Bacterial host factors regulate the infection cycle of bacteriophages. Except for some well-studied host factors (e.g., receptors or restriction-modification systems), the contribution of the rest of the host genome on phage infection remains poorly understood. We developed PHAGEPACK, a pooled assay that systematically and comprehensively measures each host-gene impact on phage fitness. PHAGEPACK combines CRISPR interference with phage packaging to link host perturbation to phage fitness during active infection. Using PHAGEPACK, we constructed a genome-wide map of genes impacting T7 phage fitness in permissive E. coli, revealing pathways previously unknown to affect phage packaging. When applied to the non-permissive E. coli O121, PHAGEPACK identified pathways leading to host resistance; their removal increased phage susceptibility up to a billion-fold. Bioinformatic analysis indicates phage genomes carry homologs or truncations of key host factors, potentially for fitness advantage. In summary, PHAGEPACK offers valuable insights into phage-host interactions, phage evolution, and bacterial resistance.
Single-cell genetic heterogeneity is ubiquitous in microbial populations and an important aspect of microbial biology; however, we lack a broadly applicable and accessible method to study this heterogeneity in microbial populations. Here, we show a simple, robust and generalizable method for high-throughput single-cell sequencing of target genetic loci in diverse microbes using simple droplet microfluidics devices (droplet targeted amplicon sequencing; DoTA-seq). DoTA-seq serves as a platform to perform diverse assays for single-cell genetic analysis of microbial populations. Using DoTA-seq, we demonstrate the ability to simultaneously track the prevalence and taxonomic associations of >10 antibiotic-resistance genes and plasmids within human and mouse gut microbial communities. This workflow is a powerful and accessible platform for high-throughput single-cell sequencing of diverse microbial populations.
Viruses are increasingly being recognized as important components of human and environmental microbiomes. However, viruses in microbiomes remain difficult to study because of the difficulty in culturing them and the lack of sufficient model systems. As a result, computational methods for identifying and analyzing uncultivated viral genomes from metagenomes have attracted significant attention. Such bioinformatics approaches facilitate the screening of viruses from enormous sequencing datasets originating from various environments. Though many tools and databases have been developed for advancing the study of viruses from metagenomes, there is a lack of integrated tools enabling a comprehensive workflow and analyses platform encompassing all the diverse segments of virus studies. Here, we developed ViWrap, a modular pipeline written in Python. ViWrap combines the power of multiple tools into a single platform to enable various steps of virus analysis, including identification, annotation, genome binning, species- and genus-level clustering, assignment of taxonomy, prediction of hosts, characterization of genome quality, comprehensive summaries, and intuitive visualization of results. Overall, ViWrap enables a standardized and reproducible pipeline for both extensive and stringent characterization of viruses from metagenomes, viromes, and microbial genomes. Our approach has flexibility in using various options for diverse applications and scenarios, and its modular structure can be easily amended with additional functions as necessary. ViWrap is designed to be easily and widely used to study viruses in human and environmental systems. ViWrap is publicly available via GitHub (https://github.com/AnantharamanLab/ViWrap). A detailed description of the software, its usage, and interpretation of results can be found on the website.
The arginine dihydrolase pathway ( arc operon) present in a subset of diverse human gut species enables arginine catabolism. This specialized metabolic pathway can alter environmental pH and nitrogen availability, which in turn could shape gut microbiota inter-species interactions. By exploiting synthetic control of gene expression, we investigated the role of the arc operon in probiotic Escherichia coli Nissle 1917 on human gut community assembly and health-relevant metabolite profiles in vitro and in the murine gut. By stabilizing environmental pH, the arc operon reduced variability in community composition across different initial pH perturbations. The abundance of butyrate producing bacteria were altered in response to arc operon activity and butyrate production was enhanced in a physiologically relevant pH range. While the presence of the arc operon altered community dynamics, it did not impact production of short chain fatty acids. Dynamic computational modeling of pH-mediated interactions reveals the quantitative contribution of this mechanism to community assembly. In sum, our framework to quantify the contribution of molecular pathways and mechanism modalities on microbial community dynamics and functions could be applied more broadly.
Viruses, the majority of which are uncultivated, are among the most abundant biological entities on Earth. From altering microbial physiology to driving community dynamics, viruses are fundamental members of microbiomes. While the number of studies leveraging viral metagenomics (viromics) for studying uncultivated viruses is growing, standards for viromics research are lacking. Viromics can utilize computational discovery of viruses from total metagenomes of all community members (hereafter metagenomes) or use physical separation of virus-specific fractions (hereafter viromes). However, differences in the recovery and interpretation of viruses from metagenomes and viromes obtained from the same samples remain understudied.
Marine biofilms are complex communities of microorganisms that play a crucial ecological role in oceans. Although prokaryotes are the dominant members of these biofilms, little is known about their interactions with viruses. By analysing publicly available and newly sequenced metagenomic data, we identified 2446 virus-prokaryote connections in 84 marine biofilms. Most of these connections were between the bacteriophages in the Uroviricota phylum and the bacteria of Proteobacteria, Cyanobacteria and Bacteroidota. The network of virus-host pairs is complex; a single virus can infect multiple prokaryotic populations or a single prokaryote is susceptible to several viral populations. Analysis of genomes of paired prokaryotes and viruses revealed the presence of 425 putative auxiliary metabolic genes (AMGs), 239 viral genes related to restriction-modification (RM) systems and 38,538 prokaryotic anti-viral defence-related genes involved in 15 defence systems. Transcriptomic evidence from newly established biofilms revealed the expression of viral genes, including AMGs and RM, and prokaryotic defence systems, indicating the active interplay between viruses and prokaryotes. A comparison between biofilms and seawater showed that biofilm prokaryotes have more abundant defence genes than seawater prokaryotes, and the defence gene composition differs between biofilms and the surrounding seawater. Overall, our study unveiled active viruses in natural biofilms and their complex interplay with prokaryotes, which may result in the blooming of defence strategists in biofilms. The detachment of bloomed defence strategists may reduce the infectivity of viruses in seawater and result in the emergence of a novel role of marine biofilms.
No abstract available.
Sulfate/sulfite-reducing microorganisms (SRM) are ubiquitous in nature, driving the global sulfur cycle. A hallmark of SRM is the dissimilatory sulfite reductase encoded by the genes dsrAB. Based on analysis of 950 mainly metagenome-derived dsrAB-carrying genomes, we redefine the global diversity of microorganisms with the potential for dissimilatory sulfate/sulfite reduction and uncover genetic repertoires that challenge earlier generalizations regarding their mode of energy metabolism. We show: (i) 19 out of 23 bacterial and 2 out of 4 archaeal phyla harbor uncharacterized SRM, (ii) four phyla including the Desulfobacterota harbor microorganisms with the genetic potential to switch between sulfate/sulfite reduction and sulfur oxidation, and (iii) the combination as well as presence/absence of different dsrAB-types, dsrL-types and dsrD provides guidance on the inferred direction of dissimilatory sulfur metabolism. We further provide an updated dsrAB database including > 60% taxonomically resolved, uncultured family-level lineages and recommendations on existing dsrAB-targeted primers for environmental surveys. Our work summarizes insights into the inferred ecophysiology of newly discovered SRM, puts SRM diversity into context of the major recent changes in bacterial and archaeal taxonomy, and provides an up-to-date framework to study SRM in a global context.
No abstract available.
Multiple sclerosis (MS) is a complex autoimmune disease in which both the roles of genetic susceptibility and environmental/microbial factors have been investigated. More than 200 genetic susceptibility variants have been identified along with the dysbiosis of gut microbiota, both independently have been shown to be associated with MS. We hypothesize that MS patients harboring genetic susceptibility variants along with gut microbiome dysbiosis are at a greater risk of exhibiting the disease. We investigated the genetic risk score for MS in conjunction with gut microbiota in the same cohort of 117 relapsing remitting MS (RRMS) and 26 healthy controls. DNA samples were genotyped using Illumina's Infinium Immuno array-24 v2 chip followed by calculating genetic risk score and the microbiota was determined by sequencing the V4 hypervariable region of the 16S rRNA gene. We identified two clusters of MS patients, Cluster A and B, both having a higher genetic risk score than the control group. However, the MS cases in cluster B not only had a higher genetic risk score but also showed a distinct gut microbiome than that of cluster A. Interestingly, cluster A which included both healthy control and MS cases had similar gut microbiome composition. This could be due to (i) the non-active state of the disease in that group of MS patients at the time of fecal sample collection and/or (ii) the restoration of the gut microbiome post disease modifying therapy to treat the MS. Our study showed that there seems to be an association between genetic risk score and gut microbiome dysbiosis in triggering the disease in a small cohort of MS patients. The MS Cluster A who have a higher genetic risk score but microbiome profile similar to that of healthy controls could be due to the remitting phase of the disease or due to the effect of disease modifying therapies.
Visible surface films, termed slicks, can extensively cover freshwater and marine ecosystems, with coastal regions being particularly susceptible to their presence. The sea-surface microlayer (SML), the upper 1-mm at the air-water interface in slicks (herein slick SML) harbors a distinctive bacterial community, but generally little is known about SML viruses. Using flow cytometry, metagenomics, and cultivation, we characterized viruses and bacteria in a brackish slick SML in comparison to non-slick SML as well as seawater below slick and non-slick areas (subsurface water = SSW). Size-fractionated filtration of all samples distinguished viral attachment to hosts and particles. The slick SML contained higher abundances of virus-like particles, prokaryotic cells, and dissolved organic carbon compared to non-slick SML and SSW. The community of 428 viral operational taxonomic units (vOTUs), 426 predicted as lytic, distinctly differed across all size fractions in the slick SML compared to non-slick SML and SSW. Specific metabolic profiles of bacterial metagenome-assembled genomes and isolates in the slick SML included a prevalence of genes encoding motility and carbohydrate-active enzymes (CAZymes). Several vOTUs were enriched in slick SML, and many virus variants were associated with particles. Nine vOTUs were only found in slick SML, six of them being targeted by slick SML-specific clustered-regularly interspaced short palindromic repeats (CRISPR) spacers likely originating from Gammaproteobacteria. Moreover, isolation of three previously unknown lytic phages for Alishewanella sp. and Pseudoalteromonas tunicata, abundant and actively replicating slick SML bacteria, suggests that viral activity in slicks contributes to biogeochemical cycling in coastal ecosystems.
Streptococcus agalactiae (Group B Streptococcus , GBS) is a commensal Gram-positive bacterium found in the human gastrointestinal and urogenital tracts. Much of what is known about GBS relates to the diseases it causes in pregnant people and neonates. However, GBS is a common cause of disease in the general population with 90% of GBS mortality occurring in non-pregnant people. There are limited data about the predisposing factors for GBS and the reservoirs in the body. To gain an understanding of the determinants of gastrointestinal GBS carriage, we used stool samples and associated metadata to determine the prevalence and abundance of GBS in the gut microbiome of adults and find risk factors for GBS status.
Restriction of methionine (MR), a sulfur-containing essential amino acid, has been reported to repress cancer growth and improve therapeutic responses in several preclinical settings. However, how MR impacts cancer progression in the context of the intact immune system is unknown. Here we report that while inhibiting cancer growth in immunocompromised mice, MR reduces T cell abundance, exacerbates tumour growth and impairs tumour response to immunotherapy in immunocompetent male and female mice. Mechanistically, MR reduces microbial production of hydrogen sulfide, which is critical for immune cell survival/activation. Dietary supplementation of a hydrogen sulfide donor or a precursor, or methionine, stimulates antitumour immunity and suppresses tumour progression. Our findings reveal an unexpected negative interaction between MR, sulfur deficiency and antitumour immunity and further uncover a vital role of gut microbiota in mediating this interaction. Our study suggests that any possible anticancer benefits of MR require careful consideration of both the microbiota and the immune system.
Many universally and conditionally important genes are genomically aggregated within clusters. Here, we introduce fai and zol, which together enable large-scale comparative analysis of different types of gene clusters and mobile-genetic elements (MGEs), such as biosynthetic gene clusters (BGCs) or viruses. Fundamentally, they overcome a current bottleneck to reliably perform comprehensive orthology inference at large scale across broad taxonomic contexts and thousands of genomes. First, fai allows the identification of orthologous or homologous instances of a query gene cluster of interest amongst a database of target genomes. Subsequently, zol enables reliable, context-specific inference of protein-encoding ortholog groups for individual genes across gene cluster instances. In addition, zol performs functional annotation and computes a variety of statistics for each inferred ortholog group. These programs are showcased through application to: (i) longitudinal tracking of a virus in metagenomes, (ii) discovering novel population-genetic insights of two common BGCs in a fungal species, and (iii) uncovering large-scale evolutionary trends of a virulence-associated gene cluster across thousands of genomes from a diverse bacterial genus.
The sulfur-containing amino acid cysteine is abundant in the environment, including in freshwater lakes. Biological cysteine degradation can result in hydrogen sulfide (HS), a toxic and ecologically relevant compound that is a central player in biogeochemical cycling in aquatic environments. Here, we investigated the ecological significance of cysteine in oxic freshwater, using isolated cultures, controlled experiments, and multiomics. We screened bacterial isolates enriched from natural lake water for their ability to produce HS when provided cysteine. We identified 29 isolates (Bacteroidota, Proteobacteria, and Actinobacteria) that produced HS. To understand the genomic and genetic basis for cysteine degradation and HS production, we further characterized three isolates using whole-genome sequencing (using a combination of short-read and long-read sequencing) and tracked cysteine and HS levels over their growth ranges: Stenotrophomonas maltophilia (Gammaproteobacteria), S. bentonitica (Gammaproteobacteria), and Chryseobacterium piscium (Bacteroidota). Cysteine decreased and HS increased, and all three genomes had genes involved in cysteine degradation. Finally, to assess the presence of these organisms and genes in the environment, we surveyed a 5-year time series of metagenomic data from the same isolation source (Lake Mendota, Madison, WI, USA) and identified their presence throughout the time series. Overall, our study shows that diverse isolated bacterial strains can use cysteine and produce HS under oxic conditions, and we show evidence using metagenomic data that this process may occur more broadly in natural freshwater lakes. Future considerations of sulfur cycling and biogeochemistry in oxic environments should account for HS production from the degradation of organosulfur compounds. IMPORTANCE Hydrogen sulfide (HS), a naturally occurring gas with both biological and abiotic origins, can be toxic to living organisms. In aquatic environments, HS production typically originates from anoxic (lacking oxygen) environments, such as sediments, or the bottom layers of thermally stratified lakes. However, the degradation of sulfur-containing amino acids such as cysteine, which all cells and life forms rely on, can be a source of ammonia and HS in the environment. Unlike other approaches for biological HS production such as dissimilatory sulfate reduction, cysteine degradation can occur in the presence of oxygen. Yet, little is known about how cysteine degradation influences sulfur availability and cycling in freshwater lakes. In our study, we identified diverse bacteria from a freshwater lake that can produce HS in the presence of O. Our study highlights the ecological importance of oxic HS production in natural ecosystems and necessitates a change in our outlook on sulfur biogeochemistry.
Viral metagenomics enables the detection, characterization, and quantification of viral sequences present in shotgun-sequenced datasets of purified virus-like particles and whole metagenomes. Next generation sequencing (Illumina) derived short single or paired-end read runs are a principal platform for metagenomics, and assembly of short reads allows for the identification of distinguishing viral signatures and complex genomic features for taxonomy and functional annotation. Here we describe the identification and characterization of viral genome sequences, bacteriophages, and eukaryotic viruses, from a cohort of human stool samples, using multiple methods. Following the purification of virus-like particles, sequencing, quality refinement, and genome assembly, we begin the protocol with raw short reads deposited in an open-source nucleotide archive. We highlight the use of VIBRANT, an automated computational tool for the characterization of microbial viruses and their viral community function. Finally, we also describe an alternative assembly-free option of mapping reads to established databases of reference genomes and previously characterized metagenome-assembled viral genomes.
In globally distributed deep-sea hydrothermal vent plumes, microbiomes are shaped by the redox energy landscapes created by reduced hydrothermal vent fluids mixing with oxidized seawater. Plumes can disperse over thousands of kilometers and their characteristics are determined by geochemical sources from vents, e.g., hydrothermal inputs, nutrients, and trace metals. However, the impacts of plume biogeochemistry on the oceans are poorly constrained due to a lack of integrated understanding of microbiomes, population genetics, and geochemistry. Here, we use microbial genomes to understand links between biogeography, evolution, and metabolic connectivity, and elucidate their impacts on biogeochemical cycling in the deep sea. Using data from 36 diverse plume samples from seven ocean basins, we show that sulfur metabolism defines the core microbiome of plumes and drives metabolic connectivity in the microbial community. Sulfur-dominated geochemistry influences energy landscapes and promotes microbial growth, while other energy sources influence local energy landscapes. We further demonstrated the consistency of links among geochemistry, function, and taxonomy. Amongst all microbial metabolisms, sulfur transformations had the highest MW-score, a measure of metabolic connectivity in microbial communities. Additionally, plume microbial populations have low diversity, short migration history, and gene-specific sweep patterns after migrating from background seawater. Selected functions include nutrient uptake, aerobic oxidation, sulfur oxidation for higher energy yields, and stress responses for adaptation. Our findings provide the ecological and evolutionary bases of change in sulfur-driven microbial communities and their population genetics in adaptation to changing geochemical gradients in the oceans.
Deep-sea hydrothermal vents are abundant on the ocean floor and play important roles in ocean biogeochemistry. In vent ecosystems such as hydrothermal plumes, microorganisms rely on reduced chemicals and gases in hydrothermal fluids to fuel primary production and form diverse and complex microbial communities. However, microbial interactions that drive these complex microbiomes remain poorly understood. Here, we use microbiomes from the Guaymas Basin hydrothermal system in the Pacific Ocean to shed more light on the key species in these communities and their interactions. We built metabolic models from metagenomically assembled genomes (MAGs) and infer possible metabolic exchanges and horizontal gene transfer (HGT) events within the community. We highlight possible archaea-archaea and archaea-bacteria interactions and their contributions to the robustness of the community. Cellobiose, D-Mannose 1-phosphate, O, CO, and HS were among the most exchanged metabolites. These interactions enhanced the metabolic capabilities of the community by exchange of metabolites that cannot be produced by any other community member. Archaea from the DPANN group stood out as key microbes, benefiting significantly as acceptors in the community. Overall, our study provides key insights into the microbial interactions that drive community structure and organisation in complex hydrothermal plume microbiomes.
Bacteriophages can adapt to new hosts by altering sequence motifs through recombination or convergent evolution. Where such motifs exist and what fitness advantage they confer remains largely unknown. We report a new method, Bacteriophage Library Informed Sequence Scoring (BLISS), to discover sequence motifs in metagenomic datasets governing phage activity. BLISS uses experimental deep mutational scanning data to create sequence profiles to enable deep mining of metagenomes for functional motifs which are otherwise invisible to searches. We experimentally tested 10,073 BLISS-derived sequence motifs for the receptor-binding protein of the T7 phage. The screen revealed hundreds of T7 variants with novel host specificity with functional motifs sourced from distant families besides other major phyla. Position, substitution and location preferences on T7 dictated different specificities. To demonstrate therapeutic utility, we engineered highly active T7 variants against urinary tract pathogens. BLISS is a powerful tool to unlock the functional potential encoded in phage metagenomes.
Viruses are increasingly being recognized as important components of human and environmental microbiomes. However, viruses in microbiomes remain difficult to study because of difficulty in culturing them and the lack of sufficient model systems. As a result, computational methods for identifying and analyzing uncultivated viral genomes from metagenomes have attracted significant attention. Such bioinformatics approaches facilitate screening of viruses from enormous sequencing datasets originating from various environments. Though many tools and databases have been developed for advancing the study of viruses from metagenomes, there is a lack of integrated tools enabling a comprehensive workflow and analyses platform encompassing all the diverse segments of virus studies. Here, we developed ViWrap, a modular pipeline written in Python. ViWrap combines the power of multiple tools into a single platform to enable various steps of virus analysis including identification, annotation, genome binning, species- and genus-level clustering, assignment of taxonomy, prediction of hosts, characterization of genome quality, comprehensive summaries, and intuitive visualization of results. Overall, ViWrap enables a standardized and reproducible pipeline for both extensive and stringent characterization of viruses from metagenomes, viromes, and microbial genomes. Our approach has flexibility in using various options for diverse applications and scenarios, and its modular structure can be easily amended with additional functions as necessary. ViWrap is designed to be easily and widely used to study viruses in human and environmental systems. ViWrap is publicly available via GitHub ( https://github.com/AnantharamanLab/ViWrap ). A detailed description of the software, its usage, and interpretation of results can be found on the website.
The molecular and ecological factors shaping horizontal gene transfer (HGT) via natural transformation in microbial communities are largely unknown, which is critical for understanding the emergence of antibiotic-resistant pathogens. We investigate key factors shaping HGT in a microbial co-culture by quantifying extracellular DNA release, species growth, and HGT efficiency over time. In the co-culture, plasmid release and HGT efficiency are significantly enhanced than in the respective monocultures. The donor is a key determinant of HGT efficiency as plasmids induce the SOS response, enter a multimerized state, and are released in high concentrations, enabling efficient HGT. However, HGT is reduced in response to high donor lysis rates. HGT is independent of the donor viability state as both live and dead cells transfer the plasmid with high efficiency. In sum, plasmid HGT via natural transformation depends on the interplay of plasmid properties, donor stress responses and lysis rates, and interspecies interactions.
When deep-sea hydrothermal fluids mix with cold oxygenated fluids, minerals precipitate out of solution and form hydrothermal deposits. These actively venting deep-sea hydrothermal deposits support a rich diversity of thermophilic microorganisms which are involved in a range of carbon, sulfur, nitrogen, and hydrogen metabolisms. Global patterns of thermophilic microbial diversity in deep-sea hydrothermal ecosystems have illustrated the strong connectivity between geological processes and microbial colonization, but little is known about the genomic diversity and physiological potential of these novel taxa. Here we explore this genomic diversity in 42 metagenomes from four deep-sea hydrothermal vent fields and a deep-sea volcano collected from 2004 to 2018 and document their potential implications in biogeochemical cycles.
The gut microbiome of vertebrates is capable of numerous biotransformations of bile acids, which are responsible for intestinal lipid digestion and function as key nutrient-signaling molecules. The human liver produces bile acids from cholesterol predominantly in the A/B-cis orientation in which the sterol rings are "kinked", as well as small quantities of A/B-trans oriented "flat" stereoisomers known as "primary allo-bile acids". While the complex multi-step bile acid 7α-dehydroxylation pathway has been well-studied for conversion of "kinked" primary bile acids such as cholic acid (CA) and chenodeoxycholic acid (CDCA) to deoxycholic acid (DCA) and lithocholic acid (LCA), respectively, the enzymatic basis for the formation of "flat" stereoisomers allo-deoxycholic acid (allo-DCA) and allo-lithocholic acid (allo-LCA) by Firmicutes has remained unsolved for three decades. Here, we present a novel mechanism by which Firmicutes generate the "flat" bile acids allo-DCA and allo-LCA. The BaiA1 was shown to catalyze the final reduction from 3-oxo-allo-DCA to allo-DCA and 3-oxo-allo-LCA to allo-LCA. Phylogenetic and metagenomic analyses of human stool samples indicate that BaiP and BaiJ are encoded only in Firmicutes and differ from membrane-associated bile acid 5α-reductases recently reported in Bacteroidetes that indirectly generate allo-LCA from 3-oxo-Δ4-LCA. We further map the distribution of baiP and baiJ among Firmicutes in human metagenomes, demonstrating an increased abundance of the two genes in colorectal cancer (CRC) patients relative to healthy individuals.
Stroke leads to gut bacterial dysbiosis that impacts the post-stroke outcome. The gut microbiome also contains a high abundance of viruses which might play a crucial role in disease progression and recovery by modulating the metabolism of both host and host's gut bacteria. We presently analyzed the virome composition (viruses and phages) by shotgun metagenomics in the fecal samples obtained at 1 day of reperfusion following transient focal ischemia in adult mice. Viral genomes, viral auxiliary metabolic genes, and viral protein networks were compared between stroke and sham conditions (stroke vs sham, exclusive to sham and exclusive to stroke). Following focal ischemia, abundances of 2 viral taxa decreased, and 5 viral taxa increased compared with the sham. Furthermore, the abundance of Clostridia-like phages and Erysipelatoclostridiaceae-like phages were altered in the stroke compared with the sham cohorts. This is the first report to show that the gut virome responds acutely to stroke.
Genome binning has been essential for characterization of bacteria, archaea, and even eukaryotes from metagenomes. Yet, few approaches exist for viruses. We developed vRhyme, a fast and precise software for construction of viral metagenome-assembled genomes (vMAGs). vRhyme utilizes single- or multi-sample coverage effect size comparisons between scaffolds and employs supervised machine learning to identify nucleotide feature similarities, which are compiled into iterations of weighted networks and refined bins. To refine bins, vRhyme utilizes unique features of viral genomes, namely a protein redundancy scoring mechanism based on the observation that viruses seldom encode redundant genes. Using simulated viromes, we displayed superior performance of vRhyme compared to available binning tools in constructing more complete and uncontaminated vMAGs. When applied to 10,601 viral scaffolds from human skin, vRhyme advanced our understanding of resident viruses, highlighted by identification of a Herelleviridae vMAG comprised of 22 scaffolds, and another vMAG encoding a nitrate reductase metabolic gene, representing near-complete genomes post-binning. vRhyme will enable a convention of binning uncultivated viral genomes and has the potential to transform metagenome-based viral ecology.
Recent evidence implicates microbial sulfidogenesis as a potential trigger of colorectal cancer (CRC), highlighting the need for comprehensive knowledge of sulfur metabolism within the human gut. Microbial sulfidogenesis produces genotoxic hydrogen sulfide (HS) in the human colon using inorganic (sulfate) and organic (taurine/cysteine/methionine) substrates; however, the majority of studies have focused on sulfate reduction using dissimilatory sulfite reductases (Dsr).
Temperate phages (prophages) are ubiquitous in nature and persist as dormant components of host cells (lysogenic stage) before activating and lysing the host (lytic stage). Actively replicating prophages contribute to central community processes, such as enabling bacterial virulence, manipulating biogeochemical cycling, and driving microbial community diversification. Recent advances in sequencing technology have allowed for the identification and characterization of diverse phages, yet no approaches currently exist for identifying if a prophage has activated. Here, we present PropagAtE (Prophage Activity Estimator), an automated software tool for estimating if a prophage is in the lytic or lysogenic stage of infection. PropagAtE uses statistical analyses of prophage-to-host read coverage ratios to decipher actively replicating prophages, irrespective of whether prophages were induced or spontaneously activated. We demonstrate that PropagAtE is fast, accurate, and sensitive, regardless of sequencing depth. Application of PropagAtE to prophages from 348 complex metagenomes from human gut, murine gut, and soil environments identified distinct spatial and temporal prophage activation signatures, with the highest proportion of active prophages in murine gut samples. In infants treated with antibiotics or infants without treatment, we identified active prophage populations correlated with specific treatment groups. Within time series samples from the human gut, 11 prophage populations, some encoding the sulfur metabolism gene cysH or a rhuM -like virulence factor, were consistently present over time but not active. Overall, PropagAtE will facilitate accurate representations of viruses in microbiomes by associating prophages with their active roles in shaping microbial communities in nature. IMPORTANCE Viruses that infect bacteria are key components of microbiomes and ecosystems. They can kill and manipulate microorganisms, drive planetary-scale processes and biogeochemical cycling, and influence the structures of entire food networks. Prophages are viruses that can exist in a dormant state within the genome of their host (lysogenic stage) before activating in order to replicate and kill the host (lytic stage). Recent advances have allowed for the identification of diverse viruses in nature, but no approaches exist for characterizing prophages and their stages of infection (prophage activity). We develop and benchmark an automated approach, PropagAtE, to identify the stages of infection of prophages from genomic data. We provide evidence that active prophages vary in identity and abundance across multiple environments and scales. Our approach will enable accurate and unbiased analyses of viruses in microbiomes and ecosystems.
Advances in microbiome science are being driven in large part due to our ability to study and infer microbial ecology from genomes reconstructed from mixed microbial communities using metagenomics and single-cell genomics. Such omics-based techniques allow us to read genomic blueprints of microorganisms, decipher their functional capacities and activities, and reconstruct their roles in biogeochemical processes. Currently available tools for analyses of genomic data can annotate and depict metabolic functions to some extent; however, no standardized approaches are currently available for the comprehensive characterization of metabolic predictions, metabolite exchanges, microbial interactions, and microbial contributions to biogeochemical cycling.
Viruses are diverse biological entities that influence all life. Even with limited genome sizes, viruses can manipulate, drive, steal from, and kill their hosts. The field of virus genomics, using sequencing data to understand viral capabilities, has seen significant innovations in recent years. However, with advancements in metagenomic sequencing and related technologies, the bottleneck to discovering and employing the virosphere has become the analysis of genomes rather than generation. With metagenomics rapidly expanding available data, vital components of virus genomes and features are being overlooked, with the issue compounded by lagging databases and bioinformatics methods. Despite the field moving in a positive direction, there are noteworthy points to keep in mind, from how software-based virus genome predictions are interpreted to what information is overlooked by current standards. In this review, we discuss conventions and ideologies that likely need to be revised while continuing forward in the study of virus genomics.
Polysaccharide utilization loci (PULs) are co-regulated bacterial genes that sense nutrients and enable glycan digestion. Human gut microbiome members, notably Bacteroides, contain numerous PULs that enable glycan utilization and shape ecological dynamics. To investigate the role of PULs on fitness and inter-species interactions, we develop a CRISPR-based genome editing tool to study 23 PULs in Bacteroides uniformis (BU). BU PULs show distinct glycan-degrading functions and transcriptional coordination that enables the population to adapt upon loss of other PULs. Exploiting a BU mutant barcoding strategy, we demonstrate that in vitro fitness and BU colonization in the murine gut are enhanced by deletion of specific PULs and modulated by glycan availability. PULs mediate glycan-dependent interactions with butyrate producers that depend on the degradation mechanism and glycan utilization ability of the butyrate producer. Thus, PULs determine community dynamics and butyrate production and provide a selective advantage or disadvantage depending on the nutritional landscape.
No abstract available.
Viruses are ubiquitous on Earth and are keystone components of environments, ecosystems, and human health. Yet, viruses remain poorly studied because most cannot be isolated in a laboratory. In the field of biogeochemistry, which aims to understand the interactions between biology, geology, and chemistry, there is progress to be made in understanding the different roles played by viruses in nutrient cycling, food webs, and elemental transformations. In this commentary, we outline current microbial ecology frameworks for understanding biogeochemical cycling in aquatic ecosystems. Next, we review some existing experimental and computational techniques that are enabling us to study the role of viruses in biogeochemical cycling, using examples from aquatic environments. Finally, we provide a conceptual model that balances limitations of computational tools when combined with biogeochemistry and ecological data. We envision meeting the grand challenge of understanding how viruses impact biogeochemical cycling by using a multifaceted approach to viral ecology.
Viruses influence the fate of nutrients and human health by killing microorganisms and altering metabolic processes. Organosulfur metabolism and biologically derived hydrogen sulfide play dynamic roles in manifestation of diseases, infrastructure degradation, and essential biological processes. Although microbial organosulfur metabolism is well studied, the role of viruses in organosulfur metabolism is unknown. Here, we report the discovery of 39 gene families involved in organosulfur metabolism encoded by 3,749 viruses from diverse ecosystems, including human microbiomes. The viruses infect organisms from all three domains of life. Six gene families encode for enzymes that degrade organosulfur compounds into sulfide, whereas others manipulate organosulfur compounds and may influence sulfide production. We show that viral metabolic genes encode key enzymatic domains, are translated into protein, and are maintained after recombination, and sulfide provides a fitness advantage to viruses. Our results reveal viruses as drivers of organosulfur metabolism with important implications for human and environmental health.
Microbial sulfur metabolism contributes to biogeochemical cycling on global scales. Sulfur metabolizing microbes are infected by phages that can encode auxiliary metabolic genes (AMGs) to alter sulfur metabolism within host cells but remain poorly characterized. Here we identified 191 phages derived from twelve environments that encoded 227 AMGs for oxidation of sulfur and thiosulfate (dsrA, dsrC/tusE, soxC, soxD and soxYZ). Evidence for retention of AMGs during niche-differentiation of diverse phage populations provided evidence that auxiliary metabolism imparts measurable fitness benefits to phages with ramifications for ecosystem biogeochemistry. Gene abundance and expression profiles of AMGs suggested significant contributions by phages to sulfur and thiosulfate oxidation in freshwater lakes and oceans, and a sensitive response to changing sulfur concentrations in hydrothermal environments. Overall, our study provides fundamental insights on the distribution, diversity, and ecology of phage auxiliary metabolism associated with sulfur and reinforces the necessity of incorporating viral contributions into biogeochemical configurations.
Rheumatoid arthritis (RA) is an autoimmune disease characterized in seropositive individuals by the presence of anti-cyclic citrullinated protein (CCP) antibodies. RA is linked to the intestinal microbiota, yet the association of microbes with CCP serology and their contribution to RA is unclear. We describe intestinal phage communities of individuals at risk for developing RA, with or without anti-CCP antibodies, whose first-degree relatives have been diagnosed with RA. We show that at-risk individuals harbor intestinal phage compositions that diverge based on CCP serology, are dominated by Streptococcaceae, Bacteroidaceae, and Lachnospiraceae phages, and may originate from disparate ecosystems. These phages encode unique repertoires of auxiliary metabolic genes, which associate with anti-CCP status, suggesting that these phages directly influence the metabolic and immunomodulatory capability of the microbiota. This work sets the stage for the use of phages as preclinical biomarkers and provides insight into a possible microbial-based causation of RA disease development.
Bile acids are detergent molecules that solubilize dietary lipids and lipid-soluble vitamins. Humans synthesize bile acids with α-orientation hydroxyl groups which can be biotransformed by gut microbiota to toxic, hydrophobic bile acids, such as deoxycholic acid (DCA). Gut microbiota can also convert hydroxyl groups from the α-orientation through an oxo-intermediate to the β-orientation, resulting in more hydrophilic, less toxic bile acids. This interconversion is catalyzed by regio- (C-3 vs. C-7) and stereospecific (α vs. β) hydroxysteroid dehydrogenases (HSDHs). So far, genes encoding the urso- (7α-HSDH & 7β-HSDH) and iso- (3α-HSDH & 3β-HSDH) bile acid pathways have been described. Recently, multiple human gut clostridia were reported to encode 12α-HSDH, which interconverts DCA and 12-oxolithocholic acid (12-oxoLCA). 12β-HSDH completes the epi-bile acid pathway by converting 12-oxoLCA to the 12β-bile acid denoted epiDCA; however, a gene(s) encoding this enzyme has yet to be identified. We confirmed 12β-HSDH activity in cultures of Clostridium paraputrificum ATCC 25780. From six candidate C. paraputrificum ATCC 25780 oxidoreductase genes, we discovered the first gene (DR024_RS09610) encoding bile acid 12β-HSDH. Phylogenetic analysis revealed unforeseen diversity for 12β-HSDH, leading to validation of two additional bile acid 12β-HSDHs through a synthetic biology approach. By comparison to a previous phylogenetic analysis of 12α-HSDH, we identified the first potential C-12 epimerizing strains: Collinsella tanakaei YIT 12063 and Collinsella stercoris DSM 13279. A Hidden Markov Model search against human gut metagenomes located putative 12β-HSDH genes in about 30% of subjects within the cohorts analyzed, indicating this gene is relevant in the human gut microbiome.
Lake Tanganyika (LT) is the largest tropical freshwater lake, and the largest body of anoxic freshwater on Earth's surface. LT's mixed oxygenated surface waters float atop a permanently anoxic layer and host rich animal biodiversity. However, little is known about microorganisms inhabiting LT's 1470 meter deep water column and their contributions to nutrient cycling, which affect ecosystem-level function and productivity. Here, we applied genome-resolved metagenomics and environmental analyses to link specific taxa to key biogeochemical processes across a vertical depth gradient in LT. We reconstructed 523 unique metagenome-assembled genomes (MAGs) from 34 bacterial and archaeal phyla, including many rarely observed in freshwater lakes. We identified sharp contrasts in community composition and metabolic potential with an abundance of typical freshwater taxa in oxygenated mixed upper layers, and Archaea and uncultured Candidate Phyla in deep anoxic waters. Genomic capacity for nitrogen and sulfur cycling was abundant in MAGs recovered from anoxic waters, highlighting microbial contributions to the productive surface layers via recycling of upwelled nutrients, and greenhouse gases such as nitrous oxide. Overall, our study provides a blueprint for incorporation of aquatic microbial genomics in the representation of tropical freshwater lakes, especially in the context of ongoing climate change, which is predicted to bring increased stratification and anoxia to freshwater lakes.
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Deep-sea hydrothermal plumes are considered natural laboratories for understanding ecological and biogeochemical interactions. Previous studies focused on interactions between microorganisms and inorganic, reduced hydrothermal inputs including sulfur, hydrogen, iron, and manganese. However, little is known about transformations of organic compounds, especially methylated, sulfur-containing compounds, and petroleum hydrocarbons. Here, we reconstructed nine gammaproteobacterial metagenome-assembled genomes, affiliated with Methylococcales, Methylophaga, and Cycloclasticus, from three hydrothermal ecosystems. We present evidence that these three groups have high transcriptional activities of genes encoding cycling of C-compounds, petroleum hydrocarbons, and organic sulfur in hydrothermal plumes. This includes oxidation of methanethiol, the simplest thermochemically-derived organic sulfur, for energy metabolism in Methylococcales and Cycloclasticus. Together with active transcription of genes for thiosulfate and methane oxidation in Methylococcales, these results suggest an adaptive strategy of versatile and simultaneous use of multiple available electron donors. Meanwhile, the first near-complete MAG of hydrothermal Methylophaga aminisulfidivorans and its transcriptional profile point to active chemotaxis targeting small organic compounds. Petroleum hydrocarbon-degrading Cycloclasticus are abundant and active in plumes of oil spills as well as deep-sea vents, suggesting that they are indigenous and effectively respond to stimulus of hydrocarbons in the deep sea. These findings suggest that these three groups of Gammaproteobacteria transform organic carbon and sulfur compounds via versatile and opportunistic metabolism and modulate biogeochemistry in plumes of hydrothermal systems as well as oil spills, thus contributing broad ecological impact to the deep ocean globally.
Methylmercury is a potent bioaccumulating neurotoxin that is produced by specific microorganisms that methylate inorganic mercury. Methylmercury production in diverse anaerobic bacteria and archaea was recently linked to the hgcAB genes. However, the full phylogenetic and metabolic diversity of mercury-methylating microorganisms has not been fully unraveled due to the limited number of cultured experimentally verified methylators and the limitations of primer-based molecular methods. Here, we describe the phylogenetic diversity and metabolic flexibility of putative mercury-methylating microorganisms by hgcAB identification in publicly available isolate genomes and metagenome-assembled genomes (MAGs) as well as novel freshwater MAGs. We demonstrate that putative mercury methylators are much more phylogenetically diverse than previously known and that hgcAB distribution among genomes is most likely due to several independent horizontal gene transfer events. The microorganisms we identified possess diverse metabolic capabilities spanning carbon fixation, sulfate reduction, nitrogen fixation, and metal resistance pathways. We identified 111 putative mercury methylators in a set of previously published permafrost metatranscriptomes and demonstrated that different methylating taxa may contribute to hgcA expression at different depths. Overall, we provide a framework for illuminating the microbial basis of mercury methylation using genome-resolved metagenomics and metatranscriptomics to identify putative methylators based upon hgcAB presence and describe their putative functions in the environment. IMPORTANCE Accurately assessing the production of bioaccumulative neurotoxic methylmercury by characterizing the phylogenetic diversity, metabolic functions, and activity of methylators in the environment is crucial for understanding constraints on the mercury cycle. Much of our understanding of methylmercury production is based on cultured anaerobic microorganisms within the Deltaproteobacteria , Firmicutes , and Euryarchaeota. Advances in next-generation sequencing technologies have enabled large-scale cultivation-independent surveys of diverse and poorly characterized microorganisms from numerous ecosystems. We used genome-resolved metagenomics and metatranscriptomics to highlight the vast phylogenetic and metabolic diversity of putative mercury methylators and their depth-discrete activities in thawing permafrost. This work underscores the importance of using genome-resolved metagenomics to survey specific putative methylating populations of a given mercury-impacted ecosystem.
Viruses are central to microbial community structure in all environments. The ability to generate large metagenomic assemblies of mixed microbial and viral sequences provides the opportunity to tease apart complex microbiome dynamics, but these analyses are currently limited by the tools available for analyses of viral genomes and assessing their metabolic impacts on microbiomes.
The assembly of single-amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) has led to a surge in genome-based discoveries of members affiliated with Archaea and Bacteria, bringing with it a need to develop guidelines for nomenclature of uncultivated microorganisms. The International Code of Nomenclature of Prokaryotes (ICNP) only recognizes cultures as 'type material', thereby preventing the naming of uncultivated organisms. In this Consensus Statement, we propose two potential paths to solve this nomenclatural conundrum. One option is the adoption of previously proposed modifications to the ICNP to recognize DNA sequences as acceptable type material; the other option creates a nomenclatural code for uncultivated Archaea and Bacteria that could eventually be merged with the ICNP in the future. Regardless of the path taken, we believe that action is needed now within the scientific community to develop consistent rules for nomenclature of uncultivated taxa in order to provide clarity and stability, and to effectively communicate microbial diversity.
Proteobacteria constitute one of the most diverse and abundant groups of microbes on Earth. In productive marine environments like deep-sea hydrothermal systems, Proteobacteria are implicated in autotrophy coupled to sulfur, methane, and hydrogen oxidation, sulfate reduction, and denitrification. Beyond chemoautotrophy, little is known about the ecological significance of poorly studied Proteobacteria lineages that are globally distributed and active in hydrothermal systems. Here we apply multi-omics to characterize 51 metagenome-assembled genomes from three hydrothermal vent plumes in the Pacific and Atlantic Oceans that are affiliated with nine Proteobacteria lineages. Metabolic analyses revealed these organisms to contain a diverse functional repertoire including chemolithotrophic ability to utilize sulfur and C compounds, and chemoorganotrophic ability to utilize environment-derived fatty acids, aromatics, carbohydrates, and peptides. Comparative genomics with marine and terrestrial microbiomes suggests that lineage-associated functional traits could explain niche specificity. Our results shed light on the ecological functions and metabolic strategies of novel Proteobacteria in hydrothermal systems and beyond, and highlight the relationship between genome diversification and environmental adaptation.
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.
Bacteriophages typically have small genomes and depend on their bacterial hosts for replication. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is-to our knowledge-the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR-Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR-Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR-Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth's ecosystems.
Soil microbial activity drives the carbon and nitrogen cycles and is an important determinant of atmospheric trace gas turnover, yet most soils are dominated by microorganisms with unknown metabolic capacities. Even Acidobacteria, among the most abundant bacteria in soil, remain poorly characterized, and functions across groups such as Verrucomicrobia, Gemmatimonadetes, Chloroflexi and Rokubacteria are understudied. Here, we have resolved 60 metagenomic and 20 proteomic data sets from a Mediterranean grassland soil ecosystem and recovered 793 near-complete microbial genomes from 18 phyla, representing around one-third of all microorganisms detected. Importantly, this enabled extensive genomics-based metabolic predictions for these communities. Acidobacteria from multiple previously unstudied classes have genomes that encode large enzyme complements for complex carbohydrate degradation. Alternatively, most microorganisms encode carbohydrate esterases that strip readily accessible methyl and acetyl groups from polymers like pectin and xylan, forming methanol and acetate, the availability of which could explain the high prevalence of C metabolism and acetate utilization in genomes. Microorganism abundances among samples collected at three soil depths and under natural and amended rainfall regimes indicate statistically higher associations of inorganic nitrogen metabolism and carbon degradation in deep and shallow soils, respectively. This partitioning decreased in samples under extended spring rainfall, indicating that long-term climate alteration can affect both carbon and nitrogen cycling. Overall, by leveraging natural and experimental gradients with genome-resolved metabolic profiles, we link microorganisms lacking prior genomic characterization to specific roles in complex carbon, C, nitrate and ammonia transformations, and constrain factors that impact their distributions in soil.
The original version of this Article contained errors in Fig. 4. In panel a, the labels 'F420-reducing NiFe hydrogenase (group 3a)' and 'Group 2 NiFe hydrogenase' were misplaced. These errors have been corrected in both the PDF and HTML versions of the Article.
Blooms of planktonic cyanobacteria have long been of concern in lakes, but more recently, harmful impacts of riverine benthic cyanobacterial mats been recognized. As yet, we know little about how various benthic cyanobacteria are distributed in river networks, or how environmental conditions or other associated microbes in their consortia affect their biosynthetic capacities. We performed metagenomic sequencing for 22 Oscillatoriales-dominated (Cyanobacteria) microbial mats collected across the Eel River network in Northern California and investigated factors associated with anatoxin-a producing cyanobacteria. All microbial communities were dominated by one or two cyanobacterial species, so the key mat metabolisms involve oxygenic photosynthesis and carbon oxidation. Only a few metabolisms fueled the growth of the mat communities, with little evidence for anaerobic metabolic pathways. We genomically defined four cyanobacterial species, all which shared
Bacteriophages (phages) dramatically shape microbial community composition, redistribute nutrients via host lysis and drive evolution through horizontal gene transfer. Despite their importance, much remains to be learned about phages in the human microbiome. We investigated the gut microbiomes of humans from Bangladesh and Tanzania, two African baboon social groups and Danish pigs; many of these microbiomes contain phages belonging to a clade with genomes >540 kilobases in length, the largest yet reported in the human microbiome and close to the maximum size ever reported for phages. We refer to these as Lak phages. CRISPR spacer targeting indicates that Lak phages infect bacteria of the genus Prevotella. We manually curated to completion 15 distinct Lak phage genomes recovered from metagenomes. The genomes display several interesting features, including use of an alternative genetic code, large intergenic regions that are highly expressed and up to 35 putative transfer RNAs, some of which contain enigmatic introns. Different individuals have distinct phage genotypes, and shifts in variant frequencies over consecutive sampling days reflect changes in the relative abundance of phage subpopulations. Recent homologous recombination has resulted in extensive genome admixture of nine baboon Lak phage populations. We infer that Lak phages are widespread in gut communities that contain the Prevotella species, and conclude that megaphages, with fascinating and underexplored biology, may be common but largely overlooked components of human and animal gut microbiomes.
The evolution of aerobic respiration was likely linked to the origins of oxygenic Cyanobacteria. Close phylogenetic neighbors to Cyanobacteria, such as Margulisbacteria (RBX-1 and ZB3), Saganbacteria (WOR-1), Melainabacteria and Sericytochromatia, may constrain the metabolic platform in which aerobic respiration arose. Here, we analyze genomic sequences and predict that sediment-associated Margulisbacteria have a fermentation-based metabolism featuring a variety of hydrogenases, a streamlined nitrogenase, and electron bifurcating complexes involved in cycling of reducing equivalents. The genomes of ocean-associated Margulisbacteria encode an electron transport chain that may support aerobic growth. Some Saganbacteria genomes encode various hydrogenases, and others may be able to use O under certain conditions via a putative novel type of heme copper O reductase. Similarly, Melainabacteria have diverse energy metabolisms and are capable of fermentation and aerobic or anaerobic respiration. The ancestor of all these groups may have been an anaerobe in which fermentation and H metabolism were central metabolic features. The ability to use O as a terminal electron acceptor must have been subsequently acquired by these lineages.
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The subterranean world hosts up to one-fifth of all biomass, including microbial communities that drive transformations central to Earth's biogeochemical cycles. However, little is known about how complex microbial communities in such environments are structured, and how inter-organism interactions shape ecosystem function. Here we apply terabase-scale cultivation-independent metagenomics to aquifer sediments and groundwater, and reconstruct 2,540 draft-quality, near-complete and complete strain-resolved genomes that represent the majority of known bacterial phyla as well as 47 newly discovered phylum-level lineages. Metabolic analyses spanning this vast phylogenetic diversity and representing up to 36% of organisms detected in the system are used to document the distribution of pathways in coexisting organisms. Consistent with prior findings indicating metabolic handoffs in simple consortia, we find that few organisms within the community can conduct multiple sequential redox transformations. As environmental conditions change, different assemblages of organisms are selected for, altering linkages among the major biogeochemical cycles.
The tree of life is one of the most important organizing principles in biology(1). Gene surveys suggest the existence of an enormous number of branches(2), but even an approximation of the full scale of the tree has remained elusive. Recent depictions of the tree of life have focused either on the nature of deep evolutionary relationships(3-5) or on the known, well-classified diversity of life with an emphasis on eukaryotes(6). These approaches overlook the dramatic change in our understanding of life's diversity resulting from genomic sampling of previously unexamined environments. New methods to generate genome sequences illuminate the identity of organisms and their metabolic capacities, placing them in community and ecosystem contexts(7,8). Here, we use new genomic data from over 1,000 uncultivated and little known organisms, together with published sequences, to infer a dramatically expanded version of the tree of life, with Bacteria, Archaea and Eukarya included. The depiction is both a global overview and a snapshot of the diversity within each major lineage. The results reveal the dominance of bacterial diversification and underline the importance of organisms lacking isolated representatives, with substantial evolution concentrated in a major radiation of such organisms. This tree highlights major lineages currently underrepresented in biogeochemical models and identifies radiations that are probably important for future evolutionary analyses.
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Five closely related populations of bacteria from the Candidate Phylum (CP) Peregrinibacteria, part of the bacterial Candidate Phyla Radiation (CPR), were sampled from filtered groundwater obtained from an aquifer adjacent to the Colorado River near the town of Rifle, CO, USA. Here, we present the first complete genome sequences for organisms from this phylum. These bacteria have small genomes and, unlike most organisms from other lineages in the CPR, have the capacity for nucleotide synthesis. They invest significantly in biosynthesis of cell wall and cell envelope components, including peptidoglycan, isoprenoids via the mevalonate pathway, and a variety of amino sugars including perosamine and rhamnose. The genomes encode an intriguing set of large extracellular proteins, some of which are very cysteine-rich and may function in attachment, possibly to other cells. Strain variation in these proteins is an important source of genotypic variety. Overall, the cell envelope features, combined with the lack of biosynthesis capacities for many required cofactors, fatty acids, and most amino acids point to a symbiotic lifestyle. Phylogenetic analyses indicate that these bacteria likely represent a new class within the Peregrinibacteria phylum, although they ultimately may be recognized as members of a separate phylum. We propose the provisional taxonomic assignment as 'Candidatus Peribacter riflensis', Genus Peribacter, Family Peribacteraceae, Order Peribacterales, Class Peribacteria in the phylum Peregrinibacteria.
Current understanding of microorganism-virus interactions, which shape the evolution and functioning of Earth's ecosystems, is based primarily on cultivated organisms. Here we investigate thousands of viral and microbial genomes recovered using a cultivation-independent approach to study the frequency, variety and taxonomic distribution of viral defence mechanisms. CRISPR-Cas systems that confer microorganisms with immunity to viruses are present in only 10% of 1,724 sampled microorganisms, compared with previous reports of 40% occurrence in bacteria and 81% in archaea. We attribute this large difference to the lack of CRISPR-Cas systems across major bacterial lineages that have no cultivated representatives. We correlate absence of CRISPR-Cas with lack of nucleotide biosynthesis capacity and a symbiotic lifestyle. Restriction systems are well represented in these lineages and might provide both non-specific viral defence and access to nucleotides.
Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a critical yet severely inefficient enzyme that catalyses the fixation of virtually all of the carbon found on Earth. Here, we report a functional metagenomic selection that recovers physiologically active RubisCO molecules directly from uncultivated and largely unknown members of natural microbial communities. Selection is based on CO2 -dependent growth in a host strain capable of expressing environmental deoxyribonucleic acid (DNA), precluding the need for pure cultures or screening of recombinant clones for enzymatic activity. Seventeen functional RubisCO-encoded sequences were selected using DNA extracted from soil and river autotrophic enrichments, a photosynthetic biofilm and a subsurface groundwater aquifer. Notably, three related form II RubisCOs were recovered which share high sequence similarity with metagenomic scaffolds from uncultivated members of the Gallionellaceae family. One of the Gallionellaceae RubisCOs was purified and shown to possess CO2 /O2 specificity typical of form II enzymes. X-ray crystallography determined that this enzyme is a hexamer, only the second form II multimer ever solved and the first RubisCO structure obtained from an uncultivated bacterium. Functional metagenomic selection leverages natural biological diversity and billions of years of evolution inherent in environmental communities, providing a new window into the discovery of CO2 -fixing enzymes not previously characterized.
Microbial processes within deep-sea hydrothermal plumes affect ocean biogeochemistry on global scales. In rising hydrothermal plumes, a combination of microbial metabolism and particle formation processes initiate the transformation of reduced chemicals like hydrogen sulfide, hydrogen, methane, iron, manganese and ammonia that are abundant in hydrothermal vent fluids. Despite the biogeochemical importance of this rising portion of plumes, it is understudied in comparison to neutrally buoyant plumes. Here we use metagenomics and bioenergetic modeling to describe the abundance and genetic potential of microorganisms in relation to available electron donors in five different hydrothermal plumes and three associated background deep-sea waters from the Eastern Lau Spreading Center located in the Western Pacific Ocean. Three hundred and thirty one distinct genomic 'bins' were identified, comprising an estimated 951 genomes of archaea, bacteria, eukarya and viruses. A significant proportion of these genomes is from novel microorganisms and thus reveals insights into the energy metabolism of heretofore unknown microbial groups. Community-wide analyses of genes encoding enzymes that oxidize inorganic energy sources showed that sulfur oxidation was the most abundant and diverse chemolithotrophic microbial metabolism in the community. Genes for sulfur oxidation were commonly present in genomic bins that also contained genes for oxidation of hydrogen and methane, suggesting metabolic versatility in these microbial groups. The relative diversity and abundance of genes encoding hydrogen oxidation was moderate, whereas that of genes for methane and ammonia oxidation was low in comparison to sulfur oxidation. Bioenergetic-thermodynamic modeling supports the metagenomic analyses, showing that oxidation of elemental sulfur with oxygen is the most dominant catabolic reaction in the hydrothermal plumes. We conclude that the energy metabolism of microbial communities inhabiting rising hydrothermal plumes is dictated by the underlying plume chemistry, with a dominant role for sulfur-based chemolithoautotrophy.
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