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.

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.

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.

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.

Unusual biology across a group comprising more than 15% of domain Bacteria.

A prominent feature of the bacterial domain is a radiation of major lineages that are defined as candidate phyla because they lack isolated representatives. Bacteria from these phyla occur in diverse environments and are thought to mediate carbon and hydrogen cycles. Genomic analyses of a few representatives suggested that metabolic limitations have prevented their cultivation. Here we reconstructed 8 complete and 789 draft genomes from bacteria representing >35 phyla and documented features that consistently distinguish these organisms from other bacteria. We infer that this group, which may comprise >15% of the bacterial domain, has shared evolutionary history, and describe it as the candidate phyla radiation (CPR). All CPR genomes are small and most lack numerous biosynthetic pathways. Owing to divergent 16S ribosomal RNA (rRNA) gene sequences, 50-100% of organisms sampled from specific phyla would evade detection in typical cultivation-independent surveys. CPR organisms often have self-splicing introns and proteins encoded within their rRNA genes, a feature rarely reported in bacteria. Furthermore, they have unusual ribosome compositions. All are missing a ribosomal protein often absent in symbionts, and specific lineages are missing ribosomal proteins and biogenesis factors considered universal in bacteria. This implies different ribosome structures and biogenesis mechanisms, and underlines unusual biology across a large part of the bacterial domain.

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.

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.

Accurate, multi-kb reads resolve complex populations and detect rare microorganisms.

Accurate evaluation of microbial communities is essential for understanding global biogeochemical processes and can guide bioremediation and medical treatments. Metagenomics is most commonly used to analyze microbial diversity and metabolic potential, but assemblies of the short reads generated by current sequencing platforms may fail to recover heterogeneous strain populations and rare organisms. Here we used short (150-bp) and long (multi-kb) synthetic reads to evaluate strain heterogeneity and study microorganisms at low abundance in complex microbial communities from terrestrial sediments. The long-read data revealed multiple (probably dozens of) closely related species and strains from previously undescribed Deltaproteobacteria and Aminicenantes (candidate phylum OP8). Notably, these are the most abundant organisms in the communities, yet short-read assemblies achieved only partial genome coverage, mostly in the form of short scaffolds (N50 = ∼ 2200 bp). Genome architecture and metabolic potential for these lineages were reconstructed using a new synteny-based method. Analysis of long-read data also revealed thousands of species whose abundances were <0.1% in all samples. Most of the organisms in this "long tail" of rare organisms belong to phyla that are also represented by abundant organisms. Genes encoding glycosyl hydrolases are significantly more abundant than expected in rare genomes, suggesting that rare species may augment the capability for carbon turnover and confer resilience to changing environmental conditions. Overall, the study showed that a diversity of closely related strains and rare organisms account for a major portion of the communities. These are probably common features of many microbial communities and can be effectively studied using a combination of long and short reads.

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.

Genomic resolution of a cold subsurface aquifer community provides metabolic insights for novel microbes adapted to high CO2 concentrations.

As in many deep underground environments, the microbial communities in subsurface high-CO2 ecosystems remain relatively unexplored. Recent investigations based on single-gene assays revealed a remarkable variety of organisms from little studied phyla in Crystal Geyser (Utah, USA), a site where deeply sourced CO2 -saturated fluids are erupted at the surface. To provide genomic resolution of the metabolisms of these organisms, we used a novel metagenomic approach to recover 227 high-quality genomes from 150 microbial species affiliated with 46 different phylum-level lineages. Bacteria from two novel phylum-level lineages have the capacity for CO2 fixation. Analyses of carbon fixation pathways in all studied organisms revealed that the Wood-Ljungdahl pathway and the Calvin-Benson-Bassham Cycle occurred with the highest frequency, whereas the reverse TCA cycle was little used. We infer that this, and selection for form II RuBisCOs, are adaptions to high CO2 -concentrations. However, many autotrophs can also grow mixotrophically, a strategy that confers metabolic versatility. The assignment of 156 hydrogenases to 90 different organisms suggests that H2 is an important inter-species energy currency even under gaseous CO2 -saturation. Overall, metabolic analyses at the organism level provided insight into the biochemical cycles that support subsurface life under the extreme condition of CO2 saturation.

The aerobic respiratory chain of the acidophilic archaeon Ferroplasma acidiphilum: A membrane-bound complex oxidizing ferrous iron.

The extremely acidophilic archaeon Ferroplasma acidiphilum is found in iron-rich biomining environments and is an important micro-organism in naturally occurring microbial communities in acid mine drainage. F. acidiphilum is an iron oxidizer that belongs to the order Thermoplasmatales (Euryarchaeota), which harbors the most extremely acidophilic micro-organisms known so far. At present, little is known about the nature or the structural and functional organization of the proteins in F. acidiphilum that impact the iron biogeochemical cycle. We combine here biochemical and biophysical techniques such as enzyme purification, activity measurements, proteomics and spectroscopy to characterize the iron oxidation pathway(s) in F. acidiphilum. We isolated two respiratory membrane protein complexes: a 850 kDa complex containing an aa3-type cytochrome oxidase and a blue copper protein, which directly oxidizes ferrous iron and reduces molecular oxygen, and a 150 kDa cytochrome ba complex likely composed of a di-heme cytochrome and a Rieske protein. We tentatively propose that both of these complexes are involved in iron oxidation respiratory chains, functioning in the so-called uphill and downhill electron flow pathways, consistent with autotrophic life. The cytochrome ba complex could possibly play a role in regenerating reducing equivalents by a reverse ('uphill') electron flow. This study constitutes the first detailed biochemical investigation of the metalloproteins that are potentially directly involved in iron-mediated energy conservation in a member of the acidophilic archaea of the genus Ferroplasma.

Changes in protein expression across laboratory and field experiments in Geobacter bemidjiensis.

Bacterial extracellular metal respiration, as carried out by members of the genus Geobacter, is of interest for applications including microbial fuel cells and bioremediation. Geobacter bemidjiensis is the major species whose growth is stimulated during groundwater amendment with acetate. We have carried out label-free proteomics studies of G. bemidjiensis grown with acetate as the electron donor and either fumarate, ferric citrate, or one of two hydrous ferric oxide mineral types as electron acceptor. The major class of proteins whose expression changes across these conditions is c-type cytochromes, many of which are known to be involved in extracellular metal reduction in other, better-characterized Geobacter species. Some proteins with multiple homologues in G. bemidjiensis (OmcS, OmcB) had different expression patterns than observed for their G. sulfurreducens homologues under similar growth conditions. We also compared the proteome from our study to a prior proteomics study of biomass recovered from an aquifer in Colorado, where the microbial community was dominated by strains closely related to G. bemidjiensis. We detected an increased number of proteins with functions related to motility and chemotaxis in the Colorado field samples compared to the laboratory samples, suggesting the importance of motility for in situ extracellular metal respiration.

Single-cell genomics reveals metabolic strategies for microbial growth and survival in an oligotrophic aquifer.

Bacteria from the genus Pedobacter are a major component of microbial assemblages at Hanford Site (a largely decommissioned nuclear production complex) in eastern Washington state, USA, and have been shown to change significantly in abundance in response to the subsurface intrusion of Columbia River water. Here we employed single-cell genomics techniques to shed light on the physiological niche of these micro-organisms. Analysis of four Pedobacter single amplified genomes (SAGs) from Hanford Site sediments revealed a chemoheterotrophic lifestyle, with the potential to exist under both aerobic and microaerophilic conditions via expression of both aa3-type and cbb3-type cytochrome c oxidases. These SAGs encoded a wide range of both intra- and extracellular carbohydrate-active enzymes, potentially enabling the degradation of recalcitrant substrates such as xylan and chitin, and the utilization of more labile sugars such as mannose and fucose. Coupled to these enzymes, a diversity of transporters and sugar-binding molecules were involved in the uptake of carbon from the extracellular local environment. The SAGs were enriched in TonB-dependent receptors, which play a key role in uptake of substrates resulting from degradation of recalcitrant carbon. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas mechanisms for resisting viral infections were identified in all SAGs. These data demonstrate the potential mechanisms utilized for persistence by heterotrophic micro-organisms in a carbon-limited aquifer, and hint at potential linkages between observed Pedobacter abundance shifts within the 300 Area (in the south-eastern corner of the site) subsurface and biogeochemical shifts associated with Columbia River water intrusion.

Single-cell genomics reveals metabolic strategies for microbial growth and survival in an oligotrophic aquifer.

Bacteria from the genus Pedobacter are a major component of microbial assemblages at Hanford Site (a largely decommissioned nuclear production complex) in eastern Washington state, USA, and have been shown to change significantly in abundance in response to the subsurface intrusion of Columbia River water. Here we employed single-cell genomics techniques to shed light on the physiological niche of these micro-organisms. Analysis of four Pedobacter single amplified genomes (SAGs) from Hanford Site sediments revealed a chemoheterotrophic lifestyle, with the potential to exist under both aerobic and microaerophilic conditions via expression of both aa3-type and cbb3-type cytochrome c oxidases. These SAGs encoded a wide range of both intra- and extracellular carbohydrate-active enzymes, potentially enabling the degradation of recalcitrant substrates such as xylan and chitin, and the utilization of more labile sugars such as mannose and fucose. Coupled to these enzymes, a diversity of transporters and sugar-binding molecules were involved in the uptake of carbon from the extracellular local environment. The SAGs were enriched in TonB-dependent receptors, which play a key role in uptake of substrates resulting from degradation of recalcitrant carbon. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas mechanisms for resisting viral infections were identified in all SAGs. These data demonstrate the potential mechanisms utilized for persistence by heterotrophic micro-organisms in a carbon-limited aquifer, and hint at potential linkages between observed Pedobacter abundance shifts within the 300 Area (in the south-eastern corner of the site) subsurface and biogeochemical shifts associated with Columbia River water intrusion.

Comparative genomics in acid mine drainage biofilm communities reveals metabolic and structural differentiation of co-occurring archaea.

BACKGROUND: Metal sulfide mineral dissolution during bioleaching and acid mine drainage (AMD) formation creates an environment that is inhospitable to most life. Despite dominance by a small number of bacteria, AMD microbial biofilm communities contain a notable variety of coexisting and closely related Euryarchaea, most of which have defied cultivation efforts. For this reason, we used metagenomics to analyze variation in gene content that may contribute to niche differentiation among co-occurring AMD archaea. Our analyses targeted members of the Thermoplasmatales and related archaea. These results greatly expand genomic information available for this archaeal order. RESULTS: We reconstructed near-complete genomes for uncultivated, relatively low abundance organisms A-, E-, and Gplasma, members of Thermoplasmatales order, and for a novel organism, Iplasma. Genomic analyses of these organisms, as well as Ferroplasma type I and II, reveal that all are facultative aerobic heterotrophs with the ability to use many of the same carbon substrates, including methanol. Most of the genomes share genes for toxic metal resistance and surface-layer production. Only Aplasma and Eplasma have a full suite of flagellar genes whereas all but the Ferroplasma spp. have genes for pili production. Cryogenic-electron microscopy (cryo-EM) and tomography (cryo-ET) strengthen these metagenomics-based ultrastructural predictions. Notably, only Aplasma, Gplasma and the Ferroplasma spp. have predicted iron oxidation genes and Eplasma and Iplasma lack most genes for cobalamin, valine, (iso)leucine and histidine synthesis. CONCLUSION: The Thermoplasmatales AMD archaea share a large number of metabolic capabilities. All of the uncultivated organisms studied here (A-, E-, G-, and Iplasma) are metabolically very similar to characterized Ferroplasma spp., differentiating themselves mainly in their genetic capabilities for biosynthesis, motility, and possibly iron oxidation. These results indicate that subtle, but important genomic differences, coupled with unknown differences in gene expression, distinguish these organisms enough to allow for co-existence. Overall this study reveals shared features of organisms from the Thermoplasmatales lineage and provides new insights into the functioning of AMD communities.

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.

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.

Mineral respiration under extreme acidic conditions: from a supramolecular organization to a molecular adaptation in Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotrophic Gram-negative bacterium that can derive energy from the oxidation of ferrous iron at pH 2 using oxygen as electron acceptor. The study of this bacterium has economic and fundamental biological interest because of its use in the industrial extraction of copper and uranium from ores. For this reason, its respiratory chain has been analysed in detail in recent years. Studies have shown the presence of a functional supercomplex that spans the outer and the inner membranes and allows a direct electron transfer from the extracellular Fe2+ ions to the inner membrane cytochrome c oxidase. Iron induces the expression of two operons encoding proteins implicated in this complex as well as in the regeneration of the reducing power. Most of these are metalloproteins that have been characterized biochemically, structurally and biophysically. For some of them, the molecular basis of their adaptation to the periplasmic acidic environment has been described. Modifications in the metal surroundings have been highlighted for cytochrome c and rusticyanin, whereas, for the cytochrome c oxidase, an additional partner that maintains its stability and activity has been demonstrated recently.

An unconventional copper protein required for cytochrome c oxidase respiratory function under extreme acidic conditions.

Very little is known about the processes used by acidophile organisms to preserve stability and function of respiratory pathways. Here, we reveal a potential strategy of these organisms for protecting and keeping functional key enzymes under extreme conditions. Using Acidithiobacillus ferrooxidans, we have identified a protein belonging to a new cupredoxin subfamily, AcoP, for "acidophile CcO partner," which is required for the cytochrome c oxidase (CcO) function. We show that it is a multifunctional copper protein with at least two roles as follows: (i) as a chaperone-like protein involved in the protection of the Cu(A) center of the CcO complex and (ii) as a linker between the periplasmic cytochrome c and the inner membrane cytochrome c oxidase. It could represent an interesting model for investigating the multifunctionality of proteins known to be crucial in pathways of energy metabolism.

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.