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.

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.

The Candidate Phyla Radiation (CPR) comprises a huge group of bacteria that have small genomes that rarely encode CRISPR-Cas systems for phage defense. Consequently, questions remain about their mechanisms of phage resistance and the nature of phage that infect them. The compact CRISPR-CasY system (Cas12d) with potential value in genome editing was first discovered in these organisms. Relatively few CasY sequences have been reported to date, and little is known about the function and activity of these systems in the natural environment. Here, we conducted a genome-resolved metagenomic investigation of hot spring microbiomes and recovered CRISPR systems mostly from Roizmanbacteria that involve CasY proteins that are divergent from published sequences. Within population diversity in the spacer set indicates current in situ diversification of most of the loci. In addition to CasY, some Roizmanbacteria genomes also encode large type I-B and/or III-A systems that, based on spacer targeting, are used in phage defense. CRISPR targeting identified three phage represented by complete genomes and a prophage, which are the first reported for bacteria of the Microgenomates superphylum. Interestingly, one phage encodes a Cas4-like protein, a scenario that has been suggested to drive acquisition of self-targeting spacers. Consistent with this, the Roizmanbacteria population that it infects has a CRISPR locus that includes self-targeting spacers and a fragmented CasY gene (fCasY). Despite gene fragmentation, the PAM sequence is the same as that of other CasY reported in this study. Fragmentation of CasY may avoid the lethality of self-targeting spacers. However, the spacers may still have some biological role, possibly in genome regulation. The findings expand our understanding of CasY diversity, and more broadly, CRISPR-Cas systems and phage of CPR bacteria.

Disparate redox activities that take place beyond the bounds of the prokaryotic cell cytosol must connect to membrane or cytosolic electron pools. Proteins post-translationally flavinylated by the enzyme ApbE mediate electron transfer in several characterized extracytosolic redox systems but the breadth of functions of this modification remains unknown. Here, we present a comprehensive bioinformatic analysis of 31,910 prokaryotic genomes that provides evidence of extracytosolic ApbEs within ~50% of bacteria and the involvement of flavinylation in numerous uncharacterized biochemical processes. By mining flavinylation-associated gene clusters, we identify five protein classes responsible for transmembrane electron transfer and two domains of unknown function (DUF2271 and DUF3570) that are flavinylated by ApbE. We observe flavinylation/iron transporter gene colocalization patterns that implicate functions in iron reduction and assimilation. We find associations with characterized and uncharacterized respiratory oxidoreductases that highlight roles of flavinylation in respiratory electron transport chains. Finally, we identify interspecies gene cluster variability consistent with flavinylation/cytochrome functional redundancies and discover a class of 'multi-flavinylated proteins' that may resemble multi-heme cytochromes in facilitating longer distance electron transfer. These findings provide mechanistic insight into an important facet of bacterial physiology and establish flavinylation as a functionally diverse mediator of extracytosolic electron transfer.

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.

Bacteria isolated from soils are major sources of specialized metabolites, including antibiotics and other compounds with clinical value that likely shape interactions among microbial community members and impact biogeochemical cycles. Yet, isolated lineages represent a small fraction of all soil bacterial diversity. It remains unclear how the production of specialized metabolites varies across the phylogenetic diversity of bacterial species in soils and whether the genetic potential for production of these metabolites differs with soil depth and vegetation type within a geographic region. We sampled soils and saprolite from three sites in a northern California Critical Zone Observatory with various vegetation and bedrock characteristics and reconstructed 1,334 metagenome-assembled genomes containing diverse biosynthetic gene clusters (BGCs) for secondary metabolite production. We obtained genomes for prolific producers of secondary metabolites, including novel groups within the Actinobacteria, Chloroflexi, and candidate phylum "Candidatus Dormibacteraeota." Surprisingly, one genome of a candidate phyla radiation (CPR) bacterium coded for a ribosomally synthesized linear azole/azoline-containing peptide, a capacity we found in other publicly available CPR bacterial genomes. Overall, bacteria with higher biosynthetic potential were enriched in shallow soils and grassland soils, with patterns of abundance of BGC type varying by taxonomy.IMPORTANCE Microbes produce specialized compounds to compete or communicate with one another and their environment. Some of these compounds, such as antibiotics, are also useful in medicine and biotechnology. Historically, most antibiotics have come from soil bacteria which can be isolated and grown in the lab. Though the vast majority of soil bacteria cannot be isolated, we can extract their genetic information and search it for genes which produce these specialized compounds. These understudied soil bacteria offer a wealth of potential for the discovery of new and important microbial products. Here, we identified the ability to produce these specialized compounds in diverse and novel bacteria in a range of soil environments. This information will be useful to other researchers who wish to isolate certain products. Beyond their use to humans, understanding the distribution and function of microbial products is key to understanding microbial communities and their effects on biogeochemical cycles.

Using cryo-electron microscopy (cryo-EM), we determined the structure of the Escherichia coli 70S ribosome with a global resolution of 2.0 Å. The maps reveal unambiguous positioning of protein and RNA residues, their detailed chemical interactions, and chemical modifications. Notable features include the first examples of isopeptide and thioamide backbone substitutions in ribosomal proteins, the former likely conserved in all domains of life. The maps also reveal extensive solvation of the small (30S) ribosomal subunit, and interactions with A-site and P-site tRNAs, mRNA, and the antibiotic paromomycin. The maps and models of the bacterial ribosome presented here now allow a deeper phylogenetic analysis of ribosomal components including structural conservation to the level of solvation. The high quality of the maps should enable future structural analyses of the chemical basis for translation and aid the development of robust tools for cryo-EM structure modeling and refinement.

Arsenic is a ubiquitous toxic element, the global cycle of which is highly affected by microbial redox reactions and assimilation into organoarsenic compounds through sequential methylation reactions. While microbial biotransformation of arsenic has been studied for decades, the past years have seen the discovery of multiple new genes related to arsenic metabolism. Still, most studies focus on a small set of key genes or a small set of cultured microorganisms. Here, we leveraged the recently greatly expanded availability of microbial genomes of diverse organisms from lineages lacking cultivated representatives, including those reconstructed from metagenomes, to investigate genetic repertoires of taxonomic and environmental controls on arsenic metabolic capacities. Based on the collection of arsenic-related genes, we identified thirteen distinct metabolic guilds, four of which combine the aio and ars operons. We found that the best studied phyla have very different combinations of capacities than less well-studied phyla, including phyla lacking isolated representatives. We identified a distinct arsenic gene signature in the microbiomes of humans exposed or likely exposed to drinking water contaminated by arsenic and that arsenic methylation is important in soil and in human microbiomes. Thus, the microbiomes of humans exposed to arsenic have the potential to exacerbate arsenic toxicity. Finally, we show that machine learning can predict bacterial arsenic metabolism capacities based on their taxonomy and the environment from which they were sampled.

Mineral-respiring bacteria use a process called extracellular electron transfer to route their respiratory electron transport chain to insoluble electron acceptors on the exterior of the cell. We recently characterized a flavin-based extracellular electron transfer system that is present in the foodborne pathogen Listeria monocytogenes, as well as many other Gram-positive bacteria, and which highlights a more generalized role for extracellular electron transfer in microbial metabolism. Here we identify a family of putative extracellular reductases that possess a conserved posttranslational flavinylation modification. Phylogenetic analyses suggest that divergent flavinylated extracellular reductase subfamilies possess distinct and often unidentified substrate specificities. We show that flavinylation of a member of the fumarate reductase subfamily allows this enzyme to receive electrons from the extracellular electron transfer system and support L. monocytogenes growth. We demonstrate that this represents a generalizable mechanism by finding that a L. monocytogenes strain engineered to express a flavinylated extracellular urocanate reductase uses urocanate by a related mechanism and to a similar effect. These studies thus identify an enzyme family that exploits a modular flavin-based electron transfer strategy to reduce distinct extracellular substrates and support a multifunctional view of the role of extracellular electron transfer activities in microbial physiology.

A variety of electron transfer mechanisms link bacterial cytosolic electron pools with functionally diverse redox activities in the cell envelope and extracellular space. In Listeria monocytogenes, the ApbE-like enzyme FmnB catalyzes extracytosolic protein flavinylation, covalently linking a flavin cofactor to proteins that transfer electrons to extracellular acceptors. L. monocytogenes uses an energy-coupling factor (ECF) transporter complex that contains distinct substrate-binding, transmembrane, ATPase A, and ATPase A' subunits (RibU, EcfT, EcfA, and EcfA') to import environmental flavins, but the basis of extracytosolic flavin trafficking for FmnB flavinylation remains poorly defined. In this study, we show that the EetB and FmnA proteins are related to ECF transporter substrate-binding and transmembrane subunits, respectively, and are essential for exporting flavins from the cytosol for flavinylation. Comparisons of the flavin import versus export capabilities of L. monocytogenes strains lacking different ECF transporter subunits demonstrate a strict directionality of substrate-binding subunit transport but partial functional redundancy of transmembrane and ATPase subunits. Based on these results, we propose that ECF transporter complexes with different subunit compositions execute directional flavin import/export through a broadly conserved mechanism. Finally, we present genomic context analyses that show that related ECF exporter genes are distributed across members of the phylum Firmicutes and frequently colocalize with genes encoding flavinylated extracytosolic proteins. These findings clarify the basis of ECF transporter export and extracytosolic flavin cofactor trafficking in Firmicutes. IMPORTANCE Bacteria import vitamins and other essential compounds from their surroundings but also traffic related compounds from the cytosol to the cell envelope where they serve various functions. Studying the foodborne pathogen Listeria monocytogenes, we find that the modular use of subunits from a prominent class of bacterial transporters enables the import of environmental vitamin B₂ cofactors and the extracytosolic trafficking of a vitamin B₂-derived cofactor that facilitates redox reactions in the cell envelope. These studies clarify the basis of bidirectional small-molecule transport across the cytoplasmic membrane and the assembly of redox-active proteins within the cell envelope and extracellular space.