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In the version of this article initially published, the author A. Murat Eren was listed as A.M. Eren. The corresponding affiliation was given as the Knapp Center for Biomedical Discovery, rather than Department of Medicine, University of Chicago, Chicago, Illinois, USA, and Marine Biological Laboratory, Woods Hole, Massachusetts, USA. The errors have been corrected in the HTML and PDF versions of the article as of 29 November 2017. In the version of this article initially published, the following acknowledgment was omitted: A.L. was supported by the Russian Science Foundation (grant number 14-50-00069). The error has been corrected in the HTML and PDF versions of the article as of 7 December 2017.

Deforestation in the Brazilian Amazon occurs at an alarming rate, which has broad effects on global greenhouse gas emissions, carbon storage, and biogeochemical cycles. In this study, soil metagenomes and metagenome-assembled genomes (MAGs) were analyzed for alterations to microbial community composition, functional groups, and putative physiology as it related to land-use change and tropical soil. A total of 28 MAGs were assembled encompassing 10 phyla, including both dominant and rare biosphere lineages. Amazon Acidobacteria subdivision 3, Melainabacteria, Microgenomates, and Parcubacteria were found exclusively in pasture soil samples, while Candidatus Rokubacteria was predominant in the adjacent rainforest soil. These shifts in relative abundance between land-use types were supported by the different putative physiologies and life strategies employed by the taxa. This research provides unique biological insights into candidate phyla in tropical soil and how deforestation may impact the carbon cycle and affect climate change.

We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a Metagenome-Assembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Gene Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.

Objectives: To explore differences in the vaginal microbiome between preterm and term deliveries.Design: Nested case-control study in 3D cohort (design, develop, discover).Setting: Quebec, Canada.Sample: Ninety-four women with spontaneous preterm birth as cases [17 early (<34 weeks) and 77 late (34-36 weeks) preterm birth] and 356 women as controls with term delivery (≥37 weeks).Methods: To assess the vaginal microbiome by sequencing the V4 region of the 16S ribosomal RNA (rRNA) gene in swabs self-collected during early pregnancy.Main Outcome Measures: Comparison of relative abundance of bacterial operational taxonomic units and oligotypes and identifying vaginal community state types (CSTs) in early or late spontaneous preterm and term deliveries.Results: Lactobacillus gasseri/ Lactobacillus johnsonii (coefficient -5.36, 95% CI -8.07 to -2.65), Lactobacillus crispatus (99%)/ Lactobacillus acidophilus (99%) (-4.58, 95% CI -6.20 to -2.96), Lactobacillus iners (99%)/ Ralstonia solanacearum (99%) (-3.98, 95% CI -6.48 to -1.47) and Bifidobacterium longum/ Bifidobacterium breve (-8.84, 95% CI -12.96 to -4.73) were associated with decreased risk of early but not late preterm birth. Six vaginal CSTs were identified: four dominated by Lactobacillus; one with presence of bacterial vaginosis-associated bacteria (Gardnerella vaginalis, Atopobium vaginae and Veillonellaceae bacterium) (CST IV); and one with nondominance of Lactobacillus (CST VI). CST IV was associated with increased risk of early (4.22, 95% CI 1.24-24.85) but not late (1.63, 95% CI 0.68-5.04) preterm birth, compared with CST VI.Conclusions: Lactobacillus gasseri/L. johnsonii, L. crispatus/L. acidophilus, L. iners/R. solanacearum and B. longum/B. breve may be associated with decreased risk of early preterm birth. A bacterial vaginosis-related vaginal CST versus a CST nondominated by Lactobacillus may be associated with increased risk of early preterm birth.Tweetable Abstract: Largest study of its kind finds certain species of vaginal Lactobacillus + Bifidobacterium may relate to lower risk of preterm birth. [ABSTRACT FROM AUTHOR]

The remarkable pace of genomic data generation is rapidly transforming our understanding of life at the micron scale. Yet this data stream also creates challenges for team science. A single microbe can have multiple versions of genome architecture, functional gene annotations, and gene identifiers; additionally, the lack of mechanisms for collating and preserving advances in this knowledge raises barriers to community coalescence around shared datasets. "Digital Microbes" are frameworks for interoperable and reproducible collaborative science through open source, community-curated data packages built on a (pan)genomic foundation. Housed within an integrative software environment, Digital Microbes ensure real-time alignment of research efforts for collaborative teams and facilitate novel scientific insights as new layers of data are added. Here we describe two Digital Microbes: 1) the heterotrophic marine bacterium Ruegeria pomeroyi DSS-3 with > 100 transcriptomic datasets from lab and field studies, and 2) the pangenome of the cosmopolitan marine heterotroph Alteromonas containing 339 genomes. Examples demonstrate how an integrated framework collating public (pan)genome-informed data can generate novel and reproducible findings., (© 2024. The Author(s).)

Oil spills are a frequent perturbation to the marine environment that has rapid and significant impacts on the local microbiome. Previous studies have shown that exposure to synthetic dispersant alone did not enhance heterotrophic microbial activity or oxidation rates of specific hydrocarbon components but increased the abundance of some taxa (e.g., Colwellia ). In contrast, exposure to oil, but not dispersants, increased the abundance of other taxa (e.g., Marinobacter ) and stimulated hydrocarbon oxidation rates. Here, we advance these findings by interpreting metatranscriptomic data from this experiment to explore how and why specific components of the microbial community responded to distinct organic carbon exposure regimes. Dispersant alone was selected for a unique community and for dominant organisms that reflected treatment- and time-dependent responses. Dispersant amendment also led to diverging functional profiles among the different treatments. Similarly, oil alone was selected for a community that was distinct from treatments amended with dispersants. The presence of oil and dispersants with added nutrients led to substantial differences in microbial responses, likely suggesting increased fitness driven by the presence of additional inorganic nutrients. The oil-only additions led to a marked increase in the expression of phages, prophages, transposable elements, and plasmids (PPTEPs), suggesting that aspects of microbial community response to oil are driven by the "mobilome," potentially through viral-associated regulation of metabolic pathways in ciliates and flagellates that would otherwise throttle the microbial community through grazing.IMPORTANCEMicrocosm experiments simulated the April 2010 Deepwater Horizon oil spill by applying oil and synthetic dispersants (Corexit EC9500A and EC9527A) to deep ocean water samples. The exposure regime revealed severe negative alterations in the treatments' heterotrophic microbial activity and hydrocarbon oxidation rates. We expanded these findings by exploring metatranscriptomic signatures of the microbial communities during the chemical amendments in the microcosm experiments. Here we report how dominant organisms were uniquely associated with treatment- and time-dependent trajectories during the exposure regimes; nutrient availability was a significant factor in driving changes in metatranscriptomic responses. Remarkable signals associated with PPTEPs showed the potential role of mobilome and viral-associated survival responses. These insights underscore the time-dependent environmental perturbations of fragile marine environments under oil and anthropogenic stress., Competing Interests: The authors declare no conflict of interest.

A wide variety of human diseases are associated with loss of microbial diversity in the human gut, inspiring a great interest in the diagnostic or therapeutic potential of the microbiota. However, the ecological forces that drive diversity reduction in disease states remain unclear, rendering it difficult to ascertain the role of the microbiota in disease emergence or severity. One hypothesis to explain this phenomenon is that microbial diversity is diminished as disease states select for microbial populations that are more fit to survive environmental stress caused by inflammation or other host factors. Here, we tested this hypothesis on a large scale, by developing a software framework to quantify the enrichment of microbial metabolisms in complex metagenomes as a function of microbial diversity. We applied this framework to over 400 gut metagenomes from individuals who are healthy or diagnosed with inflammatory bowel disease (IBD). We found that high metabolic independence (HMI) is a distinguishing characteristic of microbial communities associated with individuals diagnosed with IBD. A classifier we trained using the normalized copy numbers of 33 HMI-associated metabolic modules not only distinguished states of health versus IBD, but also tracked the recovery of the gut microbiome following antibiotic treatment, suggesting that HMI is a hallmark of microbial communities in stressed gut environments.

Gene function annotations enable microbial ecologists to make inferences about metabolic potential from genomes and metagenomes. However, even tools that use the same database and general approach can differ markedly in the annotations they recover. We compare three popular methods for identifying KEGG Orthologs, applying them to genomes drawn from a range of bacterial families that occupy different host-associated and free-living biomes. Our results show that by adaptively tuning sequence similarity thresholds, sensitivity can be substantially improved while maintaining accuracy. We observe the largest improvements when few reference sequences exist for a given protein family, and when annotating genomes from non-model organisms (such as gut-dwelling Lachnospiraceae). Our results suggest that straightforward heuristic adjustments can broadly improve microbial metabolic predictions.

Gut microbiota can influence host gene expression and physiology through metabolites. Besides, the presence or absence of gut microbiome can reprogram host transcriptome and epitranscriptome as represented by N 6 -methyladenosine (m 6 A), the most abundant mammalian mRNA modification. However, which and how gut microbiota-derived metabolites reprogram host transcriptome and m 6 A epitranscriptome remain poorly understood. Here, investigation is conducted into how gut microbiota-derived metabolites impact host transcriptome and m 6 A epitranscriptome using multiple mouse models and multi-omics approaches. Various antibiotics-induced dysbiotic mice are established, followed by fecal microbiota transplantation (FMT) into germ-free mice, and the results show that bile acid metabolism is significantly altered along with the abundance change in bile acid-producing microbiota. Unbalanced gut microbiota and bile acids drastically change the host transcriptome and the m 6 A epitranscriptome in multiple tissues. Mechanistically, the expression of m 6 A writer proteins is regulated in animals treated with antibiotics and in cultured cells treated with bile acids, indicating a direct link between bile acid metabolism and m 6 A biology. Collectively, these results demonstrate that antibiotic-induced gut dysbiosis regulates the landscape of host transcriptome and m 6 A epitranscriptome via bile acid metabolism pathway. This work provides novel insights into the interplay between microbial metabolites and host gene expression., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Wolbachia is a maternally inherited intracellular bacterium that infects a wide range of arthropods including mosquitoes. The endosymbiont is widely used in biocontrol strategies due to its capacity to modulate arthropod reproduction and limit pathogen transmission. Wolbachia infections in Culex spp. are generally assumed to be monoclonal but the potential presence of genetically distinct Wolbachia subpopulations within and between individual organs has not been investigated using whole genome sequencing. Here we reconstructed Wolbachia genomes from ovary and midgut metagenomes of single naturally infected Culex pipiens mosquitoes from Southern France to investigate patterns of intra- and inter-individual differences across mosquito organs. Our analyses revealed a remarkable degree of intra-individual conservancy among Wolbachia genomes from distinct organs of the same mosquito both at the level of gene presence-absence signal and single-nucleotide polymorphisms (SNPs). Yet, we identified several synonymous and non-synonymous substitutions between individuals, demonstrating the presence of some level of genomic heterogeneity among Wolbachia that infect the same C. pipiens field population. Overall, the absence of genetic heterogeneity within Wolbachia populations in a single individual confirms the presence of a dominant Wolbachia that is maintained under strong purifying forces of evolution., Competing Interests: The authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

With almost a quadrillion individuals, the Antarctic krill processes five million tons of organic carbon every day during austral summer. This high carbon flux requires a broad range of hydrolytic enzymes to decompose the diverse food-derived biopolymers. While krill itself possesses numerous such enzymes, it is unclear, to what extent the endogenous microbiota contribute to the hydrolytic potential of the gut environment. Here we applied amplicon sequencing, shotgun metagenomics, cultivation, and physiological assays to characterize the krill gut microbiota. The broad bacterial diversity (273 families, 919 genera, and 2,309 species) also included a complex potentially anaerobic sub-community. Plate-based assays with 198 isolated pure cultures revealed widespread capacities to utilize lipids (e.g., tributyrin), followed by proteins (casein) and to a lesser extent by polysaccharides (e.g., alginate and chitin). While most isolates affiliated with the genera Pseudoalteromonas and Psychrobacter , also Rubritalea spp. (Verrucomicrobia) were observed. The krill gut microbiota growing on marine broth agar plates possess 13,012 predicted hydrolyses; 15-fold more than previously predicted from a transcriptome-proteome compendium of krill. Cultivation-independent and -dependent approaches indicated members of the families Flavobacteriaceae and Pseudoalteromonadaceae to dominate the capacities for lipid/protein hydrolysis and to provide a plethora of carbohydrate-active enzymes, sulfatases, and laminarin- or porphyrin-depolymerizing hydrolases. Notably, also the potential to hydrolyze plastics such as polyethylene terephthalate and polylactatide was observed, affiliating mostly with Moraxellaceae. Overall, this study shows extensive microbial diversity in the krill gut, and suggests that the microbiota likely play a significant role in the nutrient acquisition of the krill by enriching its hydrolytic enzyme repertoire.IMPORTANCEThe Antarctic krill ( Euphausia superba ) is a keystone species of the Antarctic marine food web, connecting the productivity of phyto- and zooplankton with the nutrition of the higher trophic levels. Accordingly, krill significantly contributes to biomass turnover, requiring the decomposition of seasonally varying plankton-derived biopolymers. This study highlights the likely role of the krill gut microbiota in this ecosystem function by revealing the great number of diverse hydrolases that microbes contribute to the krill gut environment. The here resolved repertoire of hydrolytic enzymes could contribute to the overall nutritional resilience of krill and to the general organic matter cycling under changing environmental conditions in the Antarctic sea water. Furthermore, the krill gut microbiome could serve as a valuable resource of cold-adapted hydrolytic enzymes for diverse biotechnological applications., Competing Interests: The authors declare no conflict of interest.

Plasmids alter microbial evolution and lifestyles by mobilizing genes that often confer fitness in changing environments across clades. Yet our ecological and evolutionary understanding of naturally occurring plasmids is far from complete. Here we developed a machine-learning model, PlasX, which identified 68,350 non-redundant plasmids across human gut metagenomes and organized them into 1,169 evolutionarily cohesive 'plasmid systems' using our sequence containment-aware network-partitioning algorithm, MobMess. Individual plasmids were often country specific, yet most plasmid systems spanned across geographically distinct human populations. Cargo genes in plasmid systems included well-known determinants of fitness, such as antibiotic resistance, but also many others including enzymes involved in the biosynthesis of essential nutrients and modification of transfer RNAs, revealing a wide repertoire of likely fitness determinants in complex environments. Our study introduces computational tools to recognize and organize plasmids, and uncovers the ecological and evolutionary patterns of diverse plasmids in naturally occurring habitats through plasmid systems., (© 2024. The Author(s).)

Plasmids are extrachromosomal genetic elements that often encode fitness-enhancing features. However, many bacteria carry "cryptic" plasmids that do not confer clear beneficial functions. We identified one such cryptic plasmid, pBI143, which is ubiquitous across industrialized gut microbiomes and is 14 times as numerous as crAssphage, currently established as the most abundant extrachromosomal genetic element in the human gut. The majority of mutations in pBI143 accumulate in specific positions across thousands of metagenomes, indicating strong purifying selection. pBI143 is monoclonal in most individuals, likely due to the priority effect of the version first acquired, often from one's mother. pBI143 can transfer between Bacteroidales, and although it does not appear to impact bacterial host fitness in vivo, it can transiently acquire additional genetic content. We identified important practical applications of pBI143, including its use in identifying human fecal contamination and its potential as an alternative approach to track human colonic inflammatory states., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Importance: The Gram-negative bacterium Bacteroides fragilis is a common member of the human gut microbiota that colonizes multiple host niches and can influence human physiology through a variety of mechanisms. Identification of genes that enable B. fragilis to grow across a range of host environments has been impeded in part by the relatively limited genetic tractability of this species. We have developed a high-throughput genetic resource for a B. fragilis strain isolated from a UC pouchitis patient. Bile acids limit microbial growth and are altered in abundance in UC pouches, where B. fragilis often blooms. Using this resource, we uncovered pathways and processes that impact B. fragilis fitness in bile and that may contribute to population expansions during bouts of gut inflammation., Competing Interests: The authors declare no conflict of interest.

Respiratory reductases enable microorganisms to use molecules present in anaerobic ecosystems as energy-generating respiratory electron acceptors. Here we identify three taxonomically distinct families of human gut bacteria (Burkholderiaceae, Eggerthellaceae and Erysipelotrichaceae) that encode large arsenals of tens to hundreds of respiratory-like reductases per genome. Screening species from each family (Sutterella wadsworthensis, Eggerthella lenta and Holdemania filiformis), we discover 22 metabolites used as respiratory electron acceptors in a species-specific manner. Identified reactions transform multiple classes of dietary- and host-derived metabolites, including bioactive molecules resveratrol and itaconate. Products of identified respiratory metabolisms highlight poorly characterized compounds, such as the itaconate-derived 2-methylsuccinate. Reductase substrate profiling defines enzyme-substrate pairs and reveals a complex picture of reductase evolution, providing evidence that reductases with specificities for related cinnamate substrates independently emerged at least four times. These studies thus establish an exceptionally versatile form of anaerobic respiration that directly links microbial energy metabolism to the gut metabolome., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Host-microbe interactions have been linked to health and disease states through the use of microbial taxonomic profiling, mostly via 16S ribosomal RNA gene sequencing. However, many mechanistic insights remain elusive, in part because studying the genomes of microbes associated with mammalian tissue is difficult due to the high ratio of host to microbial DNA in such samples. Here we describe a microbial-enrichment method (MEM), which we demonstrate on a wide range of sample types, including saliva, stool, intestinal scrapings, and intestinal mucosal biopsies. MEM enabled high-throughput characterization of microbial metagenomes from human intestinal biopsies by reducing host DNA more than 1,000-fold with minimal microbial community changes (roughly 90% of taxa had no significant differences between MEM-treated and untreated control groups). Shotgun sequencing of MEM-treated human intestinal biopsies enabled characterization of both high- and low-abundance microbial taxa, pathways and genes longitudinally along the gastrointestinal tract. We report the construction of metagenome-assembled genomes directly from human intestinal biopsies for bacteria and archaea at relative abundances as low as 1%. Analysis of metagenome-assembled genomes reveals distinct subpopulation structures between the small and large intestine for some taxa. MEM opens a path for the microbiome field to acquire deeper insights into host-microbe interactions by enabling in-depth characterization of host-tissue-associated microbial communities., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Environmental perturbations shape the structure and function of microbial communities. Oil spills are a major perturbation and resolving spills often requires active measures like dispersant application that can exacerbate the initial disturbance. Species-specific responses of microorganisms to oil and dispersant exposure during such perturbations remain largely unknown. We merged metatranscriptomic libraries with pangenomes to generate Core-Accessory Metatranscriptomes (CA-Metatranscriptomes) for two microbial hydrocarbon degraders that played important roles in the aftermath of the Deepwater Horizon oil spill. The Colwellia CA-Metatranscriptome illustrated pronounced dispersant-driven acceleration of core (~41%) and accessory gene (~59%) transcription, suggesting an opportunistic strategy. Marinobacter responded to oil exposure by expressing mainly accessory genes (~93%), suggesting an effective hydrocarbon-degrading lifestyle. The CA-Metatranscriptome approach offers a robust way to identify the underlying mechanisms of key microbial functions and highlights differences of specialist-vs-opportunistic responses to environmental disturbance., (© 2023. ISME Publications B.V.)

Background and Aims: Exclusive enteral nutrition [EEN] is a dietary intervention to induce clinical remission in children with active luminal Crohn's disease [CD]. While changes in the gut microbial communities have been implicated in achieving this remission, a precise understanding of the role of microbial ecology in the restoration of gut homeostasis is lacking., Methods: Here we reconstructed genomes from the gut metagenomes of 12 paediatric subjects who were sampled before, during and after EEN. We then classified each microbial population into distinct 'phenotypes' or patterns of response based on changes in their relative abundances throughout the therapy on a per-individual basis., Results: Our data show that children achieving clinical remission during therapy were enriched with microbial populations that were either suppressed or that demonstrated a transient bloom as a function of EEN. In contrast, this ecosystem-level response was not observed in cases of EEN failure. Further analysis revealed that populations that were suppressed during EEN were significantly more prevalent in healthy children and adults across the globe compared with those that bloomed ephemerally during the therapy., Conclusions: These observations taken together suggest that successful outcomes of EEN are marked by a temporary emergence of microbial populations that are rare in healthy individuals, and a concomitant reduction in microbes that are commonly associated with gut homeostasis. Our work is a first attempt to highlight individual-specific, complex environmental factors that influence microbial response in EEN. This model offers a novel, alternative viewpoint to traditional taxonomic strategies used to characterize associations with health and disease states., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Mosquitoes represent the most important pathogen vectors and are responsible for the spread of a wide variety of poorly treatable diseases. Wolbachia are obligate intracellular bacteria that are widely distributed among arthropods and collectively represents one of the most promising solutions for vector control. In particular, Wolbachia has been shown to limit the transmission of pathogens, and to dramatically affect the reproductive behavior of their host through its phage WO. While much research has focused on deciphering and exploring the biocontrol applications of these WO-related phenotypes, the extent and potential impact of the Wolbachia mobilome remain poorly appreciated. Notably, several Wolbachia plasmids, carrying WO-like genes and Insertion Sequences (IS), thus possibly interrelated to other genetic units of the endosymbiont, have been recently discovered. Here we investigated the diversity and biogeography of the first described plasmid of Wolbachia in Culex pipiens (pWCP) in several islands and continental countries around the world-including Cambodia, Guadeloupe, Martinique, Thailand, and Mexico-together with mosquito strains from colonies that evolved for 2 to 30 years in the laboratory. We used PCR and qPCR to determine the presence and copy number of pWCP in individual mosquitoes, and highly accurate Sanger sequencing to evaluate potential variations. Together with earlier observation, our results show that pWCP is omnipresent and strikingly conserved among Wolbachia populations within mosquitoes from distant geographies and environmental conditions. These data suggest a critical role for the plasmid in Wolbachia ecology and evolution, and the potential of a great tool for further genetic dissection and possible manipulation of this endosymbiont., (© 2023. The Author(s).)

Background: Changes in microbial community composition as a function of human health and disease states have sparked remarkable interest in the human gut microbiome. However, establishing reproducible insights into the determinants of microbial succession in disease has been a formidable challenge., Results: Here we use fecal microbiota transplantation (FMT) as an in natura experimental model to investigate the association between metabolic independence and resilience in stressed gut environments. Our genome-resolved metagenomics survey suggests that FMT serves as an environmental filter that favors populations with higher metabolic independence, the genomes of which encode complete metabolic modules to synthesize critical metabolites, including amino acids, nucleotides, and vitamins. Interestingly, we observe higher completion of the same biosynthetic pathways in microbes enriched in IBD patients., Conclusions: These observations suggest a general mechanism that underlies changes in diversity in perturbed gut environments and reveal taxon-independent markers of "dysbiosis" that may explain why widespread yet typically low-abundance members of healthy gut microbiomes can dominate under inflammatory conditions without any causal association with disease., (© 2023. The Author(s).)

Comprehensive sampling of natural genetic diversity with metagenomics enables highly resolved insights into the interplay between ecology and evolution. However, resolving adaptive, neutral, or purifying processes of evolution from intrapopulation genomic variation remains a challenge, partly due to the sole reliance on gene sequences to interpret variants. Here, we describe an approach to analyze genetic variation in the context of predicted protein structures and apply it to a marine microbial population within the SAR11 subclade 1a.3.V, which dominates low-latitude surface oceans. Our analyses reveal a tight association between genetic variation and protein structure. In a central gene in nitrogen metabolism, we observe decreased occurrence of nonsynonymous variants from ligand-binding sites as a function of nitrate concentrations, revealing genetic targets of distinct evolutionary pressures maintained by nutrient availability. Our work yields insights into the governing principles of evolution and enables structure-aware investigations of microbial population genetics.

H37Rv is the most widely used Mycobacterium tuberculosis strain, and its genome is globally used as the M. tuberculosis reference sequence. Here, we present Bact-Builder, a pipeline that uses consensus building to generate complete and accurate bacterial genome sequences and apply it to three independently cultured and sequenced H37Rv aliquots of a single laboratory stock. Two of the 4,417,942 base-pair long H37Rv assemblies are 100% identical, with the third differing by a single nucleotide. Compared to the existing H37Rv reference, the new sequence contains ~6.4 kb additional base pairs, encoding ten new regions that include insertions in PE/PPE genes and new paralogs of esxN and esxJ, which are differentially expressed compared to the reference genes. New sequencing and de novo assemblies with Bact-Builder confirm that all 10 regions, plus small additional polymorphisms, are also present in the commonly used H37Rv strains NR123, TMC102, and H37Rv1998. Thus, Bact-Builder shows promise as an improved method to perform accurate and reproducible de novo assemblies of bacterial genomes, and our work provides important updates to the primary M. tuberculosis reference genome., (© 2022. The Author(s).)

Coastal marine macrophytes exhibit some of the highest rates of primary productivity in the world. They have been found to host a diverse set of microbes, many of which may impact the biology of their hosts through metabolisms that are unique to microbial taxa. Here, we characterized the metabolic functions of macrophyte-associated microbial communities using metagenomes collected from 2 species of kelp ( Laminaria setchellii and Nereocystis luetkeana ) and 3 marine angiosperms ( Phyllospadix scouleri , P. serrulatus , and Zostera marina ), including the rhizomes of two surfgrass species ( Phyllospadix spp.), the seagrass Zostera marina , and the sediments surrounding P. scouleri and Z. marina . Using metagenomic sequencing, we describe 63 metagenome-assembled genomes (MAGs) that potentially benefit from being associated with macrophytes and may contribute to macrophyte fitness through their metabolic activity. Host-associated metagenomes contained genes for the use of dissolved organic matter from hosts and vitamin (B 1 , B 2 , B 7 , B 12 ) biosynthesis in addition to a range of nitrogen and sulfur metabolisms that recycle dissolved inorganic nutrients into forms more available to the host. The rhizosphere of surfgrass and seagrass contained genes for anaerobic microbial metabolisms, including nifH genes associated with nitrogen fixation, despite residing in a well-mixed and oxygenated environment. The range of oxygen environments engineered by macrophytes likely explains the diversity of both oxidizing and reducing microbial metabolisms and contributes to the functional capabilities of microbes and their influences on carbon and nitrogen cycling in nearshore ecosystems. IMPORTANCE Kelps, seagrasses, and surfgrasses are ecosystem engineers on rocky shorelines, where they show remarkably high levels of primary production. Through analysis of their associated microbial communities, we found a variety of microbial metabolisms that may benefit the host, including nitrogen metabolisms, sulfur oxidation, and the production of B vitamins. In turn, these microbes have the genetic capabilities to assimilate the dissolved organic compounds released by their macrophyte hosts. We describe a range of oxygen environments associated with surfgrass, including low-oxygen microhabitats in their rhizomes that host genes for nitrogen fixation. The tremendous productivity of coastal seaweeds and seagrasses is likely due in part to the activities of associated microbes, and an increased understanding of these associations is needed.

Microbes have inhabited the oceans and soils for millions of years and are uniquely adapted to their habitat. In contrast, sewer infrastructure in modern cities dates back only ~150 years. Sewer pipes transport human waste and provide a view into public health, but the resident organisms that likely modulate these features are relatively unexplored. Here, we show that the bacterial assemblages sequenced from untreated wastewater in 71 U.S. cities were highly coherent at a fine sequence level, suggesting that urban infrastructure separated by great spatial distances can give rise to strikingly similar communities. Within the overall microbial community structure, temperature had a discernible impact on the distribution patterns of closely related amplicon sequence variants, resulting in warm and cold ecotypes. Two bacterial genera were dominant in most cities regardless of their size or geographic location; on average, Arcobacter accounted for 11% and Acinetobacter 10% of the entire community. Metagenomic analysis of six cities revealed these highly abundant resident organisms carry clinically important antibiotic resistant genes bla CTX-M , bla OXA , and bla TEM . In contrast, human fecal bacteria account for only ~13% of the community; therefore, antibiotic resistance gene inputs from human sources to the sewer system could be comparatively small, which will impact measurement capabilities when monitoring human populations using wastewater. With growing awareness of the metabolic potential of microbes within these vast networks of pipes and the ability to examine the health of human populations, it is timely to increase our understanding of the ecology of these systems. IMPORTANCE Sewer infrastructure is a relatively new habitat comprised of thousands of kilometers of pipes beneath cities. These wastewater conveyance systems contain large reservoirs of microbial biomass with a wide range of metabolic potential and are significant reservoirs of antibiotic resistant organisms; however, we lack an adequate understanding of the ecology or activity of these communities beyond wastewater treatment plants. The striking coherence of the sewer microbiome across the United States demonstrates that the sewer environment is highly selective for a particular microbial community composition. Therefore, results from more in-depth studies or proven engineering controls in one system could be extrapolated more broadly. Understanding the complex ecology of sewer infrastructure is critical for not only improving our ability to treat human waste and increasing the sustainability of our cities but also to create scalable and effective sewage microbial observatories, which are inevitable investments of the future to monitor health in human populations.

By offering extremely long contiguous characterization of individual DNA molecules, rapidly emerging long-read sequencing strategies offer comprehensive insights into the organization of genetic information in genomes and metagenomes. However, successful long-read sequencing experiments demand high concentrations of highly purified DNA of high molecular weight (HMW), which limits the utility of established DNA extraction kits designed for short-read sequencing. The challenges associated with input DNA quality intensify further when working with complex environmental samples of low microbial biomass, which requires new protocols that are tailored to study metagenomes with long-read sequencing. Here, we use human tongue scrapings to benchmark six HMW DNA extraction strategies that are based on commercially available kits, phenol-chloroform (PC) extraction and agarose encasement followed by agarase digestion. A typical end goal of HMW DNA extractions is to obtain the longest possible reads during sequencing, which is often achieved by PC extractions, as demonstrated in sequencing of cultured cells. Yet our analyses that consider overall read-size distribution, assembly performance and the number of circularized elements found in sequencing results suggest that column-based kits with enzyme supplementation, rather than PC methods, may be more appropriate for long-read sequencing of metagenomes., (© 2022 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Despite the popularity of kombucha tea, the distribution of different microbes across kombucha ferments and how those microbes interact within communities are not well characterized. Using metagenomics, comparative genomics, synthetic community experiments, and metabolomics, we determined the taxonomic, ecological, and functional diversity of 23 distinct kombuchas from across the United States. Shotgun metagenomic sequencing demonstrated that the bacterium Komagataeibacter rhaeticus and the yeast Brettanomyces bruxellensis were the most common microbes in the sampled kombucha communities. To determine the specificity of bacterium-yeast interactions, we experimentally quantified microbial interactions within kombucha biofilms by measuring densities of interacting species and biofilm production. In pairwise combinations of bacteria and yeast, B. bruxellensis and individual strains of Komagataeibacter spp. were sufficient to form kombucha fermentations with robust biofilms, but Zygosaccharomyces bisporus, another yeast found in kombucha, did not stimulate bacteria to produce biofilms. Profiling the spent media of both yeast species using nuclear magnetic resonance spectroscopy suggested that the enhanced ability of B. bruxellensis to ferment and produce key metabolites in sucrose-sweetened tea may explain why it stimulates biofilm formation. Comparative genomics demonstrated that Komagataeibacter spp. with >99% genomic similarity can still have dramatic differences in biofilm production, with strong producers yielding five times more biofilm than the weakest producers. IMPORTANCE Through an integration of metagenomic and experimental approaches, our work reveals the diversity and nature of interactions among key taxa in kombucha microbiomes through the construction of synthetic microbial pairs. Manipulation of these microbes in kombucha has the potential to shape both the fermentation qualities of kombucha and the production of biofilms and is valuable for kombucha beverage producers, biofilm engineers, and synthetic ecologists.

Marine planktonic eukaryotes play critical roles in global biogeochemical cycles and climate. However, their poor representation in culture collections limits our understanding of the evolutionary history and genomic underpinnings of planktonic ecosystems. Here, we used 280 billion Tara Oceans metagenomic reads from polar, temperate, and tropical sunlit oceans to reconstruct and manually curate more than 700 abundant and widespread eukaryotic environmental genomes ranging from 10 Mbp to 1.3 Gbp. This genomic resource covers a wide range of poorly characterized eukaryotic lineages that complement long-standing contributions from culture collections while better representing plankton in the upper layer of the oceans. We performed the first, to our knowledge, comprehensive genome-wide functional classification of abundant unicellular eukaryotic plankton, revealing four major groups connecting distantly related lineages. Neither trophic modes of plankton nor its vertical evolutionary history could completely explain the functional repertoire convergence of major eukaryotic lineages that coexisted within oceanic currents for millions of years., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Biological nitrogen fixation contributes significantly to marine primary productivity. The current view depicts few cyanobacterial diazotrophs as the main marine nitrogen fixers. Here, we used 891 Tara Oceans metagenomes derived from surface waters of five oceans and two seas to generate a manually curated genomic database corresponding to free-living, filamentous, colony-forming, particle-attached, and symbiotic bacterial and archaeal populations. The database provides the genomic content of eight cyanobacterial diazotrophs including a newly discovered population related to known heterocystous symbionts of diatoms, as well as 40 heterotrophic bacterial diazotrophs that considerably expand the known diversity of abundant marine nitrogen fixers. These 48 populations encapsulate 92% of metagenomic signal for known nifH genes in the sunlit ocean, suggesting that the genomic characterization of the most abundant marine diazotrophs may be nearing completion. Newly identified heterotrophic bacterial diazotrophs are widespread, express their nifH genes in situ, and also occur in large planktonic size fractions where they might form aggregates that provide the low-oxygen microenvironments required for nitrogen fixation. Critically, we found heterotrophic bacterial diazotrophs to be more abundant than cyanobacterial diazotrophs in most metagenomes from the open oceans and seas, emphasizing the importance of a wide range of heterotrophic populations in the marine nitrogen balance., (© 2021. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

One-quarter of photosynthesis-derived carbon on Earth rapidly cycles through a set of short-lived seawater metabolites that are generated from the activities of marine phytoplankton, bacteria, grazers and viruses. Here we discuss the sources of microbial metabolites in the surface ocean, their roles in ecology and biogeochemistry, and approaches that can be used to analyse them from chemistry, biology, modelling and data science. Although microbial-derived metabolites account for only a minor fraction of the total reservoir of marine dissolved organic carbon, their flux and fate underpins the central role of the ocean in sustaining life on Earth., (© 2022. Springer Nature Limited.)

Genes of unknown function are among the biggest challenges in molecular biology, especially in microbial systems, where 40-60% of the predicted genes are unknown. Despite previous attempts, systematic approaches to include the unknown fraction into analytical workflows are still lacking. Here, we present a conceptual framework, its translation into the computational workflow AGNOSTOS and a demonstration on how we can bridge the known-unknown gap in genomes and metagenomes. By analyzing 415,971,742 genes predicted from 1749 metagenomes and 28,941 bacterial and archaeal genomes, we quantify the extent of the unknown fraction, its diversity, and its relevance across multiple organisms and environments. The unknown sequence space is exceptionally diverse, phylogenetically more conserved than the known fraction and predominantly taxonomically restricted at the species level. From the 71 M genes identified to be of unknown function, we compiled a collection of 283,874 lineage-specific genes of unknown function for Cand . Patescibacteria (also known as Candidate Phyla Radiation, CPR), which provides a significant resource to expand our understanding of their unusual biology. Finally, by identifying a target gene of unknown function for antibiotic resistance, we demonstrate how we can enable the generation of hypotheses that can be used to augment experimental data., Competing Interests: CV, MS, SA, AB, PB, EC, TD, CD, AE, RF, RK, AM, PS, KS, MS, FG, AF No competing interests declared, (© 2022, Vanni et al.)

tRNA is the most extensively modified RNA in cells. On average, a bacterial tRNA contains 8 modifications per molecule and a eukaryotic tRNA contains 13 modifications per molecule. Recent studies reveal that tRNA modifications are highly dynamic and respond extensively to environmental conditions. Functions of tRNA modification dynamics include enhanced, on-demand decoding of specific codons in response genes and regulation of tRNA fragment biogenesis. This review summarizes recent advances in the studies of tRNA modification dynamics in biological processes, tRNA modification erasers, and human-associated bacteria. Furthermore, we use the term "metaepitranscriptomics" to describe the potential and approach of tRNA modification studies in natural biological communities such as microbiomes. tRNA is highly modified in cells, and tRNA modifications respond extensively to environmental conditions to enhance translation of specific genes and produce tRNA fragments on demand. We review recent advances in tRNA sequencing methods, tRNA modification dynamics in biological processes, and tRNA modification studies in natural communities such as the microbiomes., Competing Interests: Declaration of interests Tao Pan is a co-founder of 4SR Biosciences., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Since the first discovery of reverse transcriptase in bacteria, and later in archaea, bacterial and archaeal retroelements have been defined by their common enzyme that coordinates diverse functions. Yet, evolutionary refinement has produced distinct retroelements across the tree of microbial life that are perhaps best described in terms of their programmed RNA-a compact sequence that preserves core information for a sophisticated mechanism. From this perspective, reverse transcriptase has been selected as the modular tool for carrying out nature's instructions in various RNA templates. Beneficial retroelements-those that can provide a fitness advantage to their host-evolved to their extant forms in a wide array of microorganisms and their viruses, spanning nearly all habitats. Within each specialized retroelement class, several universal features seem to be shared across diverse taxa, while specific functional and mechanistic insights are based on only a few model retroelement systems from clinical isolates. Currently, little is known about the diversity of cellular functions and ecological significance of retroelements across different biomes. With increasing availability of isolate, metagenome-assembled, and single-amplified genomes, the taxonomic and functional breadth of prokaryotic retroelements is coming into clearer view. This review explores the recently characterized classes of beneficial, yet accessory retroelements of bacteria and archaea. We describe how these specialized mechanisms exploit a form of fixed mobility, whereby the retroelements do not appear to proliferate selfishly throughout the genome. Moreover, we discuss computational approaches for systematic identification of retroelements from vast sequence repositories and highlight recent discoveries in terms of their apparent distribution and ecological significance in nature. Lastly, we present a new perspective on the eco-evolutionary significance of these genetic elements in marine bacteria and demonstrate approaches that enable the characterization of their environmental diversity through metagenomics., (© 2022. The Author(s).)

Pseudouridine (Ψ) is an abundant mRNA modification in mammalian transcriptome, but its functions have remained elusive due to the difficulty of transcriptome-wide mapping. We develop a nanopore native RNA sequencing method for quantitative Ψ prediction (NanoPsu) that utilizes native content training, machine learning modeling, and single-read linkage analysis. Biologically, we find interferon inducible Ψ modifications in interferon-stimulated gene transcripts which are consistent with a role of Ψ in enabling efficacy of mRNA vaccines., (© 2021. The Author(s).)

Background & Aims: Perturbations in the early-life gut microbiome are associated with increased risk for complex immune disorders like inflammatory bowel diseases. We previously showed that maternal antibiotic-induced gut dysbiosis vertically transmitted to offspring increases experimental colitis risk in interleukin (IL) 10 gene deficient (IL10 -/- ) mice, a finding that may result from the loss/lack of essential microbes needed for appropriate immunologic education early in life. Here, we aimed to identify key microbes required for proper development of the early-life gut microbiome that decrease colitis risk in genetically susceptible animals., Methods: Metagenomic sequencing followed by reconstruction of metagenome-assembled genomes was performed on fecal samples of IL10 -/- mice with and without antibiotic-induced dysbiosis to identify potential missing microbial members needed for immunologic education. One high-value target strain was then engrafted early and/or late into the gut microbiomes of IL10 -/- mice with antibiotic-induced dysbiosis., Results: Early-, but not late-, life engraftment of a single dominant Bacteroides strain of non-antibiotic-treated IL10 -/- mice was sufficient to restore the development of the gut microbiome, promote immune tolerance, and prevent colitis in IL10 -/- mice that had antibiotic-induced dysbiosis., Conclusions: Restitution of a keystone microbial strain missing in the early-life antibiotic-induced gut dysbiosis results in recovery of the microbiome, proper development of immune tolerance, and reduced risk for colitis in genetically prone hosts., (Copyright © 2021 AGA Institute. All rights reserved.)

We introduce STrain Resolution ON assembly Graphs (STRONG), which identifies strains de novo, from multiple metagenome samples. STRONG performs coassembly, and binning into metagenome assembled genomes (MAGs), and stores the coassembly graph prior to variant simplification. This enables the subgraphs and their unitig per-sample coverages, for individual single-copy core genes (SCGs) in each MAG, to be extracted. A Bayesian algorithm, BayesPaths, determines the number of strains present, their haplotypes or sequences on the SCGs, and abundances. STRONG is validated using synthetic communities and for a real anaerobic digestor time series generates haplotypes that match those observed from long Nanopore reads., (© 2021. The Author(s).)

Antimicrobial resistance (AMR) in drinking water has received less attention than its counterparts in the urban water cycle. While culture-based techniques or gene-centric PCR have been used to probe the impact of treatment approaches (e.g., disinfection) on AMR in drinking water, to our knowledge there is no systematic comparison of AMR trait distribution and prevalence between disinfected and disinfectant residual-free drinking water systems. We used metagenomics to assess the associations between disinfectant residuals and AMR prevalence and its host association in full-scale drinking water distribution systems (DWDSs) with and without disinfectant residuals. While the differences in AMR profiles between DWDSs were associated with the presence or absence of disinfectant, they were also associated with overall water chemistry and more importantly with microbial community structure. AMR genes and mechanisms differentially abundant in disinfected systems were primarily associated with nontuberculous mycobacteria (NTM). Finally, evaluation of metagenome assembled genomes (MAGs) also suggests that NTM possessing AMR genes conferring intrinsic resistance to key antibiotics were prevalent in disinfected systems, whereas such NTM genomes were not detected in disinfectant residual free DWDSs. Altogether, our findings provide insights into the drinking water resistome and its association with potential opportunistic pathogens, particularly in systems with disinfectant residual., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: The increasing availability of microbial genomes and environmental shotgun metagenomes provides unprecedented access to the genomic differences within related bacteria. The human oral microbiome with its diverse habitats and abundant, relatively well-characterized microbial inhabitants presents an opportunity to investigate bacterial population structures at an ecosystem scale., Results: Here, we employ a metapangenomic approach that combines public genomes with Human Microbiome Project (HMP) metagenomes to study the diversity of microbial residents of three oral habitats: tongue dorsum, buccal mucosa, and supragingival plaque. For two exemplar taxa, Haemophilus parainfluenzae and the genus Rothia, metapangenomes reveal distinct genomic groups based on shared genome content. H. parainfluenzae genomes separate into three distinct subgroups with differential abundance between oral habitats. Functional enrichment analyses identify an operon encoding oxaloacetate decarboxylase as diagnostic for the tongue-abundant subgroup. For the genus Rothia, grouping by shared genome content recapitulates species-level taxonomy and habitat preferences. However, while most R. mucilaginosa are restricted to the tongue as expected, two genomes represent a cryptic population of R. mucilaginosa in many buccal mucosa samples. For both H. parainfluenzae and the genus Rothia, we identify not only limitations in the ability of cultivated organisms to represent populations in their native environment, but also specifically which cultivar gene sequences are absent or ubiquitous., Conclusions: Our findings provide insights into population structure and biogeography in the mouth and form specific hypotheses about habitat adaptation. These results illustrate the power of combining metagenomes and pangenomes to investigate the ecology and evolution of bacteria across analytical scales.

Introduction: Microbial residents of the human oral cavity have long been a major focus of microbiology due to their influence on host health and intriguing patterns of site specificity amidst the lack of dispersal limitation. However, the determinants of niche partitioning in this habitat are yet to be fully understood, especially among taxa that belong to recently discovered branches of microbial life., Results: Here, we assemble metagenomes from tongue and dental plaque samples from multiple individuals and reconstruct 790 non-redundant genomes, 43 of which resolve to TM7, a member of the Candidate Phyla Radiation, forming six monophyletic clades that distinctly associate with either plaque or tongue. Both pangenomic and phylogenomic analyses group tongue-specific clades with other host-associated TM7 genomes. In contrast, plaque-specific TM7 group with environmental TM7 genomes. Besides offering deeper insights into the ecology, evolution, and mobilome of cryptic members of the oral microbiome, our study reveals an intriguing resemblance between dental plaque and non-host environments indicated by the TM7 evolution, suggesting that plaque may have served as a stepping stone for environmental microbes to adapt to host environments for some clades of microbes. Additionally, we report that prophages are widespread among oral-associated TM7, while absent from environmental TM7, suggesting that prophages may have played a role in adaptation of TM7 to the host environment., Conclusions: Our data illuminate niche partitioning of enigmatic members of the oral cavity, including TM7, SR1, and GN02, and provide genomes for poorly characterized yet prevalent members of this biome, such as uncultivated Flavobacteriaceae.

Traditional cultivation approaches in microbiology are labor-intensive, low-throughput, and yield biased sampling of environmental microbes due to ecological and evolutionary factors. New strategies are needed for ample representation of rare taxa and slow-growers that are often outcompeted by fast-growers in cultivation experiments. Here we describe a microfluidic platform that anaerobically isolates and cultivates microbial cells in millions of picoliter droplets and automatically sorts them based on colony density to enhance slow-growing organisms. We applied our strategy to a fecal microbiota transplant (FMT) donor stool using multiple growth media, and found significant increase in taxonomic richness and larger representation of rare and clinically relevant taxa among droplet-grown cells compared to conventional plates. Furthermore, screening the FMT donor stool for antibiotic resistance revealed 21 populations that evaded detection in plate-based assessment of antibiotic resistance. Our method improves cultivation-based surveys of diverse microbiomes to gain deeper insights into microbial functioning and lifestyles., Competing Interests: WW, MT, AW, CC, YS, EC, AE No competing interests declared, ST Savaş Tay is a founder and equity holder of BiomeSense Inc., (© 2020, Watterson et al.)

Limiting microbial growth during drinking water distribution is achieved either by maintaining a disinfectant residual or through nutrient limitation without using a disinfectant. The impact of these contrasting approaches on the drinking water microbiome is not systematically understood. We use genome-resolved metagenomics to compare the structure, metabolic traits, and population genomes of drinking water microbiome samples from bulk drinking water across multiple full-scale disinfected and non-disinfected drinking water systems. Microbial communities cluster at the structural- and functional potential-level based on the presence/absence of a disinfectant residual. Disinfectant residual alone explained 17 and 6.5% of the variance in structure and functional potential of the drinking water microbiome, respectively, despite including multiple drinking water systems with variable source waters and source water communities and treatment strategies. The drinking water microbiome is structurally and functionally less diverse and variable across disinfected compared to non-disinfected systems. While bacteria were the most abundant domain, archaea and eukaryota were more abundant in non-disinfected and disinfected systems, respectively. Community-level differences in functional potential were driven by enrichment of genes associated with carbon and nitrogen fixation in non-disinfected systems and γ-aminobutyrate metabolism in disinfected systems likely associated with the recycling of amino acids. Genome-level analyses for a subset of phylogenetically-related microorganisms suggests that disinfection selects for microorganisms capable of using fatty acids, presumably from microbial decay products, via the glyoxylate cycle. Overall, we find that disinfection exhibits systematic selective pressures on the drinking water microbiome and may select for microorganisms able to utilize microbial decay products originating from disinfection-inactivated microorganisms. Video abstract.

Sewage overflows, agricultural runoff, and stormwater discharges introduce fecal pollution into surface waters. Distinguishing these sources is critical for evaluating water quality and formulating remediation strategies. With the falling costs of sequencing, microbial community-based water quality assessment tools are under development. However, their application is limited by the need to build reference libraries, which requires extensive sampling of sources and bioinformatic expertise. Here, we introduce FORest Enteric Source IdentifiCation (FORENSIC; https://forensic.sfs.uwm.edu/), an online, library-independent source tracking platform based on random forest classification and 16S rRNA gene amplicon sequences to identify in environmental samples common fecal contamination sources, including humans, domestic pets, and agricultural animals. FORENSIC relies on a broad reference signature database of Bacteroidales and Clostridiales , two predominant bacterial groups that have coevolved with their hosts. As a result, these groups demonstrate cohesive and reliable assemblage patterns within mammalian species or among species sharing the same diet/physiology. We created a scalable and extensible platform that we tested for global applicability using samples collected in distant geographic locations. This Web application offers a fast and intuitive approach for fecal source identification, particularly in sewage-contaminated waters. IMPORTANCE FORENSIC is an online platform to identify sources of fecal pollution without the need to create reference libraries. FORENSIC is based on the ability of random forest classification to extract cohesive source microbial signatures to create classifiers despite individual variability and to detect the signatures in environmental samples. We primarily focused on defining sewage signals, which are associated with a high human health risk in polluted waters. To test for fecal contamination sources, the platform only requires paired-end reads targeting the V4 or V6 regions of the 16S rRNA gene. We demonstrated that we could use V4V5 reads trimmed to the V4 positions to generate the reference signature. The systematic workflow we describe to create and validate the signatures could be applied to many disciplines. With the increasing gap between advancing technology and practical applications, this platform makes sequence-based water quality assessments accessible to the public health and water resource communities., (Copyright © 2020 Roguet et al.)

Genomes are an integral component of the biological information about an organism; thus, the more complete the genome, the more informative it is. Historically, bacterial and archaeal genomes were reconstructed from pure (monoclonal) cultures, and the first reported sequences were manually curated to completion. However, the bottleneck imposed by the requirement for isolates precluded genomic insights for the vast majority of microbial life. Shotgun sequencing of microbial communities, referred to initially as community genomics and subsequently as genome-resolved metagenomics, can circumvent this limitation by obtaining metagenome-assembled genomes (MAGs); but gaps, local assembly errors, chimeras, and contamination by fragments from other genomes limit the value of these genomes. Here, we discuss genome curation to improve and, in some cases, achieve complete (circularized, no gaps) MAGs (CMAGs). To date, few CMAGs have been generated, although notably some are from very complex systems such as soil and sediment. Through analysis of about 7000 published complete bacterial isolate genomes, we verify the value of cumulative GC skew in combination with other metrics to establish bacterial genome sequence accuracy. The analysis of cumulative GC skew identified potential misassemblies in some reference genomes of isolated bacteria and the repeat sequences that likely gave rise to them. We discuss methods that could be implemented in bioinformatic approaches for curation to ensure that metabolic and evolutionary analyses can be based on very high-quality genomes., (© 2020 Chen et al.; Published by Cold Spring Harbor Laboratory Press.)

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world's countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome.

Members of the SAR11 order Pelagibacterales dominate the surface oceans. Their extensive diversity challenges emerging operational boundaries defined for microbial 'species' and complicates efforts of population genetics to study their evolution. Here, we employed single-amino acid variants (SAAVs) to investigate ecological and evolutionary forces that maintain the genomic heterogeneity within ubiquitous SAR11 populations we accessed through metagenomic read recruitment using a single isolate genome. Integrating amino acid and protein biochemistry with metagenomics revealed that systematic purifying selection against deleterious variants governs non-synonymous variation among very closely related populations of SAR11. SAAVs partitioned metagenomes into two main groups matching large-scale oceanic current temperatures, and six finer proteotypes that connect distant oceanic regions. These findings suggest that environmentally-mediated selection plays a critical role in the journey of cosmopolitan surface ocean microbial populations, and the idea 'everything is everywhere but the environment selects' has credence even at the finest resolutions., Competing Interests: TD, EK, OK, OE, IU, MR, SG, AE No competing interests declared, (© 2019, Delmont et al.)

IgA is prominently secreted at mucosal surfaces and coats a fraction of the commensal microbiota, a process that is critical for intestinal homeostasis. However, the mechanisms of IgA induction and the molecular targets of these antibodies remain poorly understood, particularly in humans. Here, we demonstrate that microbiota from a subset of human individuals encode two protein "superantigens" expressed on the surface of commensal bacteria of the family Lachnospiraceae such as Ruminococcus gnavus that bind IgA variable regions and stimulate potent IgA responses in mice. These superantigens stimulate B cells expressing human VH3 or murine VH5/6/7 variable regions and subsequently bind their antibodies, allowing these microbial organisms to become highly coated with IgA in vivo. These findings demonstrate a previously unappreciated role for commensal superantigens in host-microbiota interactions. Furthermore, as superantigen-expressing strains show an uneven distribution across human populations, they should be systematically considered in studies evaluating human B cell responses and microbiota during homeostasis and disease., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)