RNA language models predict mutations that improve RNA function.

Structured RNA lies at the heart of many central biological processes, from gene expression to catalysis. While advances in deep learning enable the prediction of accurate protein structural models, RNA structure prediction is not possible at present due to a lack of abundant high-quality reference data. Furthermore, available sequence data are generally not associated with organismal phenotypes that could inform RNA function. We created GARNET (Gtdb Acquired RNa with Environmental Temperatures), a new database for RNA structural and functional analysis anchored to the Genome Taxonomy Database (GTDB). GARNET links RNA sequences derived from GTDB genomes to experimental and predicted optimal growth temperatures of GTDB reference organisms. This enables construction of deep and diverse RNA sequence alignments to be used for machine learning. Using GARNET, we define the minimal requirements for a sequence- and structure-aware RNA generative model. We also develop a GPT-like language model for RNA in which triplet tokenization provides optimal encoding. Leveraging hyperthermophilic RNAs in GARNET and these RNA generative models, we identified mutations in ribosomal RNA that confer increased thermostability to the Escherichia coli ribosome. The GTDB-derived data and deep learning models presented here provide a foundation for understanding the connections between RNA sequence, structure, and function.

JUN mRNA translation regulation is mediated by multiple 5' UTR and start codon features.

Regulation of mRNA translation by eukaryotic initiation factors (eIFs) is crucial for cell survival. In humans, eIF3 stimulates translation of the JUN mRNA which encodes the transcription factor JUN, an oncogenic transcription factor involved in cell cycle progression, apoptosis, and cell proliferation. Previous studies revealed that eIF3 activates translation of the JUN mRNA by interacting with a stem loop in the 5' untranslated region (5' UTR) and with the 5' -7-methylguanosine cap structure. In addition to its interaction site with eIF3, the JUN 5' UTR is nearly one kilobase in length, and has a high degree of secondary structure, high GC content, and an upstream start codon (uAUG). This motivated us to explore the complexity of JUN mRNA translation regulation in human cells. Here we find that JUN translation is regulated in a sequence and structure-dependent manner in regions adjacent to the eIF3-interacting site in the JUN 5' UTR. Furthermore, we identify contributions of an additional initiation factor, eIF4A, in JUN regulation. We show that enhancing the interaction of eIF4A with JUN by using the compound Rocaglamide A (RocA) represses JUN translation. We also find that both the upstream AUG (uAUG) and the main AUG (mAUG) contribute to JUN translation and that they are conserved throughout vertebrates. Our results reveal additional layers of regulation for JUN translation and show the potential of JUN as a model transcript for understanding multiple interacting modes of translation regulation.

JUN mRNA Translation Regulation is Mediated by Multiple 5' UTR and Start Codon Features.

Regulation of mRNA translation by eukaryotic initiation factors (eIFs) is crucial for cell survival. In humans, eIF3 stimulates translation of the JUN mRNA which encodes the transcription factor JUN, an oncogenic transcription factor involved in cell cycle progression, apoptosis, and cell proliferation. Previous studies revealed that eIF3 activates translation of the JUN mRNA by interacting with a stem loop in the 5' untranslated region (5' UTR) and with the 5'-7-methylguanosine cap structure. In addition to its interaction site with eIF3, the JUN 5' UTR is nearly one kilobase in length, and has a high degree of secondary structure, high GC content, and an upstream start codon (uAUG). This motivated us to explore the complexity of JUN mRNA translation regulation in human cells. Here we find that JUN translation is regulated in a sequence and structure-dependent manner in regions adjacent to the eIF3-interacting site in the JUN 5' UTR. Furthermore, we identify contributions of an additional initiation factor, eIF4A, in JUN regulation. We show that enhancing the interaction of eIF4A with JUN by using the compound Rocaglamide A (RocA) represses JUN translation. We also find that both the upstream AUG (uAUG) and the main AUG (mAUG) contribute to JUN translation and that they are conserved throughout vertebrates. Our results reveal additional layers of regulation for JUN translation and show the potential of JUN as a model transcript for understanding multiple interacting modes of translation regulation.

eIF3 engages with 3'-UTR termini of highly translated mRNAs in neural progenitor cells.

Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the roles of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA) Seq, we show eIF3 crosslinks to many neurologically relevant mRNAs in NPCs. Our data reveal eIF3 predominantly interacts with 3' untranslated region (3'-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. High eIF3 crosslinking at 3'-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling. We identify the transcriptional regulator inhibitor of DNA binding 2 (ID2) mRNA as a case in which active translation levels and eIF3 crosslinking are dramatically increased upon early NPC differentiation. Furthermore, we find that eIF3 engagement at 3'-UTR ends is dependent on polyadenylation. The results presented here show that eIF3 engages with 3'-UTR termini of highly translated mRNAs, supporting a role of mRNA circularization in the mechanisms governing mRNA translation in NPCs.

Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells.

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the targeted chromosome, including in preclinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells (NCT03399448), reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

A highly efficient human cell-free translation system.

Cell-free protein synthesis (CFPS) systems enable easy in vitro expression of proteins with many scientific, industrial, and therapeutic applications. Here we present an optimized, highly efficient human cell-free translation system that bypasses many limitations of currently used in vitro systems. This CFPS system is based on extracts from human HEK293T cells engineered to endogenously express GADD34 and K3L proteins, which suppress phosphorylation of translation initiation factor eIF2α. Overexpression of GADD34 and K3L proteins in human cells before cell lysate preparation significantly simplifies lysate preparation. We find that expression of the GADD34 and K3L accessory proteins before cell lysis maintains low levels of phosphorylation of eIF2α in the extracts. During in vitro translation reactions, eIF2α phosphorylation increases moderately in a GCN2-dependent fashion that can be inhibited by GCN2 kinase inhibitors. This new CFPS system should be useful for exploring human translation mechanisms in more physiological conditions outside the cell.

The Helix-Loop-Helix motif of human EIF3A regulates translation of proliferative cellular mRNAs.

Improper regulation of translation initiation, a vital checkpoint of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) is associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiation in vitro. Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, including MYC. We demonstrate that the HLH motif of EIF3A acts specifically on the 5' UTR of MYC mRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target.

Aminobenzoic Acid Derivatives Obstruct Induced Fit in the Catalytic Center of the Ribosome.

The Escherichia coli (E. coli) ribosome can incorporate a variety of non-l-α-amino acid monomers into polypeptide chains in vitro but with poor efficiency. Although these monomers span a diverse set of compounds, there exists no high-resolution structural information regarding their positioning within the catalytic center of the ribosome, the peptidyl transferase center (PTC). Thus, details regarding the mechanism of amide bond formation and the structural basis for differences and defects in incorporation efficiency remain unknown. Within a set of three aminobenzoic acid derivatives-3-aminopyridine-4-carboxylic acid (Apy), ortho-aminobenzoic acid (oABZ), and meta-aminobenzoic acid (mABZ)-the ribosome incorporates Apy into polypeptide chains with the highest efficiency, followed by oABZ and then mABZ, a trend that does not track with the nucleophilicity of the reactive amines. Here, we report high-resolution cryo-EM structures of the ribosome with each of these three aminobenzoic acid derivatives charged on tRNA bound in the aminoacyl-tRNA site (A-site). The structures reveal how the aromatic ring of each monomer sterically blocks the positioning of nucleotide U2506, thereby preventing rearrangement of nucleotide U2585 and the resulting induced fit in the PTC required for efficient amide bond formation. They also reveal disruptions to the bound water network that is believed to facilitate formation and breakdown of the tetrahedral intermediate. Together, the cryo-EM structures reported here provide a mechanistic rationale for differences in reactivity of aminobenzoic acid derivatives relative to l-α-amino acids and each other and identify stereochemical constraints on the size and geometry of non-monomers that can be accepted efficiently by wild-type ribosomes.

Interactions between terminal ribosomal RNA helices stabilize the E. coli large ribosomal subunit.

The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding. In vivo assembled ribosomes have been isolated using MS2 tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and function in vitro. RNA tags in the Escherichia coli 50S subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth or in vitro ribosome activity. Here, we find that E. coli 50S subunits with MS2 tags inserted in H98 are destabilized compared to wild-type (WT) 50S subunits. We identify the loss of RNA-RNA tertiary contacts that bridge helices H1, H94, and H98 as the cause of destabilization. Using cryogenic electron microscopy (cryo-EM), we show that this interaction is disrupted by the addition of the MS2 tag and can be restored through the insertion of a single adenosine in the extended H98 helix. This work establishes ways to improve MS2 tags in the 50S subunit that maintain ribosome stability and investigates a complex RNA tertiary structure that may be important for stability in various bacterial ribosomes.

Atomistic simulations of the Escherichia coli ribosome provide selection criteria for translationally active substrates.

As genetic code expansion advances beyond L-α-amino acids to backbone modifications and new polymerization chemistries, delineating what substrates the ribosome can accommodate remains a challenge. The Escherichia coli ribosome tolerates non-L-α-amino acids in vitro, but few structural insights that explain how are available, and the boundary conditions for efficient bond formation are so far unknown. Here we determine a high-resolution cryogenic electron microscopy structure of the E. coli ribosome containing α-amino acid monomers and use metadynamics simulations to define energy surface minima and understand incorporation efficiencies. Reactive monomers across diverse structural classes favour a conformational space where the aminoacyl-tRNA nucleophile is <4 Å from the peptidyl-tRNA carbonyl with a Bürgi-Dunitz angle of 76-115°. Monomers with free energy minima that fall outside this conformational space do not react efficiently. This insight should accelerate the in vivo and in vitro ribosomal synthesis of sequence-defined, non-peptide heterooligomers.

Anionic G•U pairs in bacterial ribosomal rRNAs.

Wobble GU pairs (or G•U) occur frequently within double-stranded RNA helices interspersed between standard G=C and A-U Watson-Crick pairs. Another type of G•U pair interacting via their Watson-Crick edges has been observed in the A site of ribosome structures between a modified U34 in the tRNA anticodon triplet and G + 3 in the mRNA. In such pairs, the electronic structure of the U is changed with a negative charge on N3(U), resulting in two H-bonds between N1(G)…O4(U) and N2(G)…N3(U). Here, we report that such pairs occur in other highly conserved positions in ribosomal RNAs of bacteria in the absence of U modification. An anionic cis Watson-Crick G•G pair is also observed and well conserved in the small subunit. These pairs are observed in tightly folded regions.

"Candidatus Nealsonbacteria" Are Likely Biomass Recycling Ectosymbionts of Methanogenic Archaea in a Stable Benzene-Degrading Enrichment Culture.

The Candidate Phyla Radiation (CPR), also referred to as superphylum Patescibacteria, is a very large group of bacteria with no pure culture representatives discovered by 16S rRNA sequencing or genome-resolved metagenomic analyses of environmental samples. Within the CPR, candidate phylum Parcubacteria, previously referred to as OD1, is prevalent in anoxic sediments and groundwater. Previously, we had identified a specific member of the Parcubacteria (referred to as DGGOD1a) as an important member of a methanogenic benzene-degrading consortium. Phylogenetic analyses herein place DGGOD1a within the clade "Candidatus Nealsonbacteria." Because of its persistence over many years, we hypothesized that "Ca. Nealsonbacteria" DGGOD1a must play an important role in sustaining anaerobic benzene metabolism in the consortium. To try to identify its growth substrate, we amended the culture with a variety of defined compounds (pyruvate, acetate, hydrogen, DNA, and phospholipid), as well as crude culture lysate and three subfractions thereof. We observed the greatest (10-fold) increase in the absolute abundance of "Ca. Nealsonbacteria" DGGOD1a only when the consortium was amended with crude cell lysate. These results implicate "Ca. Nealsonbacteria" in biomass recycling. Fluorescence in situ hybridization and cryogenic transmission electron microscope images revealed that "Ca. Nealsonbacteria" DGGOD1a cells were attached to larger archaeal Methanothrix cells. This apparent epibiont lifestyle was supported by metabolic predictions from a manually curated complete genome. This is one of the first examples of bacterial-archaeal episymbiosis and may be a feature of other "Ca. Nealsonbacteria" found in anoxic environments. IMPORTANCE An anaerobic microbial enrichment culture was used to study members of candidate phyla that are difficult to grow in the lab. We were able to visualize tiny "Candidatus Nealsonbacteria" cells attached to a large Methanothrix cell, revealing a novel episymbiosis.

Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells.

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the chromosome, including in pre-clinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells, 1 dramatically reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

A highly efficient human cell-free translation system.

Cell-free protein synthesis (CFPS) systems enable easy in vitro expression of proteins with many scientific, industrial, and therapeutic applications. Here we present an optimized, highly efficient human cell-free translation system that bypasses many limitations of currently used in vitro systems. This CFPS system is based on extracts from human HEK293T cells engineered to endogenously express GADD34 and K3L proteins, which suppress phosphorylation of translation initiation factor eIF2α. Overexpression of GADD34 and K3L proteins in human cells significantly simplifies cell lysate preparation. The new CFPS system improves the translation of 5' cap-dependent mRNAs as well as those that use internal ribosome entry site (IRES) mediated translation initiation. We find that expression of the GADD34 and K3L accessory proteins before cell lysis maintains low levels of phosphorylation of eIF2α in the extracts. During in vitro translation reactions, eIF2α phosphorylation increases moderately in a GCN2-dependent fashion that can be inhibited by GCN2 kinase inhibitors. We also find evidence for activation of regulatory pathways related to eukaryotic elongation factor 2 (eEF2) phosphorylation and ribosome quality control in the extracts. This new CFPS system should be useful for exploring human translation mechanisms in more physiological conditions outside the cell.

Rare ribosomal RNA sequences from archaea stabilize the bacterial ribosome.

The ribosome serves as the universally conserved translator of the genetic code into proteins and supports life across diverse temperatures ranging from below freezing to above 120°C. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3'-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not affect cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation across archaeal ribosomes in the peptidyl transferase center that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.

Optimization of highly efficient exogenous-DNA-free Cas9-ribonucleoprotein mediated gene editing in disease susceptibility loci in wheat (Triticum aestivum L.).

The advancement of precision engineering for crop trait improvement is important in the face of rapid population growth, climate change, and disease. To this end, targeted double-stranded break technology using RNA-guided Cas9 has been adopted widely for genome editing in plants. Agrobacterium or particle bombardment-based delivery of plasmids encoding Cas9 and guide RNA (gRNA) is common, but requires optimization of expression and often results in random integration of plasmid DNA into the plant genome. Recent advances have described gene editing by the delivery of Cas9 and gRNA as pre-assembled ribonucleoproteins (RNPs) into various plant tissues, but with moderate efficiency in resulting regenerated plants. In this report we describe significant improvements to Cas9-RNP mediated gene editing in wheat. We demonstrate that Cas9-RNP assays in protoplasts are a fast and effective tool for rational selection of optimal gRNAs for gene editing in regenerable immature embryos (IEs), and that high temperature treatment enhances gene editing rates in both tissue types. We also show that Cas9-mediated editing persists for at least 14 days in gold particle bombarded wheat IEs. The regenerated edited wheat plants in this work are recovered at high rates in the absence of exogenous DNA and selection. With this method, we produce knockouts of a set of three homoeologous genes and two pathogenic effector susceptibility genes, engineering insensitivity to corresponding necrotrophic effectors produced by Parastagonospora nodorum. The establishment of highly efficient, exogenous DNA-free gene editing technology holds promise for accelerated trait diversity production in an expansive array of crops.

Genome-resolved metagenomics reveals site-specific diversity of episymbiotic CPR bacteria and DPANN archaea in groundwater ecosystems.

Candidate phyla radiation (CPR) bacteria and DPANN archaea are unisolated, small-celled symbionts that are often detected in groundwater. The effects of groundwater geochemistry on the abundance, distribution, taxonomic diversity and host association of CPR bacteria and DPANN archaea has not been studied. Here, we performed genome-resolved metagenomic analysis of one agricultural and seven pristine groundwater microbial communities and recovered 746 CPR and DPANN genomes in total. The pristine sites, which serve as local sources of drinking water, contained up to 31% CPR bacteria and 4% DPANN archaea. We observed little species-level overlap of metagenome-assembled genomes (MAGs) across the groundwater sites, indicating that CPR and DPANN communities may be differentiated according to physicochemical conditions and host populations. Cryogenic transmission electron microscopy imaging and genomic analyses enabled us to identify CPR and DPANN lineages that reproducibly attach to host cells and showed that the growth of CPR bacteria seems to be stimulated by attachment to host-cell surfaces. Our analysis reveals site-specific diversity of CPR bacteria and DPANN archaea that coexist with diverse hosts in groundwater aquifers. Given that CPR and DPANN organisms have been identified in human microbiomes and their presence is correlated with diseases such as periodontitis, our findings are relevant to considerations of drinking water quality and human health.

Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays.

BACKGROUND: An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day. RESULTS: Here we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo. CONCLUSION: The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.

Selective inhibition of human translation termination by a drug-like compound.

Methods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation. These molecules also inhibit translation termination by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 inhibits translation termination by arresting the nascent chain (NC) in the ribosome exit tunnel. The arrested NC adopts a compact α-helical conformation that induces 28 S rRNA nucleotide rearrangements that suppress the peptidyl transferase center (PTC) catalytic activity stimulated by eukaryotic release factor 1 (eRF1). These data support a mechanism of action for a small molecule targeting translation that suppresses peptidyl-tRNA hydrolysis promoted by eRF1, revealing principles of eukaryotic translation termination and laying the foundation for new therapeutic strategies.

Clades of huge phages from across Earth's ecosystems.

Bacteriophages typically have small genomes1 and depend on their bacterial hosts for replication2. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is-to our knowledge-the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR-Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR-Cas systems of phages 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 phages may repurpose bacterial CRISPR-Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth's ecosystems.