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

CRISPR-Cas9 systems have been causing a revolution in biology. Harrington et al. describe the discovery and technological implementation of an additional type of CRISPR system based on an extracompact effector protein, Cas14. Metagenomics data, particularly from uncultivated samples, uncovered the CRISPR-Cas14 systems containing all the components necessary for adaptive immunity in prokaryotes. At half the size of class 2 CRISPR effectors, Cas14 appears to target single-stranded DNA without class 2 sequence restrictions. By leveraging this activity, a fast and high-fidelity nucleic acid detection system enabled detection of single-nucleotide polymorphisms. Science , this issue p. 839 Identification, characterization, and technological implementation of additional archaea-derived CRISPR-Cas14 systems are described. CRISPR-Cas systems provide microbes with adaptive immunity to infectious nucleic acids and are widely employed as genome editing tools. These tools use RNA-guided Cas proteins whose large size (950 to 1400 amino acids) has been considered essential to their specific DNA- or RNA-targeting activities. Here we present a set of CRISPR-Cas systems from uncultivated archaea that contain Cas14, a family of exceptionally compact RNA-guided nucleases (400 to 700 amino acids). Despite their small size, Cas14 proteins are capable of targeted single-stranded DNA (ssDNA) cleavage without restrictive sequence requirements. Moreover, target recognition by Cas14 triggers nonspecific cutting of ssDNA molecules, an activity that enables high-fidelity single-nucleotide polymorphism genotyping (Cas14-DETECTR). Metagenomic data show that multiple CRISPR-Cas14 systems evolved independently and suggest a potential evolutionary origin of single-effector CRISPR-based adaptive immunity.

Replication rates of bacteria in both human and environmental microbiomes are measured without reference genome sequences. Culture-independent microbiome studies have increased our understanding of the complexity and metabolic potential of microbial communities. However, to understand the contribution of individual microbiome members to community functions, it is important to determine which bacteria are actively replicating. We developed an algorithm, iRep, that uses draft-quality genome sequences and single time-point metagenome sequencing to infer microbial population replication rates. The algorithm calculates an index of replication (iRep) based on the sequencing coverage trend that results from bi-directional genome replication from a single origin of replication. We apply this method to show that microbial replication rates increase after antibiotic administration in human infants. We also show that uncultivated, groundwater-associated, Candidate Phyla Radiation bacteria only rarely replicate quickly in subsurface communities undergoing substantial changes in geochemistry. Our method can be applied to any genome-resolved microbiome study to track organism responses to varying conditions, identify actively growing populations and measure replication rates for use in modeling studies.

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. Recent studies have identified a large, phylogenetically distinct clade of bacteria, the candidate phyla radiation (CPR). Here, Méheust and colleagues analyze almost 3600 genomes to characterize the protein family content of CPR versus other bacteria and archaea.

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.

Metagenomic approaches are rapidly expanding our knowledge of microbial metabolic potential. Evaluation of the functional capacities of microorganisms long relied on laboratory cultivation of individual species. About a decade ago, recovery of draft genomes for a few uncultivated bacteria and archaea from natural communities opened the way for physiological prediction of their environmental roles. Further development of the metagenomics methods used in those early studies now allows the rapid delivery of accurately reconstructed microbial genomes from diverse environmental samples. The resulting knowledge has the potential to revolutionize our understanding of the topology of the tree of life and the metabolic capacities distributed across it. Advances in bioinformatics promise a new era in which comprehensive genetic characterization is sufficiently rapid to find application in diagnostics for medicine, agriculture, forensic science, and biotechnology.

In microbial ecology, a fundamental question relates to how community diversity and composition change in response to perturbation. Most studies have had limited ability to deeply sample community structure (e.g. Sanger-sequenced 16S rRNA libraries), or have had limited taxonomic resolution (e.g. studies based on 16S rRNA hypervariable region sequencing). Here, we combine the higher taxonomic resolution of near-full-length 16S rRNA gene amplicons with the economics and sensitivity of short-read sequencing to assay the abundance and identity of organisms that represent as little as 0.01% of sediment bacterial communities. We used a new version of EMIRGE optimized for large data size to reconstruct near-full-length 16S rRNA genes from amplicons sheared and sequenced with Illumina technology. The approach allowed us to differentiate the community composition among samples acquired before perturbation, after acetate amendment shifted the predominant metabolism to iron reduction, and once sulfate reduction began. Results were highly reproducible across technical replicates, and identified specific taxa that responded to the perturbation. All samples contain very high alpha diversity and abundant organisms from phyla without cultivated representatives. Surprisingly, at the time points measured, there was no strong loss of evenness, despite the selective pressure of acetate amendment and change in the terminal electron accepting process. However, community membership was altered significantly. The method allows for sensitive, accurate profiling of the \"long tail\" of low abundance organisms that exist in many microbial communities, and can resolve population dynamics in response to environmental change.

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. Inside cells, proteins are produced by complex molecular machines called ribosomes. Techniques that allow scientists to visualize ribosomes at the atomic level, such as cryogenic electron microscopy (cryo-EM), help shed light on the structure of these molecular machines, revealing details of how they build proteins. Understanding how ribosomes work has many benefits, including the development of new antibiotics that can kill bacteria without affecting animal cells. Watson et al. used cryo-EM techniques with increased resolution to examine the ribosomes of the bacterium Escherichia coli in a higher level of detail than has been seen before. The results revealed two chemical modifications in proteins that form the ribosome that had not been observed in ribosomes previously. Additionally, a protein segment with a previously undescribed structure was identified close to the site where the ribosome reads the genetic instructions needed to make proteins. Further genetic analyses suggested these structures are in many related species, and may play important roles in how the ribosome works. Watson et al. were also able to see how paromomycin, an antibiotic used to treat parasitic infections, is positioned in the ribosome. The antibiotic interacts with a site near where the genetic code is read out, which might explain why certain changes to the antibiotic can interfere with its potency. Finally, the new ribosome structure reveals thousands of water molecules and metal ions that help keep the ribosome together as it produces proteins. This study shows the value of advances in cryo-EM technology and illustrates the importance of applying these techniques to other cell components. The results also reveal details of the ribosome useful for further research into this essential molecular machine.

More than 15% of the bacterial domain consists of a radiation of phyla about which very little is known; here, metagenomics is used to reconstruct 8 complete and 789 draft genomes from more than 35 of these phyla, revealing a shared evolutionary history, metabolic limitations, and unusual ribosome compositions. Knowing unknown bacteria More than 15% of all bacteria are members of a radiation of phyla about which very little is known, and from which no members have been cultivated. Jillian Banfield and colleagues have used metagenomics to reconstruct 8 complete and 789 draft genomes from more than 35 of these phyla obtained from groundwater samples from an aquifer adjacent to the Colorado River. The data reveal a shared evolutionary history that leads the authors to group these phyla under the name candidate phyla radiation (CPR) as a subdivision within domain Bacteria. Characteristics of the CPR bacteria include small genomes lacking many biosynthetic pathways, self-splicing introns and proteins encoded within their rRNA genes, as well as an unusual ribosome composition, lacking proteins previously considered universal in all bacteria. CPR bacteria are likely to be obligate fermenters dependent on other organisms for survival. 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 1 and are thought to mediate carbon and hydrogen cycles 2 . Genomic analyses of a few representatives suggested that metabolic limitations have prevented their cultivation 2 , 3 , 4 , 5 , 6 . 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.

Recent studies have demonstrated that drought leads to dramatic, highly conserved shifts in the root microbiome. At present, the molecular mechanisms underlying these responses remain largely uncharacterized. Here we employ genome-resolved metagenomics and comparative genomics to demonstrate that carbohydrate and secondary metabolite transport functionalities are overrepresented within drought-enriched taxa. These data also reveal that bacterial iron transport and metabolism functionality is highly correlated with drought enrichment. Using time-series root RNA-Seq data, we demonstrate that iron homeostasis within the root is impacted by drought stress, and that loss of a plant phytosiderophore iron transporter impacts microbial community composition, leading to significant increases in the drought-enriched lineage, Actinobacteria. Finally, we show that exogenous application of iron disrupts the drought-induced enrichment of Actinobacteria, as well as their improvement in host phenotype during drought stress. Collectively, our findings implicate iron metabolism in the root microbiome’s response to drought and may inform efforts to improve plant drought tolerance to increase food security. Advances in omics provide a tool to understand mechanisms for plant–microbial interactions under stress. Here the authors apply genome-resolved metagenomics to investigate sorghum and its microbiome responses to drought, identifying an unexpected role of iron metabolism.