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All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Signatures of Selection for Resistance/Tolerance to Perkinsus olseni in Grooved Carpet Shell Clam ( Ruditapes decussatus ) Using a Population Genomics Approach

ABSTRACT The grooved carpet shell clam ( Ruditapes decussatus ) is a bivalve of high commercial value distributed throughout the European coast. Its production has suffered a decline caused by different factors, especially by the parasite Perkinsus olsenii . Improving production of R . decussatus requires genomic resources to ascertain the genetic factors underlying resistance/tolerance to P. olseni i . In this study, the first reference genome of R . decussatus was assembled through long‐ and short‐read sequencing (1677 contigs; 1.386 Mb) and further scaffolded at chromosome level with Hi‐C (19 superscaffolds; 95.4% of assembly). Repetitive elements were identified (32%) and masked for annotation of 38,276 coding‐ and 13,056 non‐coding genes. This genome was used as a reference to develop a 2bRAD‐Seq 13,438 SNP panel for a genomic screening on six shellfish beds distributed across the Atlantic Ocean and Mediterranean Sea. Beds were selected by perkinsosis prevalence and the infection level was individually evaluated in all the samples. Genetic diversity was significantly higher in the Mediterranean than in the Atlantic region. The main genetic breakage was detected between those regions (F ST = 0.224), being the Mediterranean more heterogeneous than the Atlantic. Several loci under divergent selection (394 outliers; 261 genomic windows) were detected across shellfish beds. Samples were also inspected to detect signals of selection for resistance/tolerance to P. olseni i by using infection‐level and population‐genomics approaches, and 90 common divergent outliers for resistance/tolerance to perkinsosis were identified and used for gene mining. Candidate genes and markers identified provide invaluable information for controlling perkinsosis and for improving production of the grooved carpet shell clam.

Sambade, Inés M. [Department of Zoology, Genetics ↗
DOE OSTI2026

Hyporheic zone, river, and groundwater metagenome resolved genomes and rpS3 genes in East River Watershed, Colorado USA Summer 2020, 2021

Here we present metagenome assembled genomes (MAGs) for the bacterial and archaeal communities from water filter collected across 8 locations along the East River Watershed, CO, and 1 nearby groundwater well. The purpose was to look for connectivity and similarities across the network and to see the impact of the groundwater. As a part of Lawrence Berkeley National Laboratory (LBNL) Watershed Science Focus Area (SFA), we assessed community composition and strain similarities between the sites and we also compared it to previous metagenomic studies within the watershed looking at floodplain (Matheus Carnevali et al. 2021) and hillslope (Lavy et al. 2019) microbiomes. Here we present metagenome assembled genomes (MAGs) for the bacterial and archaeal communities from filters across 8 locations during August 2020 and July 2021. This resulted in 32 samples. The groundwater sample was sequenced at UC Berkley's QB3. The other 31 samples were sequenced at University of Maryland. Metagenomes were assembled using four autobinners and the best bins were selected using dasTool. The genomes were dereplicated at 95% with dRep and the subset of winning genomes were manually curated based on visual inspection of taxonomic profile, GC content, coverage, and a set of 51 bacterial single copy genes (BSCG), and 38 archaeal signal copy genes (ASCG). The dataset includes a zip file of 311 genomes (HZ_River_SW_MAGS_Dereplicated_95.zip). The dataset additionally includes a zipped file of ribosomal protein small subunit 3 (rpS3) proteins from the hyporheic zone and river data (rpS3_Proteins_HZ_River.zip), a metadata file used to register associated samples with IGSNs (International Generic Sample Numbers) (samples.csv), a location metadata file (locations.csv). This work was supported by the Watershed Function Science Focus Area at Lawrence Berkeley National Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-05CH11231.

DNA↗
DOE OSTI2025

Poplar: a phylogenomics pipeline

Motivation Generating phylogenomic trees from the genomic data is essential in understanding biological systems. Each step of this complex process has received extensive attention and has been significantly streamlined over the years. Given the public availability of data, obtaining genomes for a wide selection of species is straightforward. However, analyzing that data to generate a phylogenomic tree is a multistep process with legitimate scientific and technical challenges, often requiring a significant input from a domain-area scientist. Results We present Poplar, a new, streamlined computational pipeline, to address the computational logistical issues that arise when constructing the phylogenomic trees. It provides a framework that runs state-of-the-art software for essential steps in the phylogenomic pipeline, beginning from a genome with or without an annotation, and resulting in a species tree. Running Poplar requires no external databases. In the execution, it enables parallelism for execution for clusters and cloud computing. The trees generated by Poplar match closely with state-of-the-art published trees. The usage and performance of Poplar is far simpler and quicker than manually running a phylogenomic pipeline. Availability and implementation Freely available on GitHub at https://github.com/sandialabs/poplar. Implemented using Python and supported on Linux.

Koning, Elizabeth [Sandia National Laboratories (S↗
DOE OSTI2026

Single nucleotide variants drive evolutionary phage-host arms race in anaerobic carbon dioxide-converting microbiome

Microbial bioconversions are shaped by environmental perturbations and the adaptation of resident microbiomes. Prokaryotes coexist with bacteriophages, yet their coevolutionary trajectories remain underexplored. Here, we investigate the effects of a cultivation vessel leak on an anaerobic consortium performing carbon dioxide reduction. Using time-series shotgun metagenomic sequencing, we reconstruct microbial and viral genomes to track community shifts. We further apply single-nucleotide variant profiling and CRISPR array analysis to monitor viral microdiversity and host defense mechanisms. After bioaugmentation restores bioconversion efficiency, the consortium undergoes pronounced restructuring, with new dominant taxa emerging from the rare biosphere. We identify patterns consistent with phage predation selectively removing certain species, while others exhibit resilience to infection. This shift aligns with a widespread viral outbreak and a transient increased frequency of single nucleotide variants in bacterial CRISPR–Cas defense genes. Expansion of CRISPR spacers further supports that CRISPR-mediated processes influence microbial resilience. Concurrently, phages infecting resilient hosts exhibited adaptive evolution, marked by high genetic heterogeneity. Selective pressure varies across their genomes, targeting infectivity genes and protospacer-adjacent motifs. These findings highlight a dynamic evolutionary arms race driven by the selection of beneficial genetic variants, providing a mechanistic framework for multi-omics investigations, and informing biotechnological applications, including phage-based microbiome manipulation.

Ghiotto, G↗
DOE OSTI2026

Genome collection processing for “Conserved upper thermal limits and small safety margins in soil copiotrophic bacteria”

We extracted the genomic DNA of 400 randomly selected isolates using a Quick-DNA Microprep Kit (Zymo Research D3020) according to the manufacturer’s protocol. We then submitted the extracted gDNA samples for short-read Illumina sequencing (200 Mbp) at SeqCoast Genomics (Portsmouth, NH, USA). After preprocessing the sequences using Trimmommatic (Bolger et al. 2014), we assembled the genomes using SPADES (Bankevich et al. 2012) and checked the quality of each assembly using QUAST (Gurevich et al. 2013). We processed the genome assemblies using a KBase (v1.4.0) pipeline (Allen et al. 2017; Arkin et al. 2018). Briefly, we used DRAM (v0.1.2) with default settings to annotate the genome assemblies. We then evaluated genome quality and possible contamination levels using CheckM (v1.0.18) (Parks et al. 2015) and retained genomes with completeness above 98% and contamination below 5% (n = 354), following the authors' guidelines. We then obtained taxonomic assignments for all remaining isolates using the Genome Taxonomy Database tool GTDB-Tk (v2.3.2, database version r214) (Chaumeil et al. 2019). We constructed a phylogenetic tree using the tool SpeciesTree (v2.2.0). We then trimmed the tree (using Trim SpeciesTree to GenomeSet- v1.4.0), retaining only tips within our collection with measured thermal performance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Testing for the Genomic Footprint of Conflict Between Life Stages in an Angiosperm and Moss Species

Abstract The maintenance of genetic variation by balancing selection is of considerable interest to evolutionary biologists. An important but understudied potential driver of balancing selection is antagonistic pleiotropy between diploid and haploid stages of the plant life cycle. Despite sharing a common genome, sporophytes (2n) and gametophytes (n) may undergo differential or even opposing selection. Theoretical work suggests antagonistic pleiotropy between life stages can generate balancing selection and maintain genetic variation. Despite the potential for far-reaching consequences of gametophytic selection, empirical tests of its pleiotropic effects (neutral, synergistic, or antagonistic) on sporophytes are generally lacking. Here, we examined the population genomic signals of selection across life stages in the angiosperm Rumex hastatulus and the moss Ceratodon purpureus. We compared gene expression between life stages and sexes, combined with neutral diversity statistics and the analysis of the distribution of fitness effects. In contrast to what would be predicted under balancing selection due to antagonistic pleiotropy, we found that unbiased genes between life stages were under stronger purifying selection, likely explained by a predominance of synergistic pleiotropy between life stages and strong purifying selection on broadly expressed genes. In addition, we found that 30% of candidate genes under balancing selection in R. hastatulus were located within inversion polymorphisms. Our findings provide novel insights into the genome-wide characteristics and consequences of plant gametophytic selection.

Evolutionary Biology↗
DOE OSTI2025

The Marchantia polymorpha pangenome reveals ancient mechanisms of plant adaptation to the environment

Plant adaptation to terrestrial life started 450 million years ago and has played a major role in the evolution of life on Earth. The genetic mechanisms allowing this adaptation to a diversity of terrestrial constraints have been mostly studied by focusing on flowering plants. Here, we gathered a collection of 133 accessions of the model bryophyte Marchantia polymorpha and studied its intraspecific diversity using selection signature analyses, a genome-environment association study and a pangenome. We identified adaptive features, such as peroxidases or nucleotide-binding and leucine-rich repeats (NLRs), also observed in flowering plants, likely inherited from the first land plants. The M. polymorpha pangenome also harbors lineage-specific accessory genes absent from seed plants. We conclude that different land plant lineages still share many elements from the genetic toolkit evolved by their most recent common ancestor to adapt to the terrestrial habitat, refined by lineage-specific polymorphisms and gene family evolution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Targeted genetic manipulation and yeast-like evolutionary genomics in the green alga Auxenochlorella

Auxenochlorella spp. are diploid oleaginous green algae whose streamlined genomes can be readily manipulated by homologous recombination, making them highly amenable to discovery research and bioengineering. Vegetatively diploid organisms experience specific evolutionary phenomena, including allodiploid hybridization, mitotic recombination, loss-of-heterozygosity, and aneuploidy; however, studies of these forces have largely focused on yeasts. Here, we present a telomere-to-telomere phased diploid genome assembly of Auxenochlorella UTEX 250-A (haploid length 22 Mb) and introduce a genetic toolkit for site-specific manipulation of the nuclear genome in multiple strains, featuring several selectable markers, inducible promoters, and fluorescent reporters for protein localization. UTEX 250-A is an allodiploid hybrid of Auxenochlorella protothecoides and Auxenochlorella symbiontica, two species differentiated by extensive chromosomal rearrangements. UTEX 250-A haplotypes are a mosaic of each parental species following mitotic recombination, and two chromosomes are trisomic. Loss-of-heterozygosity events are pervasive across Auxenochlorella and can evolve rapidly in the laboratory. High-quality structural annotation yielded ∼7,500 genes per haplotype. Auxenochlorella have experienced gene family loss and reduction, including core photosynthesis genes, and exhibit periodic adenine and cytosine methylation at promoters and gene bodies, respectively. Approximately 10% of genes, especially those involved in DNA repair and sex, overlap antisense long noncoding RNAs, which may participate in a regulatory mechanism. We demonstrate the utility of Auxenochlorella for fundamental research by knockout of a chlorophyll biosynthesis enzyme, and confirm one trisomy by allele-specific transformation. These results demonstrate the generality of several evolutionary forces associated with vegetative diploidy and provide a foundation for the use of Auxenochlorella as a reference organism.

CHL27↗
DOE OSTI2026

Characterization of Multiple Trichloroethene, cis-Dichloroethene and 1,1-Dichloroethene Degrading Propanotrophic Communities

Aerobic cometabolism offers a viable strategy for the remediation of chlorinated solvent plumes at oxic sites where anaerobic approaches are limited. In this study, propane-enriched mixed cultures (derived from agricultural soils and an impacted site sediment) which previously degraded 1,4-dioxane, were evaluated for their capacity to also degrade trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), and 1,1-dichloroethene (1,1-DCE) over successive transfers. Sustained biodegradation of TCE and cDCE was observed across multiple enrichments, and cultures enriched on one compound generally degraded the other. In contrast, 1,1-DCE biodegradation was restricted to a subset of cultures and removal times increased over transfers. Further, 1,1-DCE removal was absent at elevated concentrations, both trends consistent with inhibitory or toxic effects. Whole genome sequencing analyses revealed pronounced substrate-dependent selection of microbial communities, with cDCE-degrading cultures being dominated by Mycobacterium and Mycolicibacterium, whereas TCE-degrading cultures were dominated by Rhodococcus. Rhodococcus metagenome-assembled genomes (MAGs) in the TCE degrading cultures classified as R. opacus or R. wratislaviensis. 1,1-DCE degrading cultures were dominated by Pseudonocardia, although the associated MAGs contained a truncated propane monooxygenase alpha subunit. Functional gene analysis identified both group 5 (prmABCD) and putative group 6 propane monooxygenases. The following KBase narratives contain the quality controlled reads, MAGs (fasta assemblies) and the prokka annotations for each assembly TCE Site 1A and B Propanotrophic MAGs (https://narrative.kbase.us/narrative/254918) TCE Soil 2A and B Propanotrophic MAGs (https://narrative.kbase.us/narrative/254919) TCE Soil T3 A and B Propanotrophic MAGs (https://narrative.kbase.us/narrative/254920) TCE Soil T4 A and B Propanotrophic MAGs (https://narrative.kbase.us/narrative/254921) cDCE Site 1A 1B Propanotrophic MAGs (https://narrative.kbase.us/narrative/254915) cDCE Soils T2 A and B Propanotrophic MAGs (https://narrative.kbase.us/narrative/254927) cDCE Soil T3 A and B Propanotrophic MAGs (https://narrative.kbase.us/narrative/254928) cDCE Soil 4A and B Propanotrophic MAGs (https://narrative.kbase.us/narrative/254942) 1,1-DCE T2 T3 Propanotrophic MAGs (https://narrative.kbase.us/narrative/254903)

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Omics-driven onboarding of the carotenoid producing red yeast Xanthophyllomyces dendrorhous CBS 6938

Transcriptomics is a powerful approach for functional genomics and systems biology, yet it can also be used for genetic part discovery. Here, we derive constitutive and light-regulated promoters directly from transcriptomics data of the basidiomycete red yeast Xanthophyllomyces dendrorhous CBS 6938 (anamorph Phaffia rhodozyma) and use these promoters with other genetic elements to create a modular synthetic biology parts collection for this organism. X. dendrorhous is currently the sole biotechnologically relevant yeast in the Tremellomycete class-it produces large amounts of astaxanthin, especially under oxidative stress and exposure to light. Thus, we performed transcriptomics on X. dendrorhous under different wavelengths of light (red, green, blue, and ultraviolet) and oxidative stress. Differential gene expression analysis (DGE) revealed that terpenoid biosynthesis was primarily upregulated by light through crtI, while oxidative stress upregulated several genes in the pathway. Further gene ontology (GO) analysis revealed a complex survival response to ultraviolet (UV) where X. dendrorhous upregulates aromatic amino acid and tetraterpenoid biosynthesis and downregulates central carbon metabolism and respiration. The DGE data was also used to identify 26 constitutive and regulated genes, and then, putative promoters for each of the 26 genes were derived from the genome. Simultaneously, a modular cloning system for X. dendrorhous was developed, including integration sites, terminators, selection markers, and reporters. Each of the 26 putative promoters were integrated into the genome and characterized by luciferase assay in the dark and under UV light. The putative constitutive promoters were constitutive in the synthetic genetic context, but so were many of the putative regulated promoters. Notably, one putative promoter, derived from a hypothetical gene, showed ninefold activation upon UV exposure. Thus, this study reveals metabolic pathway regulation and develops a genetic parts collection for X. dendrorhous from transcriptomic data. Therefore, this study demonstrates that combining systems biology and synthetic biology into an omics-to-parts workflow can simultaneously provide useful biological insight and genetic tools for nonconventional microbes, particularly those without a related model organism. This approach can enhance current efforts to engineer diverse microbes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Genomic signatures in Variovorax enabling colonization of the Populus endosphere

Microbial colonization of plant roots involves strong selective pressures that shape the structure and function of root-associated communities. In particular, the endosphere represents a highly selective environment requiring host entry and in planta persistence. However, strain-specific microbial traits that enable endosphere colonization remain poorly understood. Here, we use a defined, genome-resolved community of 28 Variovorax strains isolated from the roots of Populus deltoides and Populus trichocarpa (poplar trees) to determine which strains partition between rhizosphere and endosphere compartments and to identify the genomic traits associated with endosphere specialization. By combining strain-resolved metagenomic profiling, comparative genomics, and functional assays, we demonstrate that dominant endosphere colonizers are enriched in genes related to nutrient metabolism, redox balance, transcriptional regulation, and a conserved L-fucose utilization pathway experimentally shown to enhance root colonization. Not all strains succeed through the same strategy. Community-wide functional profiling revealed a distinct and reduced set of traits in the endosphere, including orthogroups associated with low-abundance strains that were overlooked in strain-level analyses. These findings reveal that multiple ecological strategies, such as metabolic competition, regulatory adaptation, and niche specialization, can support endosphere colonization. Our results advance the understanding of how bacterial colonization traits are distributed and deployed within a plant microbiome and suggest that host filtering selects for distinct, and sometimes complementary, microbial strategies. This work supports a shift toward mechanistic, genome-resolved models of microbiome assembly and offers a framework for linking microbial function to host colonization success.

comparative genomics↗
DOE OSTI2025

Genomic insights into local adaptation and migration success in reintroduced Coho Salmon of the Wenatchee River basin

ABSTRACT Objective Reintroduction of salmonids into regions where they have been extirpated is a common conservation strategy that is often implemented through natural recolonization, translocation of natural populations, or hatchery-based programs. Locally adapting to specific environmental conditions is critical for long-term population viability, particularly for species like Coho Salmon Oncorhynchus kisutch, which face diverse selective pressures during their migration. This study focused on the mid-Columbia River Coho Salmon reintroduction program managed by Yakama Nation Fisheries, which has successfully reintroduced Coho Salmon into the Wenatchee and Methow River basins, Washington. Notably, these populations have adapted to the longer migration route than those in the founding stock, with selection favoring individuals with an earlier arrival time and that can navigate a 15-km, high-gradient canyon to reach optimal spawning grounds. The objectives of this study were to investigate whether specific genomic regions are under selection for traits associated with return location and timing in Coho Salmon. Methods Low-coverage whole-genome resequencing data were used to screen for genomic regions associated with the phenotypes of interest. Results A weak polygenic signal in female Coho Salmon was found to be associated with return group, with a subset of candidate adaptive regions occurring across eight chromosomes. Conclusions These findings provide insights into the genomic mechanisms underlying local adaptation in reintroduced salmon populations and inform broodstock selection strategies aimed at promoting natural production and long-term population sustainability.

Horn, Rebekah L.↗
DOE OSTI2026

BSMV-mediated genome editing exhibits host-specific heritability: germline transmission in barley and somatic edits in Nicotiana benthamiana

Plant RNA virus–mediated guide RNA (gRNA) delivery represents a transformative advance in genome editing technologies. Unlike conventional transformation methods that rely on labor-intensive tissue culture and regeneration for each individual gRNA delivery, viral vectors can rapidly and systemically transmit gRNAs into pre-established Cas-expressing plants, providing an accelerated route for functional genomics and trait discovery directly in planta . However, key design parameters, including subgenomic promoter choice, transcript architecture, and their effects on viral fitness and editing outcomes, remain to be elucidated for most viral platforms. We developed five Barley stripe mosaic virus (BSMV) vectors, each with distinct subgenomic promoter elements to drive single gRNA expression. These were initially evaluated in Cas9-expressing transgenic Nicotiana benthamiana plants targeting the Phytoene desaturase ( PDS ) gene to compare their editing efficiencies. Single gRNAs expressed under the duplicated γb subgenomic promoter or when fused directly to the γb genome achieved the highest mutation frequencies (up to 90% at 60 days post-inoculation), whereas β1- and β2-driven sgRNAs produced delayed and reduced editing. Thus, promoter selection critically determines gRNA accumulation and the efficacy of BSMV-mediated genome editing. The top-performing design was then applied to Cas9-expressing barley ( Hordeum vulgare ) targeting HvCMF7 (conferring green-white variegation) and HvGW2.1 (impacts grain width and weight). BSMV spread systemically throughout barley, inducing somatic and heritable mutations at frequencies up to 100%, with virus-free edited progeny. In contrast, despite robust somatic editing in N. benthamiana, no heritable mutations were detected indicating species-dependent limitations in germline transmission. Our systematic comparison of subgenomic promoter architectures establishes clear design principles for optimizing viral vector–mediated delivery. Promoter choice and transcript structure critically shape editing efficiency and viral stability. The host-specific boundary for germline editing, defined by efficient heritable editing in barley but not N. benthamiana , highlights where BSMV offers advantages and where alternative vectors or hybrid strategies are required, guiding rational platform selection for diverse crop species and applications. Collectively, these findings establish BSMV as a promising next-generation vector for rapid, tissue culture–free, and transformation-independent genome editing in cereals and other recalcitrant monocots.

barley↗
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↗
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

CRISPRi-ART enables functional genomics of diverse bacteriophages using RNA-binding dCas13d

Bacteriophages constitute one of the largest reservoirs of genes of unknown function in the biosphere. Even in well-characterized phages, the functions of most genes remain unknown. Experimental approaches to study phage gene fitness and function at genome scale are lacking, partly because phages subvert many modern functional genomics tools. Here we leverage RNA-targeting dCas13d to selectively interfere with protein translation and to measure phage gene fitness at a transcriptome-wide scale. We find CRISPR Interference through Antisense RNA-Targeting (CRISPRi-ART) to be effective across phage phylogeny, from model ssRNA, ssDNA and dsDNA phages to nucleus-forming jumbo phages. Using CRISPRi-ART, we determine a conserved role of diverse rII homologues in subverting phage Lambda RexAB-mediated immunity to superinfection and identify genes critical for phage fitness. CRISPRi-ART establishes a broad-spectrum phage functional genomics platform, revealing more than 90 previously unknown genes important for phage fitness.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Hybridization breaks species barriers in long-term coevolution of a cyanobacterial population

Bacterial species often undergo rampant recombination yet maintain cohesive genomic identity. Ecological differences can generate recombination barriers between species and sustain genomic clusters in the short term. But can these forces prevent genomic mixing during long-term coevolution? Cyanobacteria in Yellowstone hot springs comprise several diverse species that have coevolved for hundreds of thousands of years, providing a rare natural experiment. By analyzing more than 300 single-cell genomes, we show that despite each species forming a distinct genomic cluster, much of the diversity within species is the result of hybridization driven by selection, which has mixed their ancestral genotypes. This widespread mixing is contrary to the prevailing view that ecological barriers can maintain cohesive bacterial species and highlights the importance of hybridization as a source of genomic diversity.

Evolutionary Biology↗
DOE OSTI2024

Metagenome-assembled genomes of freshwater Hyphomicrobium sp. G-191 and Methylophilus sp. enriched from Cedar Swamp, Woods Hole, MA

ABSTRACT Hyphomicrobium are facultative denitrifying anaerobes capable of using one-carbon compounds as a sole carbon source. Hyphomicrobium sp. G-191 was enriched from Cedar Swamp, Woods Hole, Massachusetts, using a selective medium for methanol-utilizing bacteria. We present two draft metagenome-assembled genomes (MAGs) of a Hyphomicrobium and a Methylophilus species.

Huang, Yolanda (ORCID:0000000312631515)↗