Joint Genome Institute

1. Abstract Metagenomic binning and single-cell assembly produce draft genomes whose completeness and contamination vary with experimental and computational choices. Comparing whole bin sets remains difficult because most quality assessment tools report per-bin metrics and operate either with ground truth labels or with inference estimates. GradeBins evaluates complete bin sets under two execution modes while producing matched per-bin and bin-set summaries. For real metagenomes, inference mode integrates bin statistics, mapping depth, taxonomy, and external quality estimates from tools such as CheckM2 and EukCC to standardize per-bin and bin-set quality reporting across Bacteria, Archaea, and Eukaryotes. For synthetic or otherwise labeled datasets, ground truth mode computes base-resolved completeness, contamination, and misbinning from labeled contigs or CAMI mappings, enabling objective benchmarking of binners, parameter choices, and experimental conditions, and calibration of inference-based estimates. Across synthetic metagenomes of 10, 50, 100, 500 and 1,000 Bacteria and Archaea, and a mixed metagenome containing also Eukaryotes, GradeBins separated binner and parameter effects using Total Score and a quality-weighted bin count, together with quality tier distributions, recovery fractions, and label-aware diagnostics. Inference-mode completeness generally tracked ground truth, whereas contamination and clean-bin rates showed mode-dependent shifts that were most pronounced in the mixed community. GradeBins added low overhead in these benchmarks, with peak memory below 8 GB and runtimes typically below 30 seconds. GradeBins enables reproducible protocol comparison, regression testing, and consistent quality reporting for genome-resolved metagenomics in both benchmarking and real-data settings. The full software package is open-source and available for download at https://bbmap.org/tools/gradebins .

SummaryLeveraging predicted origin sequences from a previously characterized groundwater plasmidome, we constructed a barcoded plasmid library to screen for previously unknown origins. Testing this library against a panel of representative bacterial strains led to the identification of 3 previously unknown origins that replicate in gram-negative bacteria not previously associated with these origin sequences. Experimental validation confirmed that a plasmid bearing origin 6911 as the sole origin could replicate with a copy number of 9 (±2) in Pantoea sp. MT58, a fast growing and metal tolerant, environmentally important bacterium. Plasmids based on this new origin were used to express the reporter protein GFP, and non-native metabolite pathways for the natural product indigoidine and the terpenoid compound isoprenol. Functional previously unknown origins of replication in such non-model organisms can expand the toolkit for genetic manipulations of both model and less-studied bacteria.

The genomes of white-rot fungi hold extended repertoires of enzymes active on virtually all the chemical bonds that intertwine lignocellulose polymers, and several Trametes species have been identified as powerful tools for biorefinery or bioremediation. However, only few studies have addressed the intra-species polymorphism one would expect from fungal strains collected in contrasted environments. We compared the genome sequence of pairs of strains collected in different geographic areas, for each of three fungal species. Using an updated list of the predicted functions for fungal ligno- and cellulolytic enzymes (CAZymes), we observed a high conservation of the gene repertoires among the six strains. We compared the adaptative response of the fungi grown on crystalline cellulose, wheat straw, aspen or pine sawdust by transcriptomics and secretomics. The gene regulation profiles were determined by the species and the substrates, rather than the strain. The secretomes did not show marked differences in the sets of secreted CAZymes after 3day-growth on the substrates. We identified five transcription factor genes and two sesquiterpenoid synthesis genes induced during growth on lignocellulose. Wider studies using larger sets of strains will be necessary to evaluate the genericity of our findings, and to assess the phenotype diversity one could expect from geographic diversity as compared to taxonomic diversity in Trametes fungi.

Snowmelt triggers a soil microbial bloom and crash that affects nitrogen (N) export in high-elevation watersheds. The mechanisms underlying these microbial dynamics are uncertain, making soil nitrogen processes difficult to predict as snowpack declines globally. Here, integration of genome-resolved metagenomics, metatranscriptomics and metabolomics in a high-elevation watershed revealed ecologically distinct soil microorganisms linked across the snowmelt time-period by their unique nitrogen cycling capacities. The molecular properties and transformations of dissolved organic N suggested that degradation or recycling of microbial biomass provided N for biosynthesis during the microbial bloom. Winter-adapted Bradyrhizobia spp. oxidized amino acids anaerobically and had the highest gene expression for denitrification during the microbial bloom. A pulse of nitrate was driven by spring-adapted Nitrososphaerales after snowmelt, but dissimilatory nitrate reduction to ammonia (DNRA) gene expression indicated significant nitrate retention potential. These findings inform our understanding of nitrogen cycling in environments sensitive to snowpack decline due to global change.

Karenia brevis regularly forms harmful blooms along the West Florida Shelf that negatively affect marine and terrestrial organisms through toxin production. These blooms impose economic and environmental hardship, driving the need for research to understand the factors influencing their dynamics and to mitigate their impacts. A mostly unresolved issue is the potential role of viruses in bloom termination. We conducted an experiment incubating K. brevis cultures with size-fractionated bloom water samples. Flow cytometry revealed giant virus-like populations (VLPs) in replicate cultures with <1 μm-filtered and <0.2 μm-filtered bloom water. The VLPs’ abundance was paralleled by declines in photoefficiency and culture lysis. Metagenomic analyses of the lysates revealed 11 giant virus genomes (35%–100% complete) representing 7 viral operational taxonomic units (vOTUs) within the order Imitervirales (Nucleocytoviricota). Ten of these vOTUs were more abundant in the incubations with <0.2 μm-filtered bloom water, coinciding with the absence or low abundance of algicidal bacteria. The vOTUs and K. brevis cell abundances showed a positive correlation at a coastal site during bloom and nonbloom periods. The most apparent association was to vOTU6, which may owe its competitive advantage to the presence of the auxiliary metabolic genes bacteriorhodopsin, carbonic anhydrase, and dinoflagellate viral nucleoprotein. The metagenomes also contained polinton-like virus (PLV) genomes. Since many PLVs are hypothesized to depend on co-infection with Nucleocytoviricota viruses for their propagation, our results suggest complex viral interactions within K. brevis blooms. Future research to elucidate virus–bacteria–K. brevis interaction mechanisms may be key to understanding bloom dynamics and developing management tools.

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.

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.

Protists play important roles in nutrient cycling across ecosystems, yet the composition and function of their associated microbiomes remain poorly studied. Here, we use cultivation-independent single-cell isolation and genome-resolved metagenomics to investigate the microbiomes and viromes of more than 100 uncultivated ciliates and amoebae from diverse environments. Our findings reveal unique microbiome structures and complex associations with bacterial symbionts and viruses, with stark differences between ciliates and amoebae. We recover 117 microbial genomes affiliated with known eukaryotic endosymbionts, including Holosporales, Rickettsiales, Legionellales, Chlamydiae, and Babelota, and 258 genomes linked to host-associated Patescibacteriota. Many show genome reduction and genes related to toxin-antitoxin systems and nucleotide parasitism, indicating adaptation to intracellular lifestyles. We also identify more than 80 giant viruses from diverse lineages, some actively expressing genes in single-cell transcriptomes, along with other viruses predicted to infect eukaryotes or symbiotic bacteria. The frequent co-occurrence of giant viruses and microbial symbionts, especially in amoebae, suggests multipartite interactions. Together, our study highlights protists as hubs of microbial and viral associations and provides a broad view of the diversity, activity, and ecological importance of their hidden partners.