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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

A Functional Survey of the Regulatory Landscape of Estrogen Receptor–Positive Breast Cancer Evolution

Abstract Only a handful of somatic alterations have been linked to endocrine therapy resistance in hormone-dependent breast cancer, potentially explaining ∼40% of relapses. If other mechanisms underlie the evolution of hormone-dependent breast cancer under adjuvant therapy is currently unknown. In this work, we employ functional genomics to dissect the contribution of cis-regulatory elements (CRE) to cancer evolution by focusing on 12 megabases of noncoding DNA, including clonal enhancers, gene promoters, and boundaries of topologically associating domains. Parallel epigenetic perturbation (CRISPRi) in vitro reveals context-dependent roles for many of these CREs, with a specific impact on dormancy entrance and endocrine therapy resistance. Profiling of CRE somatic alterations in a unique, longitudinal cohort of patients treated with endocrine therapies identifies a limited set of noncoding changes potentially involved in therapy resistance. Overall, our data uncover how endocrine therapies trigger the emergence of transient features which could ultimately be exploited to hinder the adaptive process. Significance: This study shows that cells adapting to endocrine therapies undergo changes in the usage or regulatory regions. Dormant cells are less vulnerable to regulatory perturbation but gain transient dependencies which can be exploited to decrease the formation of dormant persisters.

Oncology↗
DOE OSTI2024

Developing a pipeline to expand the genetic code of diverse bacteria for microbial engineering

Microbial biotechnologies are key to addressing grand challenges to promote human health, reverse carbon emissions, recycle mixed plastic waste, remediate contaminated soils, and achieve sustainable economies. Synthetic biology has enabled design of diverse microbes and their proteins for useful purposes, but the narrowness of the natural genetic code limits functional diversity (e.g., biosynthesis) of engineered microbes. The natural genetic code defines the fundamental rules of translating genetic information into proteins comprised of 22 ‘canonical’ amino acids. However, using a technique called genetic code expansion (GCE), the chemical properties and therefore functions of proteins can be transformed by incorporation of one or more of ~200 chemically diverse ‘non-canonical’ amino acids. The effective application of genetic code expansion in diverse microbes has the potential to revolutionize biotechnology. However, despite over 50 years of research and its transformative potential, the application of genetic code expansion has been limited to a handful of bacterial species. In this project, we will perform three tasks to both overcome the barriers that prevent wide spread adoption of GCE as molecular tool and demonstrate its potential for biotechnological applications. Specifically, we will (1) develop a genetic engineering methodology that will enable use of GCE in a broad range of bacterial hosts, (2) use high-throughput functional genomics methods to identify physiological responses to both genetic code expansion and exposure to non-canonical amino acids in three different bacteria, and (3) demonstrate an application of GCE by selectively incorporate non-canonical amino acids into surface displayed peptides such as those used for biomining.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Interactive tools for functional annotation of bacterial genomes

Automated annotations of protein functions are error-prone because of our lack of knowledge of protein functions. For example, it is often impossible to predict the correct substrate for an enzyme or a transporter. Furthermore, much of the knowledge that we do have about the functions of proteins is missing from the underlying databases. We discuss how to use interactive tools to quickly find different kinds of information relevant to a protein’s function. Many of these tools are available via PaperBLAST (http://papers.genomics.lbl.gov). Combining these tools often allows us to infer a protein’s function. Ideally, accurate annotations would allow us to predict a bacterium’s capabilities from its genome sequence, but in practice, this remains challenging. We describe interactive tools that infer potential capabilities from a genome sequence or that search a genome to find proteins that might perform a specific function of interest.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

MVP: a modular viromics pipeline to identify, filter, cluster, annotate, and bin viruses from metagenomes

While numerous computational frameworks and workflows are available for recovering prokaryote and eukaryote genomes from metagenome data, only a limited number of pipelines are designed specifically for viromics analysis. With many viromics tools developed in the last few years alone, it can be challenging for scientists with limited bioinformatics experience to easily recover, evaluate quality, annotate genes, dereplicate, assign taxonomy, and calculate relative abundance and coverage of viral genomes using state-of-the-art methods and standards. Here, we describe Modular Viromics Pipeline (MVP) v.1.0, a user-friendly pipeline written in Python and providing a simple framework to perform standard viromics analyses. MVP combines multiple tools to enable viral genome identification, characterization of genome quality, filtering, clustering, taxonomic and functional annotation, genome binning, and comprehensive summaries of results that can be used for downstream ecological analyses. Overall, MVP provides a standardized and reproducible pipeline for both extensive and robust characterization of viruses from large-scale sequencing data including metagenomes, metatranscriptomes, viromes, and isolate genomes. As a typical use case, we show how the entire MVP pipeline can be applied to a set of 20 metagenomes from wetland sediments using only 10 modules executed via command lines, leading to the identification of 11,656 viral contigs and 8,145 viral operational taxonomic units (vOTUs) displaying a clear beta-diversity pattern. Further, acting as a dynamic wrapper, MVP is designed to continuously incorporate updates and integrate new tools, ensuring its ongoing relevance in the rapidly evolving field of viromics. MVP is available at https://gitlab.com/ccoclet/mvp and as versioned packages in PyPi and Conda.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Rethinking Suicide Thi4 Thiazole Synthases: Comparative Genomic Insights and Pilot Functional Evidence

Suicide thiazole synthases (Thi4) are mononuclear metal enzymes that form the thiazole moiety of thiamin from NAD + , glycine, and a sulfur atom that is stripped from an active-site cysteine residue, causing enzyme inactivation. Comparative genomic analysis shows that prokaryotic Thi4 genes often cluster on the chromosomal regions encoding ThiS, ThiF, and other proteins that can produce, relay, or use persulfide or thiocarboxylate sulfur. These recurring genomic associations raise the possibility that, in some microorganisms, suicide Thi4s may interact with sulfur-relay systems, i.e., they can possibly operate in a nonsuicide mode. This proof-of-concept study explores this possibility via complementation assays using Escherichia coli as a heterologous platform. A representative bacterial Thi4 that clustered with thiS and thiF complemented an E. coli ΔthiG (thiazole auxotroph) single mutant better than a ΔthiG ΔthiF ΔthiS triple mutant. Although (in)direct sulfur transfer could not be assessed in the scope of our investigation, the initial results suggest a dependence on host sulfur relay components, consistent with predicted interactions with the host sulfide transfer chain. Collectively, this new perspective provides a useful guide for future biochemical studies on alternative modes of action for “suicide Thi4s” and accessory proteins.

Bacteria↗
DOE OSTI2025

Pangenomes suggest ecological-evolutionary responses to experimental soil warming

ABSTRACT Below-ground carbon transformations that contribute to healthy soils represent a natural climate change mitigation, but newly acquired traits adaptive to climate stress may alter microbial feedback mechanisms. To better define microbial evolutionary responses to long-term climate warming, we study microorganisms from an ongoing in situ soil warming experiment where, for over three decades, temperate forest soils are continuously heated at 5°C above ambient. We hypothesize that across generations of chronic warming, genomic signatures within diverse bacterial lineages reflect adaptations related to growth and carbon utilization. From our bacterial culture collection isolated from experimental heated and control plots, we sequenced genomes representing dominant taxa sensitive to warming, including lineages of Actinobacteria, Alphaproteobacteria, and Betaproteobacteria. We investigated genomic attributes and functional gene content to identify signatures of adaptation. Comparative pangenomics revealed accessory gene clusters related to central metabolism, competition, and carbon substrate degradation, with few functional annotations explicitly associated with long-term warming. Trends in functional gene patterns suggest genomes from heated plots were relatively enriched in central carbohydrate and nitrogen metabolism pathways, while genomes from control plots were relatively enriched in amino acid and fatty acid metabolism pathways. We observed that genomes from heated plots had less codon bias, suggesting potential adaptive traits related to growth or growth efficiency. Codon usage bias varied for organisms with similar 16S rrn operon copy number, suggesting that these organisms experience different selective pressures on growth efficiency. Our work suggests the emergence of lineage-specific trends as well as common ecological-evolutionary microbial responses to climate change. IMPORTANCE Anthropogenic climate change threatens soil ecosystem health in part by altering below-ground carbon cycling carried out by microbes. Microbial evolutionary responses are often overshadowed by community-level ecological responses, but adaptive responses represent potential changes in traits and functional potential that may alter ecosystem function. We predict that microbes are adapting to climate change stressors like soil warming. To test this, we analyzed the genomes of bacteria from a soil warming experiment where soil plots have been experimentally heated 5°C above ambient for over 30 years. While genomic attributes were unchanged by long-term warming, we observed trends in functional gene content related to carbon and nitrogen usage and genomic indicators of growth efficiency. These responses may represent new parameters in how soil ecosystems feedback to the climate system.

Choudoir, Mallory J. (ORCID:0000000291175150)↗
DOE OSTI2024

A functional microbiome catalogue crowdsourced from North American rivers

Predicting elemental cycles and maintaining water quality under increasing anthropogenic influence requires knowledge of the spatial drivers of river microbiomes. However, understanding of the core microbial processes governing river biogeochemistry is hindered by a lack of genome-resolved functional insights and sampling across multiple rivers. Here we used a community science effort to accelerate the sampling, sequencing and genome-resolved analyses of river microbiomes to create the Genome Resolved Open Watersheds database (GROWdb). GROWdb profiles the identity, distribution, function and expression of microbial genomes across river surface waters covering 90% of United States watersheds. Specifically, GROWdb encompasses microbial lineages from 27 phyla, including novel members from 10 families and 128 genera, and defines the core river microbiome at the genome level. GROWdb analyses coupled to extensive geospatial information reveals local and regional drivers of microbial community structuring, while also presenting foundational hypotheses about ecosystem function. Building on the previously conceived River Continuum Concept, we layer on microbial functional trait expression, which suggests that the structure and function of river microbiomes is predictable. We make GROWdb available through various collaborative cyberinfrastructures, so that it can be widely accessed across disciplines for watershed predictive modelling and microbiome-based management practices.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Tunturi virus isolates and metagenome-assembled viral genomes provide insights into the virome of Acidobacteriota in Arctic tundra soils

Arctic soils are climate-critical areas, where microorganisms play crucial roles in nutrient cycling processes. Acidobacteriota are phylogenetically and physiologically diverse bacteria that are abundant and active in Arctic tundra soils. Still, surprisingly little is known about acidobacterial viruses in general and those residing in the Arctic in particular. Here, we applied both culture-dependent and -independent methods to study the virome of Acidobacteriota in Arctic soils. Five virus isolates, Tunturi 1–5, were obtained from Arctic tundra soils, Kilpisjärvi, Finland (69°N), using Tunturiibacter spp. strains originating from the same area as hosts. The new virus isolates have tailed particles with podo- (Tunturi 1, 2, 3), sipho- (Tunturi 4), or myovirus-like (Tunturi 5) morphologies. The dsDNA genomes of the viral isolates are 63–98 kbp long, except Tunturi 5, which is a jumbo phage with a 309-kbp genome. Tunturi 1 and Tunturi 2 share 88% overall nucleotide identity, while the other three are not related to one another. For over half of the open reading frames in Tunturi genomes, no functions could be predicted. To further assess the Acidobacteriota-associated viral diversity in Kilpisjärvi soils, bulk metagenomes from the same soils were explored and a total of 1881 viral operational taxonomic units (vOTUs) were bioinformatically predicted. Almost all vOTUs (98%) were assigned to the class Caudoviricetes. For 125 vOTUs, including five (near-)complete ones, Acidobacteriota hosts were predicted. Acidobacteriota-linked vOTUs were abundant across sites, especially in fens. Terriglobia-associated proviruses were observed in Kilpisjärvi soils, being related to proviruses from distant soils and other biomes. Approximately genus- or higher-level similarities were found between the Tunturi viruses, Kilpisjärvi vOTUs, and other soil vOTUs, suggesting some shared groups of Acidobacteriota viruses across soils. This study provides acidobacterial virus isolates as laboratory models for future research and adds insights into the diversity of viral communities associated with Acidobacteriota in tundra soils. Predicted virus-host links and viral gene functions suggest various interactions between viruses and their host microorganisms. Largely unknown sequences in the isolates and metagenome-assembled viral genomes highlight a need for more extensive sampling of Arctic soils to better understand viral functions and contributions to ecosystem-wide cycling processes in the Arctic.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Stable hypermutators revealed by the genomic landscape of genes involved in genome stability among yeast species

Mutator phenotypes are short-lived due to the rapid accumulation of deleterious mutations. Yet, recent observations reveal that certain fungi can undergo prolonged accelerated evolution after losing genes involved in DNA repair. Here, we surveyed 1,154 yeast genomes representing nearly all known yeast species of the subphylum Saccharomycotina (phylum Ascomycota) to examine the relationship between reduced gene repertoires broadly associated with genome stability functions (e.g., DNA repair, cell cycle) and elevated evolutionary rates. We identified three distantly related lineages—encompassing 12% of species—that had both the most streamlined sets of genes involved in genome stability (specifically DNA repair) and the highest evolutionary rates in the entire subphylum. Two of these “faster-evolving lineages” (FELs)—a subclade within the order Pichiales and the Wickerhamiella/Starmerella (W/S) clade (order Dipodascales)—are described here for the first time, while the third corresponds to a previously documented Hanseniaspora FEL. Examination of genome stability gene repertoires revealed a set of genes predominantly absent in these three FELs, suggesting a potential role in the observed acceleration of evolutionary rates. In the W/S clade, genomic signatures are consistent with a substantial mutational burden, including pronounced A|T bias and endogenous DNA damage. Interestingly, we found that the W/S clade also contains DNA repair genes possibly acquired through horizontal gene transfer, including a photolyase of bacterial origin. These findings highlight how hypermutators can persist across macroevolutionary timescales, potentially linked to the loss of genes related with genome stability, with horizontal gene transfer as a possible avenue for partial functional compensation.

DNA repair↗
DOE OSTI2024

High Throughput Genome Releaser

In this study, we present the development of a High Throughput Genome Releaser, an innovative device addressing common challenges in screening PCR. This genome DNA releaser is designed for rapid, cost-effective, and efficient DNA extraction, optimized for subsequent PCR reactions. Our experimentation with various synthetic materials led us to select a particular type of plastic that mirrors the properties of glass cover slides, providing a smooth surface and effective compression capabilities. We engineered a 96-well device equipped with a 96-well plate and a top rod, operable both manually and automatically, which is compatible with widely used liquid-handling robot decks. This compatibility enhances ease of use in high-throughput PCR setups. Additionally, we developed software to support its automatic functions. The genome releaser facilitates the extraction of PCR-amplifiable genomic DNA from 96 samples within minutes, eliminates the need for extraction buffers, and is adaptable to a wide range of microorganisms and cells. This versatility could significantly advance biomanufacturing processes.

42 ENGINEERING↗
DOE OSTI2025

Long-read RNA sequencing atlas of human microglia isoforms elucidates disease-associated genetic regulation of splicing

Microglia, the innate immune cells of the central nervous system, have been genetically implicated in multiple neurodegenerative diseases. Mapping the genetics of gene expression in human microglia has identified several loci associated with disease-associated genetic variants in microglia-specific regulatory elements. However, identifying genetic effects on splicing is challenging because of the use of short sequencing reads. Here, we present the isoform-centric microglia genomic atlas (isoMiGA), which leverages long-read RNA sequencing to identify 35,879 novel microglia isoforms. We show that these isoforms are involved in stimulation response and brain region specificity. We then quantified the expression of both known and novel isoforms in a multi-ancestry meta-analysis of 555 human microglia short-read RNA sequencing samples from 391 donors, and found associations with genetic risk loci in Alzheimer’s and Parkinson’s disease. We nominate several loci that may act through complex changes in isoform and splice-site usage.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Mondo: integrating disease terminology across communities

Precision medicine aims to enhance diagnosis, treatment, and prognosis by integrating multimodal data at the point of care. However, challenges arise due to the vast number of diseases, differing methods of classification, and conflicting terminological coding systems and practices used to represent molecular definitions of disease. This lack of interoperability artificially constrains the potential for diagnosis, clinical decision support, care outcome analysis, as well as data linkage across research domains to support the development or repurposing of therapeutics. There is a clear and pressing need for a unified system for managing disease entities⁠—including identifiers, synonyms, and definitions. To address these issues, we created the Mondo disease ontology—a community-driven, open-source, unified disease classification system that harmonizes diverse terminologies into a consistent, computable framework. Mondo integrates key medical and biomedical terminologies, including Online Mendelian Inheritance in Man (OMIM), Orphanet, Medical Subject Headings (MeSH), National Cancer Institute Thesaurus (NCIt), and more, to provide a comprehensive and accurate representation of disease concepts with fully provenanced and attributed links back to the sources. Mondo can be used as the handle for curation of gene–disease associations utilized in diagnostic applications, research applications such as computational phenotyping, and in clinical coding systems in clinical decision support by pointing the clinician to the numerous knowledge resources linked to the Mondo identifier. Mondo's community-centric approach, stewarded by the Monarch Initiative's expertise in ontologies, ensures that the ontology remains adaptable to the evolving needs of biomedical research and clinical communities, as well as the knowledge providers.

biomedical informatics↗
DOE OSTI2025

Hidden Allies: Decoding the Core Endohyphal Bacteriome of Aspergillus fumigatus

ABSTRACT Bacterial–fungal interactions that influence the behaviour of one or both organisms are common in nature. Well‐studied systems include endosymbiotic relationships that range from transient to long‐term associations. Diverse endohyphal bacteria associate with fungal hosts, emphasising the need to better comprehend the fungal bacteriome. We evaluated the hypothesis thatAspergillus fumigatusharbours an endohyphal community of bacteria that influence the host phenotype. We analysed whether 38A. fumigatusstrains show stable association with diverse endohyphal bacteria; all derived from single‐conidium cultures that were subjected to antibiotic and heat treatments. The fungal bacteriome, inferred through analysis of bacterial diversity within the fungal strains (short‐ and long‐ read sequencing methods), revealed the presence of core endohyphal bacterial genera. Microscopic analysis further confirmed the presence of endohyphal bacteria. The fungal strains exhibited high genetic diversity and phenotypic heterogeneity in drug susceptibility and in vivo virulence. No correlations were observed between genomic or functional traits and bacteriome diversity, but the abundance of some bacterial genera correlated with fungal virulence or posaconazole susceptibility. The observed endobacteriome may play functional roles, for example, nitrogen fixation. Our study emphasises the existence of complex interactions between fungi and endohyphal bacteria, possibly impacting the phenotype of the fungal host, including virulence.

Environmental Sciences & Ecology↗
DOE OSTI2025

Dual transposon sequencing profiles the genetic interaction landscape in bacteria

Gene redundancy complicates systematic characterization of gene function as single-gene deletions may not produce discernible phenotypes. We report dual transposon sequencing (dual Tn-seq), a platform for assaying the fitness of a comprehensive double mutant pool in parallel. Dual Tn-seq couples random barcode transposon site sequencing with the Cre-lox system, enabling deep sampling of 73% of the 1.3 million possible double gene deletions in Streptococcus pneumoniae. The genetic interactions identified span a wide range of biochemical processes, revealing new factors in presumably well-studied pathways, exemplified by a cytidine triphosphate synthase PyrJ. Moreover, this approach should permit further investigation of growth condition–specific genetic interactions. Because dual Tn-seq does not require the construction of a large array of single mutants, it should be readily adaptable to various microorganisms.

CTP synthesis↗
DOE OSTI2026

The landscape of regulatory element evolution in a C4 perennial grass

Gene regulatory evolution is a well-known source of phenotypic diversity and adaptive evolution. Although cis-regulatory elements (CREs) play a vital role in gene expression evolution, the molecular evolution of CREs remains mostly unknown due to the difficulty in identifying and characterizing these functional elements. Comparative genomic analyses of noncoding DNA can be leveraged to identify conserved noncoding sequences (CNS), many of which may harbor functional CREs conserved by purifying selection. However, purely computational inference of CREs from putative CNS can be erroneous due to the complex genomic architecture in plants. One promising experimental approach to identify CREs is by profiling accessible chromatin regions (ACRs) that are often associated with the location of CREs. In this study, we use comparative genomics along with the profiling of ACRs to study the molecular evolution of putative functional noncoding regulatory regions in Panicoid grasses. We identified sets of CNS that varied in relationship to the degree of evolutionary divergence among the studied taxa, including identifying core-Panicoid-CNS. We augmented this analysis by profiling ACRs in Panicum hallii ecotypes using ATAC-seq. ACRs had low SNP density at the summit, harbored a high frequency of core-Panicoid-CNS, and were enriched with expression QTL. These data help to annotate the P. hallii genome for putative functional elements and suggest that a large proportion of these ACRs are evolving under purifying selection. Turnover in CNS and ACR between ecotypes of P. hallii identifies a small set of putatively divergent CREs that may underlie differences in gene regulation between genotypes from inland and coastal habitats. In summary, we profiled ACRs in Panicoid grasses and integrated this data with our putative CNS prediction framework, which provides unique insight into patterns of polymorphism and divergence in CREs in C4 perennial grasses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Climate-driven succession in marine microbiome biodiversity and biogeochemical function

Abstract Seasonal and El Niño-Southern Oscillation (ENSO) warming result in similar ocean changes as predicted with climate change. Climate-driven environmental cycles have strong impacts on microbiome diversity, but impacts on microbiome function are poorly understood. Here we quantify changes in microbial genomic diversity and functioning over 11 years covering seasonal and ENSO cycles at a coastal site in the southern California Current. We observe seasonal oscillations between large-genome lineages during cold, nutrient rich conditions in winter and spring versus small-genome lineages, including Prochlorococcus and Pelagibacter , in summer and fall. Parallel interannual changes separate communities depending on ENSO condition. Biodiversity shifts translate into clear oscillations in microbiome functional potential. Ocean warming induced an ecosystem with less iron but more macronutrient stress genes, depressed organic carbon degradation potential and biomass, and elevated carbon-to-nutrient biomass ratios. The consistent microbial response observed across time-scales points towards large climate-driven changes in marine ecosystems and biogeochemical cycles.

Larkin, Alyse A. (ORCID:0000000344660791)↗
DOE OSTI2025

An RNA ligase partner for the prokaryotic protein-only RNase P: insights into the functional diversity of RNase P from genome mining

RNase P can use either an RNA- or a protein-based active site to catalyze 5'-maturation of transfer RNAs (tRNAs). This distinctive attribute in the biocatalytic repertoire raises questions about the underlying evolutionary driving forces, especially if each variant somehow affords a selective advantage under certain conditions. Upon mining all publicly available prokaryotic genomes and examining gene co-occurrence, we discovered that an RNA ligase with circularization activity was significantly overrepresented in genomes that contain the protein form of RNase P. This unexpected linkage inspires testable ideas to understand the bases for scenarios that might favor RNase P variants of different architectures/make-up.

HARP↗