanosine RNA conformation is preferentially recognized by KRIPP11 over other conformational forms, and is bound without disruption of the quadruplex structure. In combination with prior data demonstrating association of KRIPP11 with the small ribosomal subunit, these results suggest possible roles for KRIPP11 in bridging mRNA maturation and translation or in facilitating translation of unusual dual-coded open reading frames.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Paired RNA Radiocarbon and Sequencing Analyses Indicate the Importance of Autotrophy in a Shallow Alluvial Aquifer

Abstract Determining the carbon sources for active microbial populations in the subsurface is a challenging but highly informative component of subsurface microbial ecology. This work developed a method to provide ecological insights into groundwater microbial communities by characterizing community RNA through its radiocarbon and ribosomal RNA (rRNA) signatures. RNA was chosen as the biomolecule of interest because rRNA constitutes the majority of RNA in prokaryotes, represents recently active organisms, and yields detailed taxonomic information. The method was applied to a groundwater filter collected from a shallow alluvial aquifer in Colorado. RNA was extracted, radiometrically dated, and the 16S rRNA was analyzed by RNA-Seq. The RNA had a radiocarbon signature (Δ 14 C) of −193.4 ± 5.6‰. Comparison of the RNA radiocarbon signature to those of potential carbon pools in the aquifer indicated that at least 51% of the RNA was derived from autotrophy, in close agreement with the RNA-Seq data, which documented the prevalence of autotrophic taxa, such as Thiobacillus and Gallionellaceae . Overall, this hybrid method for RNA analysis provided cultivation-independent information on the in-situ carbon sources of active subsurface microbes and reinforced the importance of autotrophy and the preferential utilization of dissolved over sedimentary organic matter in alluvial aquifers.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2019

Effects of codon optimization on coagulation factor IX translation and structure: Implications for protein and gene therapies

Synonymous codons occur with different frequencies in different organisms, a phenomenon termed codon usage bias. Codon optimization, a common term for a variety of approaches used widely by the biopharmaceutical industry, involves synonymous substitutions to increase protein expression. It had long been presumed that synonymous variants, which, by definition, do not alter the primary amino acid sequence, have no effect on protein structure and function. However, a critical mass of reports suggests that synonymous codon variations may impact protein conformation. To investigate the impact of synonymous codons usage on protein expression and function, we designed an optimized coagulation factor IX (FIX) variant and used multiple methods to compare its properties to the wild-type FIX upon expression in HEK293T cells. We found that the two variants differ in their conformation, even when controlling for the difference in expression levels. Using ribosome profiling, we identified robust changes in the translational kinetics of the two variants and were able to identify a region in the gene that may have a role in altering the conformation of the protein. Our data have direct implications for codon optimization strategies, for production of recombinant proteins and gene therapies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Bioremediation of copper in sediments from a constructed wetland ex situ with the novel bacterium Cupriavidus basilensis SRS

The H-02 constructed wetland was designed to remove metals (primarily copper and zinc) to treat building process water and storm water runoff from multiple sources associated with the Tritium Facility at the DOE-Savannah River Site, Aiken, SC. The concentration of Cu and Zn in the sediments has increased over the lifetime of the wetland and is a concern. A bioremediation option was investigated at the laboratory scale utilizing a newly isolated bacterium of the copper metabolizing genus Cupriavidus isolated from Tim’s Branch Creek, a second-order stream that eventually serves as a tributary to the Savannah River, contaminated with uranium and other metals including copper, nickel, and mercury. Cupriavidus basilensis SRS is a rod-shaped, gram-negative bacterium which has been shown to have predatory tendencies. The isolate displayed resistance to the antibiotics ofloxacin, tetracycline, ciprofloxacin, select fungi, as well as Cu 2+ and Zn 2+ . Subsequent ribosomal sequencing demonstrated a 100% confidence for placement in the genus Cupriavidus and a 99.014% match to the C. basilensis type strain. When H-02 wetland samples were inoculated with Cupriavidus basilensis SRS samples showed significant (p < 0.05) decrease in Cu 2+ concentrations and variability in Zn 2+ concentrations. Over the 72-h incubation there were no significant changes in the inoculate densities (10 6 –10 8 cells/ML) indicating Cupriavidus basilensis SRS resiliency in this environment. This research expands our understanding of the Cupriavidus genus and demonstrates the potential for Cupriavidus basilensis SRS to bioremediate sites impacted with heavy metals, most notably copper.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Comparative and pangenomic analysis of the genus Streptomyces

Abstract Streptomycetes are highly metabolically gifted bacteria with the abilities to produce bioproducts that have profound economic and societal importance. These bioproducts are produced by metabolic pathways including those for the biosynthesis of secondary metabolites and catabolism of plant biomass constituents. Advancements in genome sequencing technologies have revealed a wealth of untapped metabolic potential from Streptomyces genomes. Here, we report the largest Streptomyces pangenome generated by using 205 complete genomes. Metabolic potentials of the pangenome and individual genomes were analyzed, revealing degrees of conservation of individual metabolic pathways and strains potentially suitable for metabolic engineering. Of them, Streptomyces bingchenggensis was identified as a potent degrader of plant biomass. Polyketide, non-ribosomal peptide, and gamma-butyrolactone biosynthetic enzymes are primarily strain specific while ectoine and some terpene biosynthetic pathways are highly conserved. A large number of transcription factors associated with secondary metabolism are strain-specific while those controlling basic biological processes are highly conserved. Although the majority of genes involved in morphological development are highly conserved, there are strain-specific varieties which may contribute to fine tuning the timing of cellular differentiation. Overall, these results provide insights into the metabolic potential, regulation and physiology of streptomycetes, which will facilitate further exploitation of these important bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Enzyme-mediated aminoglycoside resistance without target mimicry

Abstract The primary mode of resistance to aminoglycoside antibiotics is through chemical modification catalyzed by aminoglycoside-modifying enzymes. Numerous structural studies of these enzymes have invariably shown that they bind aminoglycosides in the same lowest-energy conformation as the intended target for these antibiotics, the A site of the bacterial ribosome. Presumably, the binding mode mimicry enables these enzymes to compete successfully with the target, thus conferring effective resistance. Here we present the first structural and functional studies of two aminoglycoside-modifying enzymes that do not use target mimicry, AAC(3)-Ia and AAC(3)-XIa. X-ray diffraction studies reveal that these enzymes bind aminoglycoside antibiotics in a conformation where the central 2-deoxystreptamine ring is in boat conformation. The effect of this non-canonical binding mode on the enzymes’ ability to modify antibiotics is assessed in silico and in vitro, and its impact for conferring resistance is assessed in vivo. Overall, the results show that target mimicry, while advantageous, is not an essential strategy for aminoglycoside-modifying enzymes to be effective in conferring resistance.

Chemistry↗
DOE OSTI2017

A compendium of multi-omic sequence information from the Saanich Inlet water column

Marine oxygen minimum zones (OMZs) are widespread regions of the ocean that are currently expanding due to global warming. While inhospitable to most metazoans, OMZs are hotspots for microbial mediated biogeochemical cycling of carbon, nitrogen and sulphur, contributing disproportionately to marine nitrogen loss and climate active trace gas production. Our current understanding of microbial community responses to OMZ expansion is limited by a lack of time-resolved data sets linking multi-omic sequence information (DNA, RNA, protein) to geochemical parameters and process rates. Here, we present six years of time-resolved multi-omic observations in Saanich Inlet, a seasonally anoxic fjord on the coast of Vancouver Island, British Columbia, Canada that undergoes recurring changes in water column oxygenation status. This compendium provides a unique multi-omic framework for studying microbial community responses to ocean deoxygenation along defined geochemical gradients in OMZ waters.

Hawley, Alyse K. (ORCID:0000000235164043)↗
DOE OSTI2016

Pan- and core- network analysis of co-expression genes in a model plant

Genome-wide gene expression experiments have been performed using the model plant Arabidopsis during the last decade. Some studies involved construction of coexpression networks, a popular technique used to identify groups of co-regulated genes, to infer unknown gene functions. One approach is to construct a single coexpression network by combining multiple expression datasets generated in different labs. We advocate a complementary approach in which we construct a large collection of 134 coexpression networks based on expression datasets reported in individual publications. To this end we reanalyzed public expression data. To describe this collection of networks we introduced concepts of ‘pan-network’ and ‘core-network’ representing union and intersection between a sizeable fractions of individual networks, respectively. Here, we showed that these two types of networks are different both in terms of their topology and biological function of interacting genes. For example, the modules of the pan-network are enriched in regulatory and signaling functions, while the modules of the core-network tend to include components of large macromolecular complexes such as ribosomes and photosynthetic machinery. Our analysis is aimed to help the plant research community to better explore the information contained within the existing vast collection of gene expression data in Arabidopsis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

The Fecal Microbial Community of Breast-fed Infants from Armenia and Georgia

Multiple factors help shape the infant intestinal microbiota early in life. Environmental conditions such as the presence of bioactive molecules from breast milk dictate gut microbial growth and survival. Infants also receive distinct, personalized, bacterial exposures leading to differential colonization. Microbial exposures and gut environmental conditions differ between infants in different locations, as does the typical microbial community structure in an infant’s gut. Here we evaluate potential influences on the infant gut microbiota through a longitudinal study on cohorts of breast-fed infants from the neighboring countries of Armenia and Georgia, an area of the world for which the infant microbiome has not been previously investigated. Marker gene sequencing of 16S ribosomal genes revealed that the gut microbial communities of infants from these countries were dominated by bifidobacteria, were different from each other, and were marginally influenced by their mother’s secretor status. Species-level differences in the bifidobacterial communities of each country and birth method were also observed. These community differences suggest that environmental variation between individuals in different locations may influence the gut microbiota of infants.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2018

Biosynthesis of the RiPP trojan horse nucleotide antibiotic microcin C is directed by the N -formyl of the peptide precursor

Microcin C7 (McC) is a peptide antibiotic modified by a linkage of the terminal isoAsn amide to AMP via a phosphoramidate bond. Post-translational modification on this ribosomally produced heptapeptide precursor is carried out by MccB, which consumes two equivalents of ATP to generate the N–P linkage. We demonstrate that MccB only efficiently processes the precursor heptapeptide that retains the Nformylated initiator Met (fMet). Binding studies and kinetic measurements evidence the role of the N-formyl moiety. Structural data show that the N-formyl peptide binding results in an ordering of residues in the MccB “crossover loop”, which dictates specificity in homologous ubiquitin activating enzymes. The N-formyl peptide exhibits substrate inhibition, and cannot be displaced from MccB by the desformyl counterpart. Such substrate inhibition may be a strategy to avert unwanted McC buildup and avert toxicity in the cytoplasm of producing organisms.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Engineering modular enzyme assembly: synthetic interface strategies for natural products biosynthesis applications

Covering: 2020 to 2025Natural products remain indispensable sources of therapeutic and bioactive compounds, yet traditional discovery strategies are constrained by compound rediscovery. Modular biosynthetic enzymes, such as type I polyketide synthases (PKSs) and type A non-ribosomal peptide synthetases (NRPSs), offer promising platforms for combinatorial biosynthesis owing to their programmable architectures. However, practical implementation is frequently limited by inter-modular incompatibility and domain-specific interactions. This review highlights recent advances in modular enzyme assembly enabled by synthetic interfaces-including cognate docking domains, synthetic coiled-coils, SpyTag/SpyCatcher, and split inteins-which function as orthogonal, standardized connectors to facilitate post-translational complex formation. These interfaces support rational investigations into substrate specificity, module compatibility, and pathway derivatization as well as general enzyme clustering applications beyond PKS and NRPS systems. Synthetic interfaces can be integrated with computational tools to support a more systematic and scalable framework for modular enzyme engineering by providing predictive insights into domain compatibility and interface design. These approaches within iterative design-build-test-learn workflows can accelerate the programmable assembly of biosynthetic systems and expand the accessible chemical space for natural products.

Kim, Gahyeon↗
DOE OSTI2014

Culture-independent detection of 'TM7' bacteria in a streptomycin-resistant acidophilic nitrifying process

Nitrification in biological wastewater treatment processes has been believed for long time to take place under neutral conditions and is inhibited under acidic conditions. However, we previously constructed acidophilic nitrifying sequencing-batch reactors (ANSBRs) being capable of nitrification at < pH 4 and harboring bacteria of the candidate phylum 'TM7' as the major constituents of the microbial community. In light of the fact that the 16S rRNA of TM7 bacteria has a highly atypical base substitution possibly responsible for resistance to streptomycin at the ribosome level, this study was undertaken to construct streptomycin-resistant acidophilic nitrifying (SRAN) reactors and to demonstrate whether TM7 bacteria are abundant in these reactors. The SRAN reactors were constructed by seeding with nitrifying sludge from an ANSBR and cultivating with ammonium-containing mineral medium (pH 4.0), to which streptomycin at a concentration of 10, 30 and 50 mg L{sup −1} was added. In all reactors, the pH varied between 2.7 and 4.0, and ammonium was completely converted to nitrate in every batch cycle. PCR-aided denaturing gradient gel electrophoresis (DGGE) targeting 16S rRNA genes revealed that some major clones assigned to TM7 bacteria and Gammaproteobacteria were constantly present during the overall period of operation. Fluorescence in situ hybridization (FISH) with specific oligonucleotide probes also showed that TM7 bacteria predominated in all SRAN reactors, accounting for 58% of the total bacterial population on average. Although the biological significance of the TM7 bacteria in the SRAN reactors are unknown, our results suggest that these bacteria are possibly streptomycin-resistant and play some important roles in the acidophilic nitrifying process.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Solitons and protein folding: An In Silico experiment

Protein folding [1] is the process of formation of a functional 3D structure from a random coil — the shape in which amino-acid chains leave the ribosome. Anfinsen’s dogma states that the native 3D shape of a protein is completely determined by protein’s amino acid sequence. Despite the progress in understanding the process rate and the success in folding prediction for some small proteins, with presently available physics-based methods it is not yet possible to reliably deduce the shape of a biologically active protein from its amino acid sequence. The protein-folding problem endures as one of the most important unresolved problems in science; it addresses the origin of life itself. Furthermore, a wrong fold is a common cause for a protein to lose its function or even endanger the living organism. Soliton solutions of a generalized discrete non-linear Schrödinger equation (GDNLSE) obtained from the energy function in terms of bond and torsion angles κ and τ provide a constructive theoretical framework for describing protein folds and folding patterns [2]. Here we study the dynamics of this process by means of molecular-dynamics simulations. The soliton manifestation is the pattern helix–loop–helix in the secondary structure of the protein, which explains the importance of understanding loop formation in helical proteins. We performed in silico experiments for unfolding one subunit of the core structure of gp41 from the HIV envelope glycoprotein (PDB ID: 1AIK [3]) by molecular-dynamics simulations with the MD package GROMACS. We analyzed 80 ns trajectories, obtained with one united-atom and two different all-atom force fields, to justify the side-chain orientation quantification scheme adopted in the studies and to eliminate force-field based artifacts. Our results are compatible with the soliton model of protein folding and provide first insight into soliton-formation dynamics.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2013

Microbial terroir for wine grapes

The viticulture industry has been selectively growing vine cultivars with different traits (grape size, shape, color, flavor, yield of fruit, and so forth) for millennia, and small variations in soil composition, water management, climate, and the aspect of vineyards have long been associated with shifts in these traits. As such, many different clonal varieties of vines exist, even within given grape varieties, such as merlot, pinot noir, and chardonnay. The commensal microbial flora that coexists with the plant may be one of the key factors that influence these traits. To date, the role of microbes has been largely ignored, outside of microbial pathogens, mainly because the technologies did not exist to allow us to look in any real depth or breadth at the community structure of the multitudes of bacterial and fungal species associated with each plant. In PNAS, Bokulich et al. (1) used next-generation sequencing of 16S rRNA and internal transcribed spacer ribosomal sequence to determine the relative abundances of bacteria and fungi, respectively, from grape must (freshly pressed grape juice, containing the skins and seeds) from plants in eight vineyards representing four of the major wine growing regions in California. The authors show that the microbiomes (bacterial and fungal taxonomic structure) associated with this early fermentation stage show defined biogeography, illustrating that different wine-growing regions maintain different microbial communities, with some influences from the grape variety and the year of production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1

The eIF4E-binding protein (4E-BP) is a phosphorylation-dependent regulator of protein synthesis. The nonphosphorylated or minimally phosphorylated form binds translation initiation factor 4E (eIF4E), preventing binding of eIF4G and the recruitment of the small ribosomal subunit. Signaling events stimulate serial phosphorylation of 4E-BP, primarily by mammalian target of rapamycin complex 1 (mTORC1) at residues T 37 /T 46 , followed by T 70 and S 65 . Hyperphosphorylated 4E-BP dissociates from eIF4E, allowing eIF4E to interact with eIF4G and translation initiation to resume. Because overexpression of eIF4E is linked to cellular transformation, 4E-BP is a tumor suppressor, and up-regulation of its activity is a goal of interest for cancer therapy. A recently discovered small molecule, eIF4E/eIF4G interaction inhibitor 1 (4EGI-1), disrupts the eIF4E/eIF4G interaction and promotes binding of 4E-BP1 to eIF4E. Structures of 14- to 16-residue 4E-BP fragments bound to eIF4E contain the eIF4E consensus binding motif, 54 YXXXXLΦ 60 (motif 1) but lack known phosphorylation sites. We report in this paper a 2.1-Å crystal structure of mouse eIF4E in complex with m 7 GTP and with a fragment of human 4E-BP1, extended C-terminally from the consensus-binding motif (4E-BP1 50–84 ). The extension, which includes a proline-turn-helix segment (motif 2) followed by a loop of irregular structure, reveals the location of two phosphorylation sites (S 65 and T 70 ). Our major finding is that the C-terminal extension (motif 3) is critical to 4E-BP1–mediated cell cycle arrest and that it partially overlaps with the binding site of 4EGI-1. Finally, the binding of 4E-BP1 and 4EGI-1 to eIF4E is therefore not mutually exclusive, and both ligands contribute to shift the equilibrium toward the inhibition of translation initiation.

59 BASIC BIOLOGICAL SCIENCES↗