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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Harnessing Fermentation May Enhance the Performance of Biological Sulfate-Reducing Bioreactors

Biological sulfate reduction (BSR) represents a promising strategy for bioremediation of sulfate-rich waste streams, yet the impact of metabolic interactions on performance is largely unexplored. Here, genome-resolved metagenomics was used to characterize 17 microbial communities in reactors treating synthetic sulfate-contaminated solutions. Reactors were supplemented with lactate or acetate and a small amount of fermentable substrate. Of the 163 genomes representing all the abundant bacteria, 130 encode 321 NiFe and FeFe hydrogenases and all genomes of the 22 sulfate-reducing microorganisms (SRM) encode genes for H 2 uptake. We observed lactate oxidation solely in the first packed bed reactor zone, with propionate and acetate oxidation in the middle and predominantly acetate oxidation in the effluent zone. The energetics of these reactions are very different, yet sulfate reduction kinetics were unaffected by the type of electron donor available. We hypothesize that the comparable rates, despite the typically slow growth of SRM on acetate, are a result of the consumption of H 2 generated by fermentation. This is supported by the sustained performance of a predominantly acetate-supplemented stirred tank reactor dominated by diverse fermentative bacteria encoding FeFe hydrogenase genes and SRM capable of acetate and hydrogen consumption and CO 2 assimilation. Thus, addition of fermentable substrates to stimulate syntrophic relationships may improve the performance of BSR reactors supplemented with inexpensive acetate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Overexpression of human ACE2 protein in mouse fibroblasts stably transfected with the intact ACE2 gene

Infection by SARS-CoV-2 is dependent on binding of the viral spike protein to angiotensin converting enzyme 2 (ACE2), a membrane glycoprotein expressed on epithelial cells in the human upper respiratory tract. Recombinant ACE2 protein has potential application for anti-viral therapy. Here we co-transfected mouse fibroblasts (A9 cells) with a cloned fragment of human genomic DNA containing the intact ACE2 gene and an unlinked neomycin phosphotransferase gene, and then selected stable neomycin-resistant transfectants. Transfectant clones expressed ACE2 protein at levels that were generally proportional to the number of ACE2 gene copies integrated in the cell genome, ranging up to approximately 50 times the level of ACE2 present of Vero-E6 cells. Cells overexpressing ACE2 were hypersensitive to infection by spike-pseudotyped vesicular stomatitis virus (VSV-S), and adsorption of VSV-S to these cells occurred at an accelerated rate compared to Vero-E6 cells. In conclusion, the transfectant cell clones described here therefore have favorable attributes as feedstocks for large-scale production of recombinant human ACE2 protein.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2026

Meta-virus resource (MetaVR): expanding the frontiers of viral diversity with 24 million uncultivated virus genomes

Viruses are ubiquitous in all environments and impact host metabolism, evolution, and ecology, although our knowledge of their biodiversity is still extremely limited. Viral diversity from genomic and metagenomic datasets has led to an explosion of uncultivated virus genomes (UViGs) and the development of specialized databases to catalog this viral diversity, though many lack comprehensive integration. Here, we introduce meta-virus resource (MetaVR), the successor of the IMG/VR database, designed to overcome previous limitations such as large-scale querying and programmatic access. Drawing on the increase of publicly available genomes and metagenomes, MetaVR significantly expands viral diversity, now comprising 24,435,662 UViGs, a 57.6% increase from its predecessor, organized into over 12 million viral operational taxonomic units. Key enhancements include the integration of curated eukaryotic host information, the integration of protein clusters and predicted structures for comparative studies, and an API for programmatic data access. Furthermore, MetaVR features an updated taxonomic framework based on ICTV release 39, assignment to Baltimore classes, and enhanced host assignment through novel computational tools like iPHoP. These advancements position MetaVR as a unique resource for exploring viral diversity, evolution, and host interactions across diverse environments. MetaVR can be freely accessed at https://www.meta-virome.org/.

Fiamenghi, Mateus B↗
DOE OSTI2026

Capacity of Arctic fjord sediments to degrade carbohydrates from permafrost active layer

The degradation of organic matter (OM) by microorganisms in thawing permafrost produces greenhouse gases. Terrestrial OM is transported into fjords through hydrological runoff, but it is unclear whether the microbial mechanisms of OM degradation on land persist after soils enter marine environments, which differ greatly in conditions and microbial communities. This question is particularly relevant for low-OM soils, which dominate Arctic landscapes and are more exposed to oxidants. Here, we compared OM-degrading capacity in permafrost-affected active layer soils and adjacent fjord sediments from Kongsfjorden, Svalbard, focusing on carbohydrate-active enzymes (CAZymes), which target some of the most abundant types of organic matter in soils. Using multi-omics approaches—metagenomics, metagenome-assembled genomes (MAGs), metabolomics, metatranscriptomics, and metaproteomics—we examined CAZyme presence, distribution, and activity. Despite environmental differences, both soils and sediments harbored diverse glycoside hydrolases and polysaccharide lyases, most of which showed evidence of activity. Verrucomicrobia expressed the highest number of CAZyme transcripts, indicating that they dominated active carbohydrate degradation in fjord sediments, while Acidobacteria and Actinobacteria were more active in soils. Notably, CAZymes in fjord sediments targeted primarily soil-derived OM, and the proportions of enzymes degrading terrestrial OM, marine OM, and microbial necromass—remnants of dead microbial cells were similar across both environments. These results suggest that microbial communities in both soils and fjord sediments are equipped to degrade carbohydrates, and that burial of terrestrial-derived OM in fjord sediments may not protect it from microbial breakdown under Arctic warming.

Chukwufumnanya, Abuah [University of Tennessee, Kn↗
DOE OSTI2022

Natural variation in growth and leaf ion homeostasis in response to salinity stress in Panicum hallii

Soil salinity can negatively impact plants growth, development and fitness. Natural plant populations restricted to coastal environments may evolve in response to saline habitats and therefore provide insights into the process of salinity adaptation. We investigated the growth and physiological responses of coastal and inland populations of Panicum hallii to experimental salinity treatments. Coastal genotypes demonstrated less growth reduction and superior ion homeostasis compared to the inland genotypes in response to saline conditions, supporting a hypothesis of local adaptation. We identified several QTL associated with the plasticity of belowground biomass, leaf sodium and potassium content, and their ratio which underscores the genetic variation present in this species for salinity responses. Genome-wide transcriptome analysis in leaf and root tissue revealed tissue specific overexpression of genes including several cation transporters in the coastal genotype. These transporters mediate sodium ion compartmentalization and potassium ion retention and thus suggests that maintenance of ionic homeostasis of the coastal genotypes might be due to the regulation of these ion transporters. These findings contribute to our understanding of the genetics and molecular mechanisms of salinity adaptation in natural populations, and widens the scope for genetic manipulation of these candidate genes to design plants more resilient to climate change.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Development and Assessment of SNP Genotyping Arrays for Citrus and Its Close Relatives

Rapid advancements in technologies provide various tools to analyze fruit crop genomes to better understand genetic diversity and relationships and aid in breeding. Genome-wide single nucleotide polymorphism (SNP) genotyping arrays offer highly multiplexed assays at a relatively low cost per data point. We report the development and validation of 1.4M SNP Axiom® Citrus HD Genotyping Array (Citrus 15AX 1 and Citrus 15AX 2) and 58K SNP Axiom® Citrus Genotyping Arrays for Citrus and close relatives. SNPs represented were chosen from a citrus variant discovery panel consisting of 41 diverse whole-genome re-sequenced accessions of Citrus and close relatives, including eight progenitor citrus species. SNPs chosen mainly target putative genic regions of the genome and are accurately called in both Citrus and its closely related genera while providing good coverage of the nuclear and chloroplast genomes. Reproducibility of the arrays was nearly 100%, with a large majority of the SNPs classified as the most stringent class of markers, “PolyHighResolution” (PHR) polymorphisms. Concordance between SNP calls in sequence data and array data average 98%. Phylogenies generated with array data were similar to those with comparable sequence data and little affected by 3 to 5% genotyping error. Both arrays are publicly available.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Cas9-derived peptides presented by MHC Class II that elicit proliferation of CD4 + T-cells

CRISPR–Cas9 mediated genome editing offers unprecedented opportunities for treating human diseases. There are several reports that demonstrate pre-existing immune responses to Cas9 which may have implications for clinical development of CRISPR-Cas9 mediated gene therapy. Here we use 209 overlapping peptides that span the entire sequence of Staphylococcus aureus Cas9 (SaCas9) and human peripheral blood mononuclear cells (PBMCs) from a cohort of donors with a distribution of Major Histocompatibility Complex (MHC) alleles comparable to that in the North American (NA) population to identify the immunodominant regions of the SaCas9 protein. We also use an MHC Associated Peptide Proteomics (MAPPs) assay to identify SaCas9 peptides presented by MHC Class II (MHC-II) proteins on dendritic cells. Using these two data sets we identify 22 SaCas9 peptides that are both presented by MHC-II proteins and stimulate CD4 + T-cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Harmonizing model organism data in the Alliance of Genome Resources

The Alliance of Genome Resources (the Alliance) is a combined effort of 7 knowledgebase projects: Saccharomyces Genome Database, WormBase, FlyBase, Mouse Genome Database, the Zebrafish Information Network, Rat Genome Database, and the Gene Ontology Resource. The Alliance seeks to provide several benefits: better service to the various communities served by these projects; a harmonized view of data for all biomedical researchers, bioinformaticians, clinicians, and students; and a more sustainable infrastructure. The Alliance has harmonized cross-organism data to provide useful comparative views of gene function, gene expression, and human disease relevance. The basis of the comparative views is shared calls of orthology relationships and the use of common ontologies. The key types of data are alleles and variants, gene function based on gene ontology annotations, phenotypes, association to human disease, gene expression, protein–protein and genetic interactions, and participation in pathways. The information is presented on uniform gene pages that allow facile summarization of information about each gene in each of the 7 organisms covered (budding yeast, roundworm Caenorhabditis elegans, fruit fly, house mouse, zebrafish, brown rat, and human). The harmonized knowledge is freely available on the alliancegenome.org portal, as downloadable files, and by APIs. We expect other existing and emerging knowledge bases to join in the effort to provide the union of useful data and features that each knowledge base currently provides.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Discovery of FoTO1 and Taxol genes enables biosynthesis of baccatin III

Abstract Plants make complex and potent therapeutic molecules 1,2 , but sourcing these molecules from natural producers or through chemical synthesis is difficult, which limits their use in the clinic. A prominent example is the anti-cancer therapeutic paclitaxel (sold under the brand name Taxol), which is derived from yew trees (Taxusspecies) 3 . Identifying the full paclitaxel biosynthetic pathway would enable heterologous production of the drug, but this has yet to be achieved despite half a century of research 4 . WithinTaxus’ large, enzyme-rich genome 5 , we suspected that the paclitaxel pathway would be difficult to resolve using conventional RNA-sequencing and co-expression analyses. Here, to improve the resolution of transcriptional analysis for pathway identification, we developed a strategy we term multiplexed perturbation × single nuclei (mpXsn) to transcriptionally profile cell states spanning tissues, cell types, developmental stages and elicitation conditions. Our data show that paclitaxel biosynthetic genes segregate into distinct expression modules that suggest consecutive subpathways. These modules resolved seven new genes, allowing a de novo 17-gene biosynthesis and isolation of baccatin III, the industrial precursor to Taxol, inNicotiana benthamianaleaves, at levels comparable with the natural abundance inTaxusneedles. Notably, we found that a nuclear transport factor 2 (NTF2)-like protein, FoTO1, is crucial for promoting the formation of the desired product during the first oxidation, resolving a long-standing bottleneck in paclitaxel pathway reconstitution. Together with a new β-phenylalanine-CoA ligase, the eight genes discovered here enable the de novo biosynthesis of 3’-N-debenzoyl-2’-deoxypaclitaxel. More broadly, we establish a generalizable approach to efficiently scale the power of co-expression analysis to match the complexity of large, uncharacterized genomes, facilitating the discovery of high-value gene sets.

Science & Technology - Other Topics↗
DOE OSTI2024

Differential DNA methylation and metabolite profiling of Atlantic killifish (Fundulus heteroclitus) from the New Bedford Harbor Superfund site

Atlantic killifish (Fundulus heteroclitus) is a valuable model in evolutionary toxicology to study how the interactions between genetic and environmental factors serve the adaptive ability of organisms to resist chemical pollution. Killifish populations inhabiting environmental toxicant-contaminated New Bedford Harbor (NBH) show phenotypes tolerant to polychlorinated biphenyls (PCBs) and differences at the transcriptional and genomic levels. However, limited research has explored epigenetic alterations and metabolic effects in NBH killifish. To identify the involvement of epigenetic and metabolic regulation in the adaptive response of killifish, we investigated tissue- and sex-specific differences in global DNA methylation and metabolomic profiles of NBH killifish populations, compared to sensitive populations from a non-polluted site, Scorton Creek (SC). The results revealed that liver-specific global DNA hypomethylation and differential metabolites were evident in fish from NBH compared with those from SC. The sex-specific differences were not greater than the tissue-specific differences. We demonstrated liver-specific enriched metabolic pathways (e.g., amino acid metabolic pathways converged into the urea cycle and glutathione metabolism), suggesting possible crosstalk between differential metabolites and DNA hypomethylation in the livers of NBH killifish. Additional investigation of methylated gene regions is necessary to understand the functional role of DNA hypomethylation in the regulation of enzyme-encoding genes associated with metabolic processes and physiological changes in NBH populations.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Multi-scale signaling and tumor evolution in high-grade gliomas

Although genomic anomalies in glioblastoma (GBM) have been well studied for over a decade, its 5-year survival rate remains lower than 5%. We seek to expand the molecular landscape of high-grade glioma, composed of IDH-wildtype GBM and IDH-mutant grade 4 astrocytoma, by integrating proteomic, metabolomic, lipidomic, and post-translational modifications (PTMs) with genomic and transcriptomic measurements to uncover multi-scale regulatory interactions governing tumor development and evolution. Applying 14 proteogenomic and metabolomic platforms to 228 tumors (212 GBM and 16 grade 4 IDH-mutant astrocytoma), including 28 at recurrence, plus 18 normal brain samples and 14 brain metastases as comparators, reveals heterogeneous upstream alterations converging on common downstream events at the proteomic and metabolomic levels and changes in protein-protein interactions and glycosylation site occupancy at recurrence. Recurrent genetic alterations and phosphorylation events on PTPN11 map to important regulatory domains in three dimensions, suggesting a central role for PTPN11 signaling across high-grade gliomas.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Proteogenomic characterization of difficult-to-treat breast cancer with tumor cells enriched through laser microdissection

Abstract Background Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer death among women globally. Despite advances, there is considerable variation in clinical outcomes for patients with non-luminal A tumors, classified as difficult-to-treat breast cancers (DTBC). This study aims to delineate the proteogenomic landscape of DTBC tumors compared to luminal A (LumA) tumors. Methods We retrospectively collected a total of 117 untreated primary breast tumor specimens, focusing on DTBC subtypes. Breast tumors were processed by laser microdissection (LMD) to enrich tumor cells. DNA, RNA, and protein were simultaneously extracted from each tumor preparation, followed by whole genome sequencing, paired-end RNA sequencing, global proteomics and phosphoproteomics. Differential feature analysis, pathway analysis and survival analysis were performed to better understand DTBC and investigate biomarkers. Results We observed distinct variations in gene mutations, structural variations, and chromosomal alterations between DTBC and LumA breast tumors. DTBC tumors predominantly had more mutations inTP53,PLXNB3, Zinc finger genes, and fewer mutations inSDC2,CDH1,PIK3CA,SVIL, andPTEN. Notably, Cytoband 1q21, which contains numerous cell proliferation-related genes, was significantly amplified in the DTBC tumors. LMD successfully minimized stromal components and increased RNA–protein concordance, as evidenced by stromal score comparisons and proteomic analysis. Distinct DTBC and LumA-enriched clusters were observed by proteomic and phosphoproteomic clustering analysis, some with survival differences. Phosphoproteomics identified two distinct phosphoproteomic profiles for high relapse-risk and low relapse-risk basal-like tumors, involving several genes known to be associated with breast cancer oncogenesis and progression, includingKIAA1522,DCK,FOXO3,MYO9B,ARID1A,EPRS,ZC3HAV1, andRBM14. Lastly, an integrated pathway analysis of multi-omics data highlighted a robust enrichment of proliferation pathways in DTBC tumors. Conclusions This study provides an integrated proteogenomic characterization of DTBC vs LumA with tumor cells enriched through laser microdissection. We identified many common features of DTBC tumors and the phosphopeptides that could serve as potential biomarkers for high/low relapse-risk basal-like BC and possibly guide treatment selections.

Oncology↗
NASA NTRS1994

Heavy ion induced mutations in mammalian cells: Cross sections and molecular analysis

Our investigations of heavy ion-induced mutations in mammalian cells, which had been begun a few years ago, were systematically continued. For the first time, it was possible to cover a large LET range with a few kinds of ions. To do this, both UNILAC and SIS were used to yield comparable data for a large energy range. This is a necessary condition for a comprehensive description of the influence of such ion parameters as energy and LET. In these experiments, the induced resistance against the poison 6-thioguanin (6-TG), which is linked to the HPRT locus on the genome, is being used as mutation system. In addition to the mutation-induction cross-section measurements, the molecular changes of the DNA are being investigated by means of Multiplex PCR ('Polymerase Chain Reaction') gene amplification. From these experiments we expect further elucidation of the mutation-inducing mechanisms composing the biological action of heavy-ion radiation.

Stoll, U.↗
NASA NTRS2004

Characterization of Two Microbial Isolates from Andean Lakes in Bolivia

We are currently investigating the biological population present in the highest and least explored perennial lakes on earth in the Bolivian and Chilean Andes, including several volcanic crater lakes of more than 6000 m elevation, in combination of microbiological and molecular biological methods. Our samples were collected in saline lakes of the Laguna Blanca Laguna Verde area in the Bolivian Altiplano and in the Licancabur volcano crater (27 deg. 47 min S/67 deg. 47 min. W) in the ongoing project studying high altitude lakes. The main goal of the project is to look for analogies with Martian paleolakes. These Bolivian lakes can be described as Andean lakes following the classification of Chong. We have attempted to isolate pure cultures and phylogenetically characterize prokaryotes that grew under laboratory conditions. Sediment samples taken from the Licancabur crater lake (LC), Laguna Verde (LV), and Laguna Blanca (LB) were analyzed and cultured using enriched liquid media under both aerobic and anaerobic conditions. All cultures were incubated at room temperature (15 to 20 C) and under light exposure. For the reported isolates, 36 hours incubation were necessary for reaching optimal optical densities to consider them viable cultures. Ten serial dilutions starting from 1% inoculum were required to obtain a suitable enriched cell culture to transfer into solid media. Cultures on solid medium were necessary to verify the formation of colonies in order to isolate pure cultures. Different solid media were prepared using several combinations of both trace minerals and carbohydrates sources in order to fit their nutrient requirements. The microorganisms formed individual colonies on solid media enriched with tryptone, yeast extract and sodium chloride. Cells morphology was studied by optical and electronic microscopy. Rodshape morphologies were observed in most cases. Total bacterial genomic DNA was isolated from 50 ml late-exponential phase culture by using the CTAB miniprep protocol. The 16S rRNA genes were amplified by PCR using both Bacteria- and Archaeauniversal primer sets: 27f and 1492r, 21f and 1492r respectively. Sequences of 16S rRNA gene were determined and initially compared with reference sequences contained in the EMBL nucleotide sequence database by using the BLAST program and were subsequently aligned with 16S rRNA reference sequences in the ARB package (http://www.mikro.biologie.tu-muenchen.de). Aligned sequences were inserted within a stable phylogenetic tree by using the ARB parsimony tool. In this work we report the morphology and phylogenetic characterization of two isolates belonged to Laguna Blanca sediments.

Demergasso, C.↗
DOE OSTI2025

Rhizosphere Microbiome Diversity Potentially Supports Robust Nature of Field Pennycress ( Thlaspi arvense L.) in Dryland Cropping Systems of Eastern Washington

ABSTRACT Field pennycress ( Thlaspi arvense L.) is an annual in the Brassicaceae family and is currently being developed as an oilseed intermediate crop suitable for renewable biodiesel and jet fuel. It displays many desirable characteristics for this role including cold tolerance, a rapid life cycle, and a seed fatty acid profile conducive to bioenergy generation. These traits make field pennycress favorable for winter oilseed cultivation in the inland Pacific Northwest (iPNW). Simultaneously, intermediate crops are an increasingly recognized component of both agronomic sustainability and soil health management. Intermediate crops enhance soil microbial diversity, which benefits both soil and plant health. To understand the impact of field pennycress on soil microbial diversity, two natural accessions and seven experimental accessions were grown at three sites in Eastern Washington. Aboveground biomass and rhizosphere soil were then collected. Soil genomic DNA was extracted from rhizosphere samples and used to generate an amplicon library for bacterial (16S) and fungal (ITS) rRNA sequences. The resulting libraries were analyzed in QIIME2, which revealed that not only did the fad2 deficient line from the Spring32‐10 background have significantly increased aboveground biomass production compared to other pennycress genotypes, but also displayed significantly higher β‐diversity in the rhizosphere community specifically at the site experiencing the driest conditions. ANCOM analysis showed that multiple sequences similar to beneficial plant and soil health enhancing organisms such as Trichoderma spirale , Pseudomonas spp., and Methylobacterium goesingense were found to be enriched in the microbiome of the fad2 Spring32‐10 background also at that site. To add additional context to rhizosphere community data, root exudates from two pennycress genotypes were captured in magenta boxes and analyzed using HPLC. Future work will expand our understanding of the mechanisms by which field pennycress creates diversity in the rhizosphere, thus expanding our ability to cultivate this crop in the iPNW.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Overestimated prediction using polygenic prediction derived from summary statistics

When polygenic risk score (PRS) is derived from summary statistics, independence between discovery and test sets cannot be monitored. We compared two types of PRS studies derived from raw genetic data (denoted as rPRS) and the summary statistics for IGAP (sPRS). Two variables with the high heritability in UK Biobank, hypertension, and height, are used to derive an exemplary scale effect of PRS. sPRS without APOE is derived from International Genomics of Alzheimer’s Project (IGAP), which records ΔAUC and ΔR 2 of 0.051 ± 0.013 and 0.063 ± 0.015 for Alzheimer’s Disease Sequencing Project (ADSP) and 0.060 and 0.086 for Accelerating Medicine Partnership - Alzheimer’s Disease (AMP-AD). On UK Biobank, rPRS performances for hypertension assuming a similar size of discovery and test sets are 0.0036 ± 0.0027 (ΔAUC) and 0.0032 ± 0.0028 (ΔR 2 ). For height, ΔR 2 is 0.029 ± 0.0037. Considering the high heritability of hypertension and height of UK Biobank and sample size of UK Biobank, sPRS results from AD databases are inflated. Independence between discovery and test sets is a well-known basic requirement for PRS studies. However, a lot of PRS studies cannot follow such requirements because of impossible direct comparisons when using summary statistics. Thus, for sPRS, potential duplications should be carefully considered within the same ethnic group.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2023

Unusual modifications of protein biomarkers expressed by plasmid, prophage, and bacterial host of pathogenic Escherichia coli identified using top‐down proteomic analysis

Rationale Pathogenic bacteria often carry prophage (bacterial viruses) and plasmids (small circular pieces of DNA) that may harbor toxin, antibacterial, and antibiotic resistance genes. Proteomic characterization of pathogenic bacteria should include the identification of host proteins and proteins produced by prophage and plasmid genomes. Methods Protein biomarkers of two strains of Shiga toxin–producingEscherichia coli(STEC) were identified using antibiotic induction, matrix‐assisted laser desorption/ionization tandem time‐of‐flight (MALDI‐TOF‐TOF) tandem mass spectrometry (MS/MS) with post‐source decay (PSD), top‐down proteomic (TDP) analysis, and plasmid sequencing. Alphafold2 was also used to compare predicted in silico structures of the identified proteins to prominent fragment ions generated using MS/MS‐PSD. Strain samples were also analyzed with and without chemical reduction treatment to detect the attachment of pendant groups bound by thioester or disulfide bonds. Results Shiga toxin was detected and/or identified in both STEC strains. For the first time, we also identified the osmotically inducible protein (OsmY) whose sequence unexpectedly had two forms: a full and a truncated sequence. The truncated OsmY terminates in the middle of an α‐helix as determined by Alphafold2. A plasmid‐encoded colicin immunity protein was also identified with and without attachment of an unidentified cysteine‐bound pendant group (~307 Da). Plasmid sequencing confirmed top‐down analysis and the identification of a promoter upstream of the immunity gene that is activated by antibiotic induction, that is, SOS box. Conclusions TDP analysis, coupled with other techniques (e.g., antibiotic induction, chemical reduction, plasmid sequencing, and in silico protein modeling), is a powerful tool to identify proteins (and their modifications), including prophage‐ and plasmid‐encoded proteins, produced by pathogenic microorganisms.

Biochemistry & Molecular Biology↗
DOE OSTI2021

Genome-wide discovery of G-quadruplexes in barley

G-quadruplexes (G4s) are four-stranded nucleic acid structures with closely spaced guanine bases forming square planar G-quartets. Aberrant formation of G4 structures has been associated with genomic instability. However, most plant species are lacking comprehensive studies of G4 motifs. In this study, genome-wide identification of G4 motifs in barley was performed, followed by a comparison of genomic distribution and molecular functions to other monocot species, such as wheat, maize, and rice. Similar to the reports on human and some plants like wheat, G4 motifs peaked around the 5' untranslated region (5' UTR), the first coding domain sequence, and the first intron start sites on antisense strands. Our comparative analyses in human, Arabidopsis, maize, rice, and sorghum demonstrated that the peak points could be erroneously merged into a single peak when large window sizes are used. We also showed that the G4 distributions around genic regions are relatively similar in the species studied, except in the case of Arabidopsis. G4 containing genes in monocots showed conserved molecular functions for transcription initiation and hydrolase activity. Additionally, we provided examples of imperfect G4 motifs.

54 ENVIRONMENTAL SCIENCES↗