Novel and Engineered Type II CRISPR Systems from Uncultivated Microbes with Broad Genome Editing Capability.

Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases have been extensively used in biotechnology and therapeutics. However, many applications are not possible owing to the size, targetability, and potential off-target effects associated with currently known systems. In this study, we identified thousands of CRISPR type II effectors by mining an extensive, genome-resolved metagenomics database encompassing hundreds of thousands of microbial genomes. We developed a high-throughput pipeline that enabled us to predict tracrRNA sequences, to design single guide RNAs, and to demonstrate nuclease activity in vitro for 41 newly described subgroups. Active systems represent an extensive diversity of protein sequences and guide RNA structures and require diverse protospacer adjacent motifs (PAMs) that collectively expand the known targeting capability of current systems. Several nucleases showed activity levels comparable to or significantly higher than SpCas9, despite being smaller in size. In addition, top systems exhibited low levels of off-target editing in mammalian cells, and PAM-interacting domain engineered chimeras further expanded their targetability. These newly discovered nucleases are attractive enzymes for translation into many applications, including therapeutics.

Habitat Transition in the Evolution of Bacteria and Archaea.

Related groups of microbes are widely distributed across Earth's habitats, implying numerous dispersal and adaptation events over evolutionary time. However, relatively little is known about the characteristics and mechanisms of these habitat transitions, particularly for populations that reside in animal microbiomes. Here, we review the literature concerning habitat transitions among a variety of bacterial and archaeal lineages, considering the frequency of migration events, potential environmental barriers, and mechanisms of adaptation to new physicochemical conditions, including the modification of protein inventories and other genomic characteristics. Cells dependent on microbial hosts, particularly bacteria from the Candidate Phyla Radiation, have undergone repeated habitat transitions from environmental sources into animal microbiomes. We compare their trajectories to those of both free-living cells-including the Melainabacteria, Elusimicrobia, and methanogenic archaea-and cellular endosymbionts and bacteriophages, which have made similar transitions. We conclude by highlighting major related topics that may be worthy of future study.

Variable impact of geochemical gradients on the functional potential of bacteria, archaea, and phages from the permanently stratified Lac Pavin.

BACKGROUND: Permanently stratified lakes contain diverse microbial communities that vary with depth and so serve as useful models for studying the relationships between microbial community structure and geochemistry. Recent work has shown that these lakes can also harbor numerous bacteria and archaea from novel lineages, including those from the Candidate Phyla Radiation (CPR). However, the extent to which geochemical stratification differentially impacts carbon metabolism and overall genetic potential in CPR bacteria compared to other organisms is not well defined. RESULTS: Here, we determine the distribution of microbial lineages along an oxygen gradient in Lac Pavin, a deep, stratified lake in central France, and examine the influence of this gradient on their metabolism. Genome-based analyses revealed an enrichment of distinct C1 and CO2 fixation pathways in the oxic lake interface and anoxic zone/sediments, suggesting that oxygen likely plays a role in structuring metabolic strategies in non-CPR bacteria and archaea. Notably, we find that the oxidation of methane and its byproducts is largely spatially separated from methane production, which is mediated by diverse communities of sediment methanogens that vary on the centimeter scale. In contrast, we detected evidence for RuBisCO throughout the water column and sediments, including form II/III and form III-related enzymes encoded by CPR bacteria in the water column and DPANN archaea in the sediments. On the whole, though, CPR bacteria and phages did not show strong signals of gene content differentiation by depth, despite the fact that distinct species groups populate different lake and sediment compartments. CONCLUSIONS: Overall, our analyses suggest that environmental gradients in Lac Pavin select for capacities of CPR bacteria and phages to a lesser extent than for other bacteria and archaea. This may be due to the fact that selection in the former groups is indirect and depends primarily on host characteristics. Video Abstract.

Conserved and lineage-specific hypothetical proteins may have played a central role in the rise and diversification of major archaeal groups.

BACKGROUND: Archaea play fundamental roles in the environment, for example by methane production and consumption, ammonia oxidation, protein degradation, carbon compound turnover, and sulfur compound transformations. Recent genomic analyses have profoundly reshaped our understanding of the distribution and functionalities of Archaea and their roles in eukaryotic evolution. RESULTS: Here, 1179 representative genomes were selected from 3197 archaeal genomes. The representative genomes clustered based on the content of 10,866 newly defined archaeal protein families (that will serve as a community resource) recapitulates archaeal phylogeny. We identified the co-occurring proteins that distinguish the major lineages. Those with metabolic roles were consistent with experimental data. However, two families specific to Asgard were determined to be new eukaryotic signature proteins. Overall, the blocks of lineage-specific families are dominated by proteins that lack functional predictions. CONCLUSIONS: Given that these hypothetical proteins are near ubiquitous within major archaeal groups, we propose that they were important in the origin of most of the major archaeal lineages. Interestingly, although there were clearly phylum-specific co-occurring proteins, no such blocks of protein families were shared across superphyla, suggesting a burst-like origin of new lineages early in archaeal evolution.

Protein Family Content Uncovers Lineage Relationships and Bacterial Pathway Maintenance Mechanisms in DPANN Archaea.

DPANN are small-celled archaea that are generally predicted to be symbionts, and in some cases are known episymbionts of other archaea. As the monophyly of the DPANN remains uncertain, we hypothesized that proteome content could reveal relationships among DPANN lineages, constrain genetic overlap with bacteria, and illustrate how organisms with hybrid bacterial and archaeal protein sets might function. We tested this hypothesis using protein family content that was defined in part using 3,197 genomes including 569 newly reconstructed genomes. Protein family content clearly separates the final set of 390 DPANN genomes from other archaea, paralleling the separation of Candidate Phyla Radiation (CPR) bacteria from all other bacteria. This separation is partly driven by hypothetical proteins, some of which may be symbiosis-related. Pacearchaeota with the most limited predicted metabolic capacities have Form II/III and III-like Rubisco, suggesting metabolisms based on scavenged nucleotides. Intriguingly, the Pacearchaeota and Woesearchaeota with the smallest genomes also tend to encode large extracellular murein-like lytic transglycosylase domain proteins that may bind and degrade components of bacterial cell walls, indicating that some might be episymbionts of bacteria. The pathway for biosynthesis of bacterial isoprenoids is widespread in Woesearchaeota genomes and is encoded in proximity to genes involved in bacterial fatty acids synthesis. Surprisingly, in some DPANN genomes we identified a pathway for synthesis of queuosine, an unusual nucleotide in tRNAs of bacteria. Other bacterial systems are predicted to be involved in protein refolding. For example, many DPANN have the complete bacterial DnaK-DnaJ-GrpE system and many Woesearchaeota and Pacearchaeota possess bacterial group I chaperones. Thus, many DPANN appear to have mechanisms to ensure efficient protein folding of both archaeal and laterally acquired bacterial proteins.

Groundwater Elusimicrobia are metabolically diverse compared to gut microbiome Elusimicrobia and some have a novel nitrogenase paralog.

Currently described members of Elusimicrobia, a relatively recently defined phylum, are animal-associated and rely on fermentation. However, free-living Elusimicrobia have been detected in sediments, soils and groundwater, raising questions regarding their metabolic capacities and evolutionary relationship to animal-associated species. Here, we analyzed 94 draft-quality, non-redundant genomes, including 30 newly reconstructed genomes, from diverse animal-associated and natural environments. Genomes group into 12 clades, 10 of which previously lacked reference genomes. Groundwater-associated Elusimicrobia are predicted to be capable of heterotrophic or autotrophic lifestyles, reliant on oxygen or nitrate/nitrite-dependent respiration, or a variety of organic compounds and Rhodobacter nitrogen fixation (Rnf) complex-dependent acetogenesis with hydrogen and carbon dioxide as the substrates. Genomes from two clades of groundwater-associated Elusimicrobia often encode a new group of nitrogenase paralogs that co-occur with an extensive suite of radical S-Adenosylmethionine (SAM) proteins. We identified similar genomic loci in genomes of bacteria from the Gracilibacteria phylum and the Myxococcales order and predict that the gene clusters reduce a tetrapyrrole, possibly to form a novel cofactor. The animal-associated Elusimicrobia clades nest phylogenetically within two free-living-associated clades. Thus, we propose an evolutionary trajectory in which some Elusimicrobia adapted to animal-associated lifestyles from free-living species via genome reduction.

The rise of diversity in metabolic platforms across the Candidate Phyla Radiation.

BACKGROUND: A unifying feature of the bacterial Candidate Phyla Radiation (CPR) is a limited and highly variable repertoire of biosynthetic capabilities. However, the distribution of metabolic traits across the CPR and the evolutionary processes underlying them are incompletely resolved. RESULTS: Here, we selected ~ 1000 genomes of CPR bacteria from diverse environments to construct a robust internal phylogeny that was consistent across two unlinked marker sets. Mapping of glycolysis, the pentose phosphate pathway, and pyruvate metabolism onto the tree showed that some components of these pathways are sparsely distributed and that similarity between metabolic platforms is only partially predicted by phylogenetic relationships. To evaluate the extent to which gene loss and lateral gene transfer have shaped trait distribution, we analyzed the patchiness of gene presence in a phylogenetic context, examined the phylogenetic depth of clades with shared traits, and compared the reference tree topology with those of specific metabolic proteins. While the central glycolytic pathway in CPR is widely conserved and has likely been shaped primarily by vertical transmission, there is evidence for both gene loss and transfer especially in steps that convert glucose into fructose 1,6-bisphosphate and glycerate 3P into pyruvate. Additionally, the distribution of Group 3 and Group 4-related NiFe hydrogenases is patchy and suggests multiple events of ancient gene transfer. CONCLUSIONS: We infer that patterns of gene gain and loss in CPR, including acquisition of accessory traits in independent transfer events, could have been driven by shifts in host-derived resources and led to sparse but varied genetic inventories.

Lipid analysis of CO2-rich subsurface aquifers suggests an autotrophy-based deep biosphere with lysolipids enriched in CPR bacteria.

Sediment-hosted CO2-rich aquifers deep below the Colorado Plateau (USA) contain a remarkable diversity of uncultivated microorganisms, including Candidate Phyla Radiation (CPR) bacteria that are putative symbionts unable to synthesize membrane lipids. The origin of organic carbon in these ecosystems is unknown and the source of CPR membrane lipids remains elusive. We collected cells from deep groundwater brought to the surface by eruptions of Crystal Geyser, sequenced the community, and analyzed the whole community lipidome over time. Characteristic stable carbon isotopic compositions of microbial lipids suggest that bacterial and archaeal CO2 fixation ongoing in the deep subsurface provides organic carbon for the complex communities that reside there. Coupled lipidomic-metagenomic analysis indicates that CPR bacteria lack complete lipid biosynthesis pathways but still possess regular lipid membranes. These lipids may therefore originate from other community members, which also adapt to high in situ pressure by increasing fatty acid unsaturation. An unusually high abundance of lysolipids attributed to CPR bacteria may represent an adaptation to membrane curvature stress induced by their small cell sizes. Our findings provide new insights into the carbon cycle in the deep subsurface and suggest the redistribution of lipids into putative symbionts within this community.

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.

Unusual Metabolism and Hypervariation in the Genome of a Gracilibacterium (BD1-5) from an Oil-Degrading Community.

The candidate phyla radiation (CPR) comprises a large monophyletic group of bacterial lineages known almost exclusively based on genomes obtained using cultivation-independent methods. Within the CPR, Gracilibacteria (BD1-5) are particularly poorly understood due to undersampling and the inherent fragmented nature of available genomes. Here, we report the first closed, curated genome of a gracilibacterium from an enrichment experiment inoculated from the Gulf of Mexico and designed to investigate hydrocarbon degradation. The gracilibacterium rose in abundance after the community switched to dominance by Colwellia Notably, we predict that this gracilibacterium completely lacks glycolysis, the pentose phosphate and Entner-Doudoroff pathways. It appears to acquire pyruvate, acetyl coenzyme A (acetyl-CoA), and oxaloacetate via degradation of externally derived citrate, malate, and amino acids and may use compound interconversion and oxidoreductases to generate and recycle reductive power. The initial genome assembly was fragmented in an unusual gene that is hypervariable within a repeat region. Such extreme local variation is rare but characteristic of genes that confer traits under pressure to diversify within a population. Notably, the four major repeated 9-mer nucleotide sequences all generate a proline-threonine-aspartic acid (PTD) repeat. The genome of an abundant Colwellia psychrerythraea population has a large extracellular protein that also contains the repeated PTD motif. Although we do not know the host for the BD1-5 cell, the high relative abundance of the C. psychrerythraea population and the shared surface protein repeat may indicate an association between these bacteria.IMPORTANCE CPR bacteria are generally predicted to be symbionts due to their extensive biosynthetic deficits. Although monophyletic, they are not monolithic in terms of their lifestyles. The organism described here appears to have evolved an unusual metabolic platform not reliant on glucose or pentose sugars. Its biology appears to be centered around bacterial host-derived compounds and/or cell detritus. Amino acids likely provide building blocks for nucleic acids, peptidoglycan, and protein synthesis. We resolved an unusual repeat region that would be invisible without genome curation. The nucleotide sequence is apparently under strong diversifying selection, but the amino acid sequence is under stabilizing selection. The amino acid repeat also occurs in a surface protein of a coexisting bacterium, suggesting colocation and possibly interdependence.

The distinction of CPR bacteria from other bacteria based on protein family content.

Candidate phyla radiation (CPR) bacteria separate phylogenetically from other bacteria, but the organismal distribution of their protein families remains unclear. Here, we leveraged sequences from thousands of uncultivated organisms and identified protein families that co-occur in genomes, thus are likely foundational for lineage capacities. Protein family presence/absence patterns cluster CPR bacteria together, and away from all other bacteria and archaea, partly due to proteins without recognizable homology to proteins in other bacteria. Some are likely involved in cell-cell interactions and potentially important for episymbiotic lifestyles. The diversity of protein family combinations in CPR may exceed that of all other bacteria. Over the bacterial tree, protein family presence/absence patterns broadly recapitulate phylogenetic structure, suggesting persistence of core sets of proteins since lineage divergence. The CPR could have arisen in an episode of dramatic but heterogeneous genome reduction or from a protogenote community and co-evolved with other bacteria.

Author Correction: Hydrogen-based metabolism as an ancestral trait in lineages sibling to the Cyanobacteria.

The original version of this Article contained errors in Fig. 4. In panel a, the labels 'F420-reducing NiFe hydrogenase (group 3a)' and 'Group 2 NiFe hydrogenase' were misplaced. These errors have been corrected in both the PDF and HTML versions of the Article.

Hydrogen-based metabolism as an ancestral trait in lineages sibling to the Cyanobacteria.

The evolution of aerobic respiration was likely linked to the origins of oxygenic Cyanobacteria. Close phylogenetic neighbors to Cyanobacteria, such as Margulisbacteria (RBX-1 and ZB3), Saganbacteria (WOR-1), Melainabacteria and Sericytochromatia, may constrain the metabolic platform in which aerobic respiration arose. Here, we analyze genomic sequences and predict that sediment-associated Margulisbacteria have a fermentation-based metabolism featuring a variety of hydrogenases, a streamlined nitrogenase, and electron bifurcating complexes involved in cycling of reducing equivalents. The genomes of ocean-associated Margulisbacteria encode an electron transport chain that may support aerobic growth. Some Saganbacteria genomes encode various hydrogenases, and others may be able to use O2 under certain conditions via a putative novel type of heme copper O2 reductase. Similarly, Melainabacteria have diverse energy metabolisms and are capable of fermentation and aerobic or anaerobic respiration. The ancestor of all these groups may have been an anaerobe in which fermentation and H2 metabolism were central metabolic features. The ability to use O2 as a terminal electron acceptor must have been subsequently acquired by these lineages.

Lateral Gene Transfer Shapes the Distribution of RuBisCO among Candidate Phyla Radiation Bacteria and DPANN Archaea.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is considered to be the most abundant enzyme on Earth. Despite this, its full diversity and distribution across the domains of life remain to be determined. Here, we leverage a large set of bacterial, archaeal, and viral genomes recovered from the environment to expand our understanding of existing RuBisCO diversity and the evolutionary processes responsible for its distribution. Specifically, we report a new type of RuBisCO present in Candidate Phyla Radiation (CPR) bacteria that is related to the archaeal Form III enzyme and contains the amino acid residues necessary for carboxylase activity. Genome-level metabolic analyses supported the inference that these RuBisCO function in a CO2-incorporating pathway that consumes nucleotides. Importantly, some Gottesmanbacteria (CPR) also encode a phosphoribulokinase that may augment carbon metabolism through a partial Calvin-Benson-Bassham cycle. Based on the scattered distribution of RuBisCO and its discordant evolutionary history, we conclude that this enzyme has been extensively laterally transferred across the CPR bacteria and DPANN archaea. We also report RuBisCO-like proteins in phage genomes from diverse environments. These sequences cluster with proteins in the Beckwithbacteria (CPR), implicating phage as a possible mechanism of RuBisCO transfer. Finally, we synthesize our metabolic and evolutionary analyses to suggest that lateral gene transfer of RuBisCO may have facilitated major shifts in carbon metabolism in several important bacterial and archaeal lineages.

Biosynthetic capacity, metabolic variety and unusual biology in the CPR and DPANN radiations.

Candidate phyla radiation (CPR) bacteria and DPANN (an acronym of the names of the first included phyla) archaea are massive radiations of organisms that are widely distributed across Earth's environments, yet we know little about them. Initial indications are that they are consistently distinct from essentially all other bacteria and archaea owing to their small cell and genome sizes, limited metabolic capacities and often episymbiotic associations with other bacteria and archaea. In this Analysis, we investigate their biology and variations in metabolic capacities by analysis of approximately 1,000 genomes reconstructed from several metagenomics-based studies. We find that they are not monolithic in terms of metabolism but rather harbour a diversity of capacities consistent with a range of lifestyles and degrees of dependence on other organisms. Notably, however, certain CPR and DPANN groups seem to have exceedingly minimal biosynthetic capacities, whereas others could potentially be free living. Understanding of these microorganisms is important from the perspective of evolutionary studies and because their interactions with other organisms are likely to shape natural microbiome function.

Genomic inference of the metabolism and evolution of the archaeal phylum Aigarchaeota.

Microbes of the phylum Aigarchaeota are widely distributed in geothermal environments, but their physiological and ecological roles are poorly understood. Here we analyze six Aigarchaeota metagenomic bins from two circumneutral hot springs in Tengchong, China, to reveal that they are either strict or facultative anaerobes, and most are chemolithotrophs that can perform sulfide oxidation. Applying comparative genomics to the Thaumarchaeota and Aigarchaeota, we find that they both originated from thermal habitats, sharing 1154 genes with their common ancestor. Horizontal gene transfer played a crucial role in shaping genetic diversity of Aigarchaeota and led to functional partitioning and ecological divergence among sympatric microbes, as several key functional innovations were endowed by Bacteria, including dissimilatory sulfite reduction and possibly carbon monoxide oxidation. Our study expands our knowledge of the possible ecological roles of the Aigarchaeota and clarifies their evolutionary relationship to their sister lineage Thaumarchaeota.

Insights into the ecology, evolution, and metabolism of the widespread Woesearchaeotal lineages.

BACKGROUND: As a recently discovered member of the DPANN superphylum, Woesearchaeota account for a wide diversity of 16S rRNA gene sequences, but their ecology, evolution, and metabolism remain largely unknown. RESULTS: Here, we assembled 133 global clone libraries/studies and 19 publicly available genomes to profile these patterns for Woesearchaeota. Phylogenetic analysis shows a high diversity with 26 proposed subgroups for this recently discovered archaeal phylum, which are widely distributed in different biotopes but primarily in inland anoxic environments. Ecological patterns analysis and ancestor state reconstruction for specific subgroups reveal that oxic status of the environments is the key factor driving the distribution and evolutionary diversity of Woesearchaeota. A selective distribution to different biotopes and an adaptive colonization from anoxic to oxic environments can be proposed and supported by evidence of the presence of ferredoxin-dependent pathways in the genomes only from anoxic biotopes but not from oxic biotopes. Metabolic reconstructions support an anaerobic heterotrophic lifestyle with conspicuous metabolic deficiencies, suggesting the requirement for metabolic complementarity with other microbes. Both lineage abundance distribution and co-occurrence network analyses across diverse biotopes confirmed metabolic complementation and revealed a potential syntrophic relationship between Woesearchaeota and methanogens, which is supported by metabolic modeling. If correct, Woesearchaeota may impact methanogenesis in inland ecosystems. CONCLUSIONS: The findings provide an ecological and evolutionary framework for Woesearchaeota at a global scale and indicate their potential ecological roles, especially in methanogenesis.

Major New Microbial Groups Expand Diversity and Alter our Understanding of the Tree of Life.

The recent recovery of genomes for organisms from phyla with no isolated representative (candidate phyla) via cultivation-independent genomics enabled delineation of major new microbial lineages, namely the bacterial candidate phyla radiation (CPR), DPANN archaea, and Asgard archaea. CPR and DPANN organisms are inferred to be mostly symbionts, and some are episymbionts of other microbial community members. Asgard genomes encode typically eukaryotic systems, and their inclusion in phylogenetic analyses results in placement of eukaryotes as a branch within Archaea. Here, we illustrate how new genomes have changed the structure of the tree of life and altered our understanding of biology, evolution, and metabolic roles in biogeochemical processes.

Correlative Cryogenic Spectromicroscopy to Investigate Selenium Bioreduction Products.

Accurate mapping of the composition and structure of minerals and associated biological materials is critical in geomicrobiology and environmental research. Here, we have developed an apparatus that allows the correlation of cryogenic transmission electron microscopy (cryo-TEM) and synchrotron hard X-ray microprobe (SHXM) data sets to precisely determine the distribution, valence state, and structure of selenium in biofilms sampled from a contaminated aquifer near Rifle, CO. Results were replicated in the laboratory via anaerobic selenate-reducing enrichment cultures. 16S rRNA analyses of field-derived biofilm indicated the dominance of Betaproteobacteria from the Comamonadaceae family and uncultivated members of the Simplicispira genus. The major product in field and culture-derived biofilms is ∼25-300 nm red amorphous Se0 aggregates of colloidal nanoparticles. Correlative analyses of the cultures provided direct evidence for the microbial dissimilatory reduction of Se(VI) to Se(IV) to Se0. Extended X-ray absorption fine-structure spectroscopy showed red amorphous Se0 with a first shell Se-Se interatomic distance of 2.339 ± 0.003 Å. Complementary scanning transmission X-ray microscopy revealed that these aggregates are strongly associated with a protein-rich biofilm matrix. These findings have important implications for predicting the stability and mobility of Se bioremediation products and understanding of Se biogeochemical cycling. The approach, involving the correlation of cryo-SHXM and cryo-TEM data sets from the same specimen area, is broadly applicable to biological and environmental samples.

Retroelement-guided protein diversification abounds in vast lineages of Bacteria and Archaea.

Major radiations of enigmatic Bacteria and Archaea with large inventories of uncharacterized proteins are a striking feature of the Tree of Life1-5. The processes that led to functional diversity in these lineages, which may contribute to a host-dependent lifestyle, are poorly understood. Here, we show that diversity-generating retroelements (DGRs), which guide site-specific protein hypervariability6-8, are prominent features of genomically reduced organisms from the bacterial candidate phyla radiation (CPR) and as yet uncultivated phyla belonging to the DPANN (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota and Nanohaloarchaea) archaeal superphylum. From reconstructed genomes we have defined monophyletic bacterial and archaeal DGR lineages that expand the known DGR range by 120% and reveal a history of horizontal retroelement transfer. Retroelement-guided diversification is further shown to be active in current CPR and DPANN populations, with an assortment of protein targets potentially involved in attachment, defence and regulation. Based on observations of DGR abundance, function and evolutionary history, we find that targeted protein diversification is a pronounced trait of CPR and DPANN phyla compared to other bacterial and archaeal phyla. This diversification mechanism may provide CPR and DPANN organisms with a versatile tool that could be used for adaptation to a dynamic, host-dependent existence.