Biology subjects

Olm, M. R.

Publications and source records attributed to Olm, M. R..

At least 19 recordsLinked to original sources

bioRxiv2026

ZipStrain Enables Rapid and Precise Strain-Resolved Metagenomics

Strain-resolved metagenomics characterizes microbial communities at nucleotide-level resolution, enabling researchers to differentiate identical from closely related organisms and characterize population structure and gene content variation. Here we introduce ZipStrain, a program that performs highly accurate strain-resolved metagenomics over 500x faster than available methods while offering superior RAM management. Applied to a dataset of 2,754 samples spanning human populations, we identify a strain-sharing gradient across social relationships, reveal striking variation in clonal structure across bacteria and bacteriophage, and pinpoint genes whose nucleotide identity deviates from genome-wide expectations. ZipStrain is distributed as an open-source Python package and accompanying Nextflow pipeline at https://github.com/OlmLab/ZipStrain.

bioinformatics↗

bioRxiv2026

IgA Targeting in the Infant Gut Is Modulated by Diet and Increasingly Directed Towards Persistent Species

BackgroundIgA is the dominant antibody in the human gut and a key regulator of host-microbe interactions. Infants begin to produce IgA at around 6 months old and receive large quantities of IgA via human milk, but technical limitations have prevented species-level characterization of IgA binding in early life. This has left basic knowledge gaps about which species are targeted by IgA in infancy, and how modifiable lifestyle factors like breastfeeding and complementary feeding impact IgA targeting. ResultsHere we adapt Metagenomic Immunoglobulin Sequencing (MIg-Seq) for low-biomass infant fecal samples and apply this optimized protocol to 32 longitudinal samples from 16 infants enrolled in the MINT trial, a four-arm randomized controlled trial comparing meat-based, dairy-based, plant-based, and reference complementary feeding patterns, with fecal sampling at 6 and 12 months (pre and post intervention). Infant IgA targeting mirrors adults at the phylum level, with both age groups showing significantly higher IgA targeting of Pseudomonadota and lower targeting of Bacteroidota relative to other phyla. During the substantial microbiome compositional shifts noted between 6 and 12 months, IgA targeting is significantly more stable than the microbiome itself. Among persistent colonizers, IgA targeting strengthens significantly from 6 to 12 months, with the most pronounced effect observed for Bifidobacterium, a finding robust across all dietary arms and feeding modes. The feeding arm to which infants were enrolled was not significantly associated with IgA binding, but several nutrient-specific associations were discovered. Animal-derived nutrients, particularly cholesterol, are strongly positively correlated with IgA targeting of Bifidobacterium longum, while plant-derived carotenoids are positively associated with IgA targeting of Flavonifractor plautii and Ruminococcus gnavus. ConclusionsThis study introduces an experimental and computational framework for species-level IgA profiling in the infant gut. The progressive strengthening of IgA targeting of Bifidobacterium and other beneficial persistent colonizers suggests a role for IgA in reinforcing beneficial microbes during infancy. The nutrient-specific dietary effects on IgA targeting reveal the immunological consequences of the complementary feeding period, and highlight a contrast between animal-versus plant-based diets. Together, these findings point to early nutritional interventions and IgA-based therapeutics as promising tools for promoting healthy immune-microbiome development.

microbiology↗

bioRxiv2025

DNA extraction and virome processing methods strongly influence recovered human gut viral community characteristics

Accurately characterising the human gut virome is critical to understanding virus-microbiome-host interactions. However, widely used methods introduce biases that complicate data interpretation and limit cross-study comparability. For instance, multiple-displacement amplification (MDA) preferentially amplifies single-stranded DNA viruses, while total metagenomes are dominated by non-viral sequences, reducing viral signal. These traditional methods have not been systematically compared to viral size-fraction metagenomes (viromes) prepared without MDA. To address this, we applied four common methods for characterising human gut viral community composition (total metagenomes, viromes with/ without DNase treatment (to remove free DNA), and MDA viromes) to a human stool sample, with technical triplicates for each approach. MDA biased viral community composition to a shocking degree: Microviridae formed [~]90% of MDA viromes compared to just 2% of non-MDA viromes. Removing ssDNA viruses from data analyses substantially reduced, but did not eliminate, MDA bias. Metagenomes were enriched for putative temperate phages and predicted Bacillota-phages, whereas predicted Bacteroidetes-phages dominated all viromes, suggesting that metagenomes and viromes select for different populations within the total viral community. DNase treatment had little-to-no effect on virome richness or community composition. This proof-of-principle experiment demonstrates that preparatory methods for viral community analysis can lead to substantially different conclusions from the same faecal sample, and we provide a comprehensive omic data analysis framework for comparing laboratory methodologies for viral ecology. With sufficient DNA yields now easily achievable from human gut viromes without the use of MDA, our results suggest that this biased amplification method should be avoided in human gut virome studies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/690293v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@36252org.highwire.dtl.DTLVardef@2c06f0org.highwire.dtl.DTLVardef@7b7cceorg.highwire.dtl.DTLVardef@13ec804_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

microbiology↗

bioRxiv2025

Prehistoric Global Migration of Vanishing Gut Microbes With Humans

The gut microbiome is crucial for health and greatly affected by lifestyle. Many microbes common in non-industrialized populations are disappearing or extinct in industrialized populations. Understanding which microbes have been long-term residents of the human gut, and may have co-evolved with humans, has implications for the importance of microbial biodiversity loss for health. However, the genetic complexities of microbial evolution and the plasticity of gut microbiome composition have made it challenging to define these long-term associations. Here, we performed deep metagenomic sequencing of the Tsimane horticulturalists of Bolivia and compared their gut microbiomes with the Hadza hunter-gatherers of Tanzania. These two populations, whose ancestors were separated for tens of thousands of years, share 1,231 microbial species, most of which are absent in industrialized populations. Population genetic analyses in 636 of these shared species revealed patterns of microbial divergence and gene flow consistent with prehistoric human co-migration, with estimated split times that approximately align with human migration out of Africa and into the Americas. Our findings indicate that a diverse gut microbiome co-migrated with humans around the globe, persisting over millennia. However, many of these species are now vanishing in industrialized populations, and the consequences for human health remain uncertain.

microbiology↗

bioRxiv2025

Continent-wide assessment of the strain-level diversity of Bradyrhizobium, a dominant soil bacterial genus

Global surveys of soil bacteria have identified several taxa that are nearly ubiquitous and often the most abundant members of soil bacterial communities. However, it remains unclear why these taxa are so dominant across a wide range of soil types and environmental conditions. Here we use genome-resolved metagenomics to test the hypothesis that strain-level differences exist in these taxa that are not adequately captured with standard marker gene sequencing, and that distinct strains harbor unique traits that reflect adaptations to different soil environments. We analyzed data from 331 natural soils spanning Australia to assess strain differentiation in Bradyrhizobium, a dominant soil bacterial genus of ecological importance. We developed a workflow for strain-level bacterial analyses of complex soil metagenomes, combining genomes from pre-existing databases with new genomes generated via targeted assembly from metagenomes to detect 181 Bradyrhizobium strains across the soil collection. In addition to a high degree of phylogenetic variation, we observed substantial variation in pangenome content and inferred traits, highlighting the breadth of diversity within this widespread genus. While members of the genus Bradyrhizobium were detected in > 80% of samples, most individual strains were restricted in their distributions. The overall strain-level community composition of Bradyrhizobium varied significantly across geographic space and environmental gradients, and was particularly associated with differences in temperature, soil pH, and soil nitrate and metal concentrations. Our work provides a general framework and methodology for studying the strain-level ecology of soil bacteria and highlights the ecological and pangenomic diversity within this dominant soil bacterial genus.

microbiology↗

bioRxiv2024

MiFoDB, a workflow for microbial food metagenomic characterization, enables high-resolution analysis of fermented food microbial dynamics

SummaryFermented foods, which contain a diverse array of microbial metabolites and microbes, are increasingly recognized as potential mediators of human immune and metabolic health. While there is growing interest in characterizing the microbial landscape of fermented foods, current characterization methods typically rely on 16S rRNA sequencing or marker gene-based methods, which have low taxonomic resolution and cannot functionally characterize microbes. Here we describe MiFoDB-workflow, a metagenomics workflow for the identification of microbes associated with food fermentation based on a primary database of 675 genomes of bacteria, yeast, fungi, and common fermented food substrates. We constructed the database using metagenome-assembled genomes (MAGs) derived from metagenomic sequencing of 90 fermented foods, combined with previously-published fermented food-derived MAGs, and relevant genomes deposited in RefSeq and GenBank genomes. We demonstrate the utility of MiFoDB for high confidence genome identification, including discovery of previously uncharacterized species, strain tracking across related foods, and functional analysis in fermented foods of different substrates. The workflow streamlines high-confidence characterization of the diversity of the fermented food landscape including novel ferments, and allows strain-level tracking of microbes across time, substrates, and producers.

microbiology↗

bioRxiv2023

Metagenomic Immunoglobulin Sequencing (MIG-Seq) Exposes Patterns of IgA Antibody Binding in the Healthy Human Gut Microbiome

IgA, the most highly produced human antibody, is continually secreted into the gut to shape the intestinal microbiota. Methodological limitations have critically hindered defining which microbial strains are targeted by IgA and why. Here, we develop a new technique, Metagenomic Immunoglobulin Sequencing (MIG-Seq), and use it to determine IgA coating levels for thousands of gut microbiome strains in healthy humans. We find that microbes associated with both health and disease have higher levels of coating, and that microbial genes are highly predictive of IgA binding levels, with mucus degradation genes especially correlated with high binding. We find a significant reduction in replication rates among microbes bound by IgA, and demonstrate that IgA binding is more correlated with host immune status than traditional microbial abundance measures. This study introduces a powerful technique for assessing strain-level IgA binding in human stool, paving the way for deeper understanding of IgA-based host microbe interactions.

microbiology↗

bioRxiv2023

Hadza Prevotella Require Diet-derived Microbiota Accessible Carbohydrates to Persist in Mice.

Industrialization has transformed the gut microbiota, reducing the prevalence of Prevotella relative to Bacteroides. Here, we isolate Bacteroides and Prevotella strains from the microbiota of Hadza hunter-gatherers of Tanzania, a population with high levels of Prevotella. We demonstrate that plant-derived microbiota-accessible carbohydrates (MACs) are required for persistence of Prevotella copri but not Bacteroides thetaiotaomicron in vivo. Differences in carbohydrate metabolism gene content, expression, and in vitro growth reveal that Hadza Prevotella strains specialize in degrading plant carbohydrates, while Hadza Bacteroides isolates use both plant and host-derived carbohydrates, a difference mirrored in Bacteroides from non-Hadza populations. When competing directly, P. copri requires plant-derived MACs to maintain colonization in the presence of B. thetaiotaomicron, as a no MAC diet eliminates P. copri colonization. Prevotellas reliance on plant-derived MACs and Bacteroides ability to use host mucus carbohydrates could explain the reduced prevalence of Prevotella in populations consuming a low-MAC, industrialized diet. Statement on work with indigenous communitiesIn order to acquire scientific knowledge that accurately represents all human populations, rather than only reflecting and benefiting those in industrialized nations, it is necessary to involve indigenous populations in research in a legal, ethical, and non-exploitative manner (Abdill et al., 2022; Green et al., 2020). Here, we isolated live bacterial strains from anonymized fecal samples collected from Hadza hunter-gatherers in 2013/2014 (Fragiadakis et al., 2019; Merrill et al., 2022; Smits et al., 2017). Samples were collected with permission from the Tanzanian government, National Institute of Medical Research (MR/53i 100/83, NIMR/HQ/R.8a/Vol.IX/1542), the Tanzania Commission for Science and Technology, and with aid from Tanzanian scientists. A material transfer agreement with the National Institute for Medical Research in Tanzania specifies that collected samples are solely to be used for academic purposes. For more information on the consent practices followed, and our ongoing work to communicate the results of these projects to the Hadza, please see (Merrill et al., 2022; Olm et al., 2022).

microbiology↗

bioRxiv2022

Robust Variation in Infant Gut Microbiome Assembly Across a Spectrum of Lifestyles

Infant microbiome assembly is intensely studied in infants from industrialized nations, but little is known about this process in populations living non-industrialized lifestyles. In this study we deeply sequenced infant stool samples from the Hadza hunter-gatherers of Tanzania and analyzed them in a global meta-analysis. Infant microbiomes develop along lifestyle-associated trajectories, with over twenty percent of genomes detected in the Hadza infant gut representing phylogenetically diverse novel species. Industrialized infants, even those who are breastfed, have microbiomes characterized by a paucity of Bifidobacterium infantis and gene cassettes involved in human milk utilization. Strains within lifestyle-associated taxonomic groups are shared between mother-infant dyads, consistent with early-life inheritance of lifestyle-shaped microbiomes. The population-specific differences in infant microbiome composition and function underscore the importance of studying microbiomes from people outside of wealthy, industrialized nations. Recognition of work on indigenous communitiesResearch involving indigenous communities is needed for a variety of reasons including to ensure that scientific discoveries and understanding appropriately represent all populations and do not only benefit those living in industrialized nations. Special considerations must be made to ensure that this research is conducted ethically and in a non-exploitative manner. In this study we performed deep metagenomic sequencing on fecal samples that were collected from Hadza hunter-gatherers in 2013/2014 and were analyzed in previous publications using different methods (1, 2). A material transfer agreement with the National Institute for Medical Research in Tanzania ensures that stool samples collected are used solely for academic purposes, permission for the study was obtained from the National Institute of Medical Research (MR/53i 100/83, NIMR/HQ/R.8a/Vol.IX/1542) and the Tanzania Commission for Science and Technology, and verbal consent was obtained from the Hadza after the studys intent and scope was described with the help of a translator. The publications that first described these samples included several scientists and Tanzanian field-guides as co-authors for the critical roles they played in sample collection, but as no new samples were collected in this study, only scientists who contributed to the analyses described here were included as co-authors in this publication. It is currently not possible for us to travel to Tanzania and present our results to the Hadza people, however we intend to do so once the conditions of the COVID-19 pandemic allow it.

microbiology↗

bioRxiv2022

Ultra-deep Sequencing of Hadza Hunter-Gatherers Recovers Vanishing Microbes

The gut microbiome is a key modulator of immune and metabolic health. Human microbiome data is biased towards industrialized populations, providing limited understanding of the distinct and diverse non-industrialized microbiomes. Here, we performed ultra-deep metagenomic sequencing and strain cultivation on 351 fecal samples from the Hadza, hunter-gatherers in Tanzania, and comparative populations in Nepal and California. We recover 94,971 total genomes of bacteria, archaea, bacteriophages, and eukaryotes, 43% of which are absent from existing unified datasets. Analysis of in situ growth rates, genetic pN/pS signatures, high-resolution strain tracking, and 124 gut-resident species vanishing in industrialized populations reveals differentiating dynamics of the Hadza gut microbiome. Industrialized gut microbes are enriched in genes associated with oxidative stress, possibly a result of microbiome adaptation to inflammatory processes. This unparalleled view of the Hadza gut microbiome provides a valuable resource that expands our understanding of microbes capable of colonizing the human gut and clarifies the extensive perturbation brought on by the industrialized lifestyle.

microbiology↗

bioRxiv2022

Using strain-resolved analysis to identify contamination in metagenomics data

Metagenomics analyses can be negatively impacted by DNA contamination. While external sources of contamination such as DNA extraction kits have been widely reported and investigated, contamination originating within the study itself remains underreported. Here we applied high-resolution strain-resolved analyses to identify contamination in two large-scale clinical metagenomics datasets. By mapping strain sharing to DNA extraction plates, we identified well-to-well contamination in both negative controls and biological samples in one dataset. Such contamination is more likely to occur among samples that are on the same or adjacent columns or rows of the extraction plate than samples that are far apart. Our strain-resolved workflow also reveals the presence of externally derived contamination, primarily in the other dataset. Overall in both datasets, contamination is more significant in samples with lower biomass. Our work demonstrates that genome-resolved strain tracking, with its essentially genome-wide nucleotide-level resolution, can be used to detect contamination in sequencing-based microbiome studies. Our results underscore the value of strain-specific methods to detect contamination and the critical importance of looking for contamination beyond negative and positive controls.

microbiology↗

bioRxiv2021

Infant gut strain persistence is associated with maternal origin, phylogeny, and functional potential including surface adhesion and iron acquisition

Gut microbiome succession impacts infant development. However, it remains unclear what factors promote persistence of initial bacterial colonists in the developing gut. Here, we performed strain-resolved metagenomic analyses to compare gut colonization of preterm and full-term infants throughout the first year of life and evaluated links between strain persistence and strain origin as well as genetic potential. Analysis of 206 fecal metagenomes collected from full-term and preterm infants and their mothers revealed that infants initially distinct microbial communities converged by age one. Approximately 11% of early colonists, primarily Bacteroides and Bifidobacterium, persisted during the first year of life, and these were more prevalent in full-term compared to preterm infants. Gut-associated strains from mothers were significantly more likely to persist in the infant gut than other strains. Enrichment in genes for surface adhesion, iron acquisition and carbohydrate degradation may explain persistence of some strains through the first year of life.

microbiology↗

bioRxiv2020

Transporter genes in biosynthetic gene clusters predict metabolite characteristics and siderophore activity

Biosynthetic gene clusters (BGCs) are operonic sets of microbial genes that synthesize specialized metabolites with diverse functions, including siderophores and antibiotics, which often require export to the extracellular environment. For this reason, genes for transport across cellular membranes are essential for the production of specialized metabolites, and are often genomically co-localized with BGCs. Here we conducted a comprehensive computational analysis of transporters associated with characterized BGCs. In addition to known exporters, in BGCs we found many importer-specific transmembrane domains that co-occur with substrate binding proteins possibly for uptake of siderophores or metabolic precursors. Machine learning models using transporter gene frequencies were predictive of known siderophore activity, molecular weights, and a measure of lipophilicity (log P) for corresponding BGC-synthesized metabolites. Transporter genes associated with BGCs were often equally or more predictive of metabolite features than biosynthetic genes. Given the importance of siderophores as pathogenicity factors, we used transporters specific for siderophore BGCs to identify both known and uncharacterized siderophore-like BGCs in genomes from metagenomes from the infant and adult gut microbiome. We find that 23% of microbial genomes from the infant gut have siderophore-like BGCs, but only 3% of those assembled from adult gut microbiomes do. While siderophore-like BGCs from the infant gut are predominantly associated with Enterobactericaee and Staphylococcus, siderophore-like BGCs can be identified from taxa in the adult gut microbiome that have rarely been recognized for siderophore production. Taken together, these results show that consideration of BGC-associated transporter genes can inform predictions of specialized metabolite structure and function.

bioinformatics↗

bioRxiv2020

Meanders as a scaling motif for understanding of floodplain soil microbiome and biogeochemical potential at the watershed scale

Biogeochemical exports of C, N, S and H2 from watersheds are modulated by the activity of microorganisms that function over micron scales. This disparity of scales presents a substantial challenge for development of predictive models describing watershed function. Here, we tested the hypothesis that meander-bound regions exhibit patterns of microbial metabolic potential that are broadly predictive of biogeochemical processes in floodplain soils along a river corridor. We intensively sampled floodplain soils located in the upper, middle, and lower reaches of the East River in Colorado and reconstructed 248 draft quality genomes representative at a sub-species level. Approximately one third of the representative genomes were detected across all three locations with similar levels of abundance, and despite the very high microbial diversity and complexity of the soils, ~15% of species were detected in two consecutive years. A core floodplain microbiome was enriched in bacterial capacities for aerobic respiration, aerobic CO oxidation, and thiosulfate oxidation with the formation of elemental sulfur. We did not detect systematic patterns of gene abundance based on sampling position relative to the river. However, at the watershed scale meander-bound floodplains appear to serve as scaling motifs that predict aggregate capacities for biogeochemical transformations in floodplain soils. Given this, we conducted a transcriptomic analysis of the middle site. Overall, the most highly transcribed genes were amoCAB and nxrAB (for nitrification) followed by genes involved in methanol and formate oxidation, and nitrogen and CO2 fixation. Low soil organic carbon correlated with high activity of genes involved in methanol, formate, sulfide, hydrogen, and ammonia oxidation, nitrite oxidoreduction, and nitrate and nitrite reduction. Thus, widely represented genetic capacities did not predict in situ activity at one time point, but rather they define a reservoir of biogeochemical potential available as conditions change.

microbiology↗

bioRxiv2020

Genetic and behavioral adaptation of Candida parapsilosis to the microbiome of hospitalized infants revealed by in situ genomics, transcriptomics and proteomics

Candida parapsilosis is a common cause of invasive candidiasis, especially in newborn infants, and infections have been increasing over the past two decades. C. parapsilosis has been primarily studied in pure culture, leaving gaps in understanding of its function in microbiome context. Here, we reconstructed five unique C. parapsilosis genomes from premature infant fecal samples and analyzed their genome structure, population diversity and in situ activity relative to reference strains in pure culture. All five genomes contain hotspots of single nucleotide variants, some of which are shared by strains from multiple hospitals. A subset of environmental and hospital-derived genomes share variants within these hotspots suggesting derivation of that region from a common ancestor. Four of the newly reconstructed C. parapsilosis genomes have four to sixteen copies of the gene RTA3, which encodes a lipid translocase and is implicated in antifungal resistance, potentially indicating adaptation to hospital antifungal use. Time course metatranscriptomics and metaproteomics on fecal samples from a premature infant with a C. parapsilosis blood infection revealed highly variable in situ expression patterns that are distinct from those of similar strains in pure cultures. For example, biofilm formation genes were relatively less expressed in situ, whereas genes linked to oxygen utilization were more highly expressed, indicative of growth in a relatively aerobic environment. In gut microbiome samples, C. parapsilosis coexisted with Enterococcus faecalis that shifted in relative abundance over time, accompanied by changes in bacterial and fungal gene expression and proteome composition. The results reveal potentially medically relevant differences in Candida function in gut vs. laboratory environments, and constrain evolutionary processes that could contribute to hospital strain persistence and transfer into premature infant microbiomes.

microbiology↗

bioRxiv2020

InStrain enables population genomic analysis from metagenomic data and rigorous detection of identical microbial strains

Coexisting microbial cells of the same species often exhibit genetic differences that can affect phenotypes ranging from nutrient preference to pathogenicity. Here we present inStrain, a program that utilizes metagenomic paired reads to profile intra-population genetic diversity (microdiversity) across whole genomes and compare populations in a microdiversity-aware manner, dramatically increasing genomic comparison accuracy when benchmarked against existing methods. We use inStrain to profile >1,000 fecal metagenomes from newborn premature infants and find that siblings share significantly more strains than unrelated infants, although identical twins share no more strains than fraternal siblings. Infants born via cesarean section harbored Klebsiella with significantly higher nucleotide diversity than infants delivered vaginally, potentially reflecting acquisition from hospital versus maternal microbiomes. Genomic loci showing diversity within an infant included variants found in other infants, possibly reflecting inoculation from diverse hospital-associated sources. InStrain can be applied to any metagenomic dataset for microdiversity analysis and rigorous strain comparison.

microbiology↗

bioRxiv2019

Consistent metagenome-derived metrics verify and define bacterial species boundaries

Longstanding questions relate to the existence of naturally distinct bacterial species and genetic approaches to distinguish them. Bacterial genomes in public databases form distinct groups, but these databases are subject to isolation and deposition biases. We compared 5,203 bacterial genomes from 1,457 environmental metagenomic samples to test for distinct clouds of diversity, and evaluated metrics that could be used to define the species boundary. Bacterial genomes from the human gut, soil, and the ocean all exhibited gaps in whole-genome average nucleotide identities (ANI) near the previously suggested species threshold of 95% ANI. While genome-wide ratios of non-synonymous and synonymous nucleotide differences (dN/dS) decrease until ANI values approach [~]98%, estimates for homologous recombination approached zero at [~]95% ANI, supporting breakdown of recombination due to sequence divergence as a species-forming force. We evaluated 107 genome-based metrics for their ability to distinguish species when full genomes are not recovered. Full length 16S rRNA genes were least useful because they were under-recovered from metagenomes, but many ribosomal proteins displayed both high metagenomic recoverability and species-discrimination power. Taken together, our results verify the existence of sequence-discrete microbial species in metagenome-derived genomes and highlight the usefulness of ribosomal genes for gene-level species discrimination.

microbiology↗

bioRxiv2019

Clades of huge phage from across Earth’s ecosystems

Phage typically have small genomes and depend on their bacterial hosts for replication. DNA sequenced from many diverse ecosystems revealed hundreds of huge phage genomes, between 200 kbp and 716 kbp in length. Thirty-four genomes were manually curated to completion, including the largest phage genomes yet reported. Expanded genetic repertoires include diverse and new CRISPR-Cas systems, tRNAs, tRNA synthetases, tRNA modification enzymes, translation initiation and elongation factors, and ribosomal proteins. Phage CRISPR-Cas systems have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phage may repurpose bacterial CRISPR-Cas systems to eliminate competing phage. We phylogenetically define major clades of huge phage from human and other animal microbiomes, oceans, lakes, sediments, soils and the built environment. We conclude that their large gene inventories reflect a conserved biological strategy, observed over a broad bacterial host range and across Earths ecosystems.

microbiology↗