Comparative metagenomics of tropical reef fishes show conserved core gut functions across hosts and diets with diet-related functional gene enrichments.

Fish gut microbial communities are important for the breakdown and energy harvesting of the host diet. Microbes within the fish gut are selected by environmental and evolutionary factors. To understand how fish gut microbial communities are shaped by diet, three tropical fish species (hawkfish, Paracirrhites arcatus; yellow tang, Zebrasoma flavescens; and triggerfish, Rhinecanthus aculeatus) were fed piscivorous (fish meal pellets), herbivorous (seaweed), and invertivorous (shrimp) diets, respectively. From fecal samples, a total of 43 metagenome assembled genomes (MAGs) were recovered from all fish diet treatments. Each host-diet treatment harbored distinct microbial communities based on taxonomy, with Proteobacteria, Bacteroidota, and Firmicutes being the most represented. Based on their metagenomes, microbial communities from all three host-diet treatments demonstrated a baseline ability to degrade proteinaceous, fatty acid, and simple carbohydrate inputs and carry out central carbon metabolism, lactate and formate fermentation, acetogenesis, nitrate respiration, and B vitamin synthesis. The herbivorous yellow tang harbored a more functionally diverse microbial community with some complex polysaccharide degradation specialists, while the piscivorous hawkfish's gut community was more specialized for the degradation of proteins. The invertivorous triggerfish's gut microbiome lacked many carbohydrate degrading capabilities, resulting in a more specialized, functionally uniform community. Across all treatments, several MAGs were able to participate in only individual steps of the degradation of complex polysaccharides, suggestive of microbial community networks that degrade complex inputs. These data suggest the existence of a functional core microbiome that is common among fish species, although the specific taxonomic identities of the associated bacteria may differ.

Reference library for microbial source tracking in the mid-Atlantic United States.

Microbial source tracking can determine fecal contamination but requires a relevant, sizable reference library for analysis. We provide a reference library of 100+ fecal microbiome samples relevant to mid-Atlantic United States ecosystems. Included are wild and domesticated fauna, wastewater, and septic samples applicable to Delaware source tracking studies.

Occurrence, Diversity, and Genomes of "Candidatus Patescibacteria" along the Early Diagenesis of Marine Sediments.

The phylum "Candidatus Patescibacteria" (or Candidate Phyla Radiation [CPR]) accounts for roughly one-quarter of microbial diversity on Earth, but the presence and diversity of these bacteria in marine sediments have been rarely charted. Here, we investigate the abundance, diversity, and metabolic capacities of CPR bacteria in three sediment sites (Mohns Ridge, North Pond, and Costa Rica Margin) with samples covering a wide range of redox zones formed during the early diagenesis of organic matter. Through metagenome sequencing, we found that all investigated sediment horizons contain "Ca. Patescibacteria" (0.4 to 28% of the total communities), which are affiliated with the classes "Ca. Paceibacteria," "Ca. Gracilibacteria," "Ca. Microgenomatia," "Ca. Saccharimonadia," "Ca. ABY1," and "Ca. WWE3." However, only a subset of the diversity of marine sediment "Ca. Patescibacteria," especially the classes "Ca. Paceibacteria" and "Ca. Gracilibacteria," can be captured by 16S rRNA gene amplicon sequencing with commonly used universal primers. We recovered 11 metagenome-assembled genomes (MAGs) of CPR from these sediments, most of which are novel at the family or genus level in the "Ca. Paceibacteria" class and are missed by the amplicon sequencing. While individual MAGs are confined to specific anoxic niches, the lack of capacities to utilize the prevailing terminal electron acceptors indicates that they may not be directly selected by the local redox conditions. These CPR bacteria lack essential biosynthesis pathways and may use a truncated glycolysis pathway to conserve energy as fermentative organotrophs. Our findings suggest that marine sediments harbor some novel yet widespread CPR bacteria during the early diagenesis of organic matter, which needs to be considered in population dynamics assessments in this vast environment. IMPORTANCE Ultrasmall-celled "Ca. Patescibacteria" have been estimated to account for one-quarter of the total microbial diversity on Earth, the parasitic lifestyle of which may exert a profound control on the overall microbial population size of the local ecosystems. However, their diversity and metabolic functions in marine sediments, one of the largest yet understudied ecosystems on Earth, remain virtually uncharacterized. By applying cultivation-independent approaches to a range of sediment redox zones, we reveal that "Ca. Patescibacteria" members are rare but widespread regardless of the prevailing geochemical conditions. These bacteria are affiliated with novel branches of "Ca. Patescibacteria" and have been largely missed in marker gene-based surveys. They do not have respiration capacity but may conserve energy by fermenting organic compounds from their episymbiotic hosts. Our findings suggest that these novel "Ca. Patescibacteria" are among the previously overlooked microbes in diverse marine sediments.

Microbial Diversity and Sulfur Cycling in an Early Earth Analogue: From Ancient Novelty to Modern Commonality.

Life emerged and diversified in the absence of molecular oxygen. The prevailing anoxia and unique sulfur chemistry in the Paleo-, Meso-, and Neoarchean and early Proterozoic eras may have supported microbial communities that differ from those currently thriving on the earth's surface. Zodletone spring in southwestern Oklahoma represents a unique habitat where spatial sampling could substitute for geological eras namely, from the anoxic, surficial light-exposed sediments simulating a preoxygenated earth to overlaid water column where air exposure simulates oxygen intrusion during the Neoproterozoic era. We document a remarkably diverse microbial community in the anoxic spring sediments, with 340/516 (65.89%) of genomes recovered in a metagenomic survey belonging to 200 bacterial and archaeal families that were either previously undescribed or that exhibit an extremely rare distribution on the current earth. Such diversity is underpinned by the widespread occurrence of sulfite, thiosulfate, tetrathionate, and sulfur reduction and the paucity of sulfate reduction machineries in these taxa. Hence, these processes greatly expand lineages mediating reductive sulfur-cycling processes in the tree of life. An analysis of the overlaying oxygenated water community demonstrated the development of a significantly less diverse community dominated by well-characterized lineages and a prevalence of oxidative sulfur-cycling processes. Such a transition from ancient novelty to modern commonality underscores the profound impact of the great oxygenation event on the earth's surficial anoxic community. It also suggests that novel and rare lineages encountered in current anaerobic habitats could represent taxa that once thrived in an anoxic earth but have failed to adapt to earth's progressive oxygenation. IMPORTANCE Life on earth evolved in an anoxic setting; however, the identity and fate of microorganisms that thrived in a preoxygenated earth are poorly understood. In Zodletone spring, the prevailing geochemical conditions are remarkably similar to conditions prevailing in surficial earth prior to oxygen buildup in the atmosphere. We identify hundreds of previously unknown microbial lineages in the spring and demonstrate that these lineages possess the metabolic machinery to mediate a wide range of reductive sulfur processes, with the capacity to respire sulfite, thiosulfate, sulfur, and tetrathionate, rather than sulfate, which is a reflection of the differences in sulfur-cycling chemistry in ancient versus modern times. Collectively, such patterns strongly suggest that microbial diversity and sulfur-cycling processes in a preoxygenated earth were drastically different from the currently observed patterns and that the Great Oxygenation Event has precipitated the near extinction of a wide range of oxygen-sensitive lineages and significantly altered the microbial reductive sulfur-cycling community on earth.

Patterns of Gene Content and Co-occurrence Constrain the Evolutionary Path toward Animal Association in Candidate Phyla Radiation Bacteria.

Candidate Phyla Radiation (CPR) bacteria are small, likely episymbiotic organisms found across Earth's ecosystems. Despite their prevalence, the distribution of CPR lineages across habitats and the genomic signatures of transitions among these habitats remain unclear. Here, we expand the genome inventory for Absconditabacteria (SR1), Gracilibacteria, and Saccharibacteria (TM7), CPR bacteria known to occur in both animal-associated and environmental microbiomes, and investigate variation in gene content with habitat of origin. By overlaying phylogeny with habitat information, we show that bacteria from these three lineages have undergone multiple transitions from environmental habitats into animal microbiomes. Based on co-occurrence analyses of hundreds of metagenomes, we extend the prior suggestion that certain Saccharibacteria have broad bacterial host ranges and constrain possible host relationships for Absconditabacteria and Gracilibacteria. Full-proteome analyses show that animal-associated Saccharibacteria have smaller gene repertoires than their environmental counterparts and are enriched in numerous protein families, including those likely functioning in amino acid metabolism, phage defense, and detoxification of peroxide. In contrast, some freshwater Saccharibacteria encode a putative rhodopsin. For protein families exhibiting the clearest patterns of differential habitat distribution, we compared protein and species phylogenies to estimate the incidence of lateral gene transfer and genomic loss occurring over the species tree. These analyses suggest that habitat transitions were likely not accompanied by large transfer or loss events but rather were associated with continuous proteome remodeling. Thus, we speculate that CPR habitat transitions were driven largely by availability of suitable host taxa and were reinforced by acquisition and loss of some capacities. IMPORTANCE Studying the genetic differences between related microorganisms from different environment types can indicate factors associated with their movement among habitats. This is particularly interesting for bacteria from the Candidate Phyla Radiation because their minimal metabolic capabilities require associations with microbial hosts. We found that shifts of Absconditabacteria, Gracilibacteria, and Saccharibacteria between environmental ecosystems and mammalian mouths/guts probably did not involve major episodes of gene gain and loss; rather, gradual genomic change likely followed habitat migration. The results inform our understanding of how little-known microorganisms establish in the human microbiota where they may ultimately impact health.

Expanding the repertoire of electron acceptors for the anaerobic oxidation of methane in carbonates in the Atlantic and Pacific Ocean.

Authigenic carbonates represent a significant microbial sink for methane, yet little is known about the microbiome responsible for the methane removal. We identify carbonate microbiomes distributed over 21 locations hosted by seven different cold seeps in the Pacific and Atlantic Oceans by carrying out a gene-based survey using 16S rRNA- and mcrA gene sequencing coupled with metagenomic analyses. Based on 16S rRNA gene amplicon analyses, these sites were dominated by bacteria affiliated to the Firmicutes, Alpha- and Gammaproteobacteria. ANME-1 and -2 archaeal clades were abundant in the carbonates yet their typical syntrophic partners, sulfate-reducing bacteria, were not significantly present. Based on mcrA amplicon analyses, the Candidatus Methanoperedens clades were also highly abundant. Our metagenome analysis indicated that methane oxidizers affiliated to the ANME-1 and -2, may be capable of performing complete methane- and potentially short-chain alkane oxidation independently using oxidized sulfur and nitrogen compounds as terminal electron acceptors. Gammaproteobacteria are hypothetically capable of utilizing oxidized nitrogen compounds and may be involved in syntrophy with methane-oxidizing archaea. Carbonate structures represent a window for a more diverse utilization of electron acceptors for anaerobic methane oxidation along the Atlantic and Pacific Margin.

"Sifarchaeota," a Novel Asgard Phylum from Costa Rican Sediment Capable of Polysaccharide Degradation and Anaerobic Methylotrophy.

The Asgard superphylum is a deeply branching monophyletic group of Archaea, recently described as some of the closest relatives of the eukaryotic ancestor. The wide application of genomic analyses from metagenome sequencing has established six distinct phyla, whose genomes encode diverse metabolic capacities and which play important biogeochemical and ecological roles in marine sediments. Here, we describe two metagenome-assembled genomes (MAGs) recovered from deep marine sediments off the Costa Rica margin, defining a novel lineage phylogenetically married to "Candidatus Thorarchaeota"; as such, we propose the name "Sifarchaeota" for this phylum. The two Sifarchaeota MAGs encode an anaerobic pathway for methylotrophy enabling the utilization of C1 to C3 compounds (methanol and methylamines) to synthesize acetyl coenzyme A (acetyl-CoA). The MAGs showed a remarkable saccharolytic capabilities compared to other Asgard lineages and encoded diverse classes of carbohydrate active enzymes (CAZymes) targeting different mono-, di-, and oligosaccharides. Comparative genomic analysis based on the full metabolic profiles of different Asgard lineages revealed the close relation between Sifarchaeota and "Candidatus Odinarchaeota" MAGs, which suggested similar metabolic potentials and ecological roles. Furthermore, we identified multiple HGT events from different bacterial donors within Sifarchaeota MAGs, which hypothetically expanded Sifarchaeota capacities for substrate utilization, energy production, and niche adaptation.IMPORTANCE The exploration of deep marine sediments has unearthed many new lineages of microbes. The finding of this novel phylum of Asgard archaea is important, since understanding the diversity and evolution of Asgard archaea may inform also about the evolution of eukaryotic cells. The comparison of metabolic potentials of the Asgard archaea can help inform about selective pressures the lineages have faced during evolution.

Genome-resolved metagenomics reveals site-specific diversity of episymbiotic CPR bacteria and DPANN archaea in groundwater ecosystems.

Candidate phyla radiation (CPR) bacteria and DPANN archaea are unisolated, small-celled symbionts that are often detected in groundwater. The effects of groundwater geochemistry on the abundance, distribution, taxonomic diversity and host association of CPR bacteria and DPANN archaea has not been studied. Here, we performed genome-resolved metagenomic analysis of one agricultural and seven pristine groundwater microbial communities and recovered 746 CPR and DPANN genomes in total. The pristine sites, which serve as local sources of drinking water, contained up to 31% CPR bacteria and 4% DPANN archaea. We observed little species-level overlap of metagenome-assembled genomes (MAGs) across the groundwater sites, indicating that CPR and DPANN communities may be differentiated according to physicochemical conditions and host populations. Cryogenic transmission electron microscopy imaging and genomic analyses enabled us to identify CPR and DPANN lineages that reproducibly attach to host cells and showed that the growth of CPR bacteria seems to be stimulated by attachment to host-cell surfaces. Our analysis reveals site-specific diversity of CPR bacteria and DPANN archaea that coexist with diverse hosts in groundwater aquifers. Given that CPR and DPANN organisms have been identified in human microbiomes and their presence is correlated with diseases such as periodontitis, our findings are relevant to considerations of drinking water quality and human health.

Groundwater Elusimicrobia are metabolically diverse compared to gut microbiome Elusimicrobia and some have a novel nitrogenase paralog.

Currently described members of Elusimicrobia, a relatively recently defined phylum, are animal-associated and rely on fermentation. However, free-living Elusimicrobia have been detected in sediments, soils and groundwater, raising questions regarding their metabolic capacities and evolutionary relationship to animal-associated species. Here, we analyzed 94 draft-quality, non-redundant genomes, including 30 newly reconstructed genomes, from diverse animal-associated and natural environments. Genomes group into 12 clades, 10 of which previously lacked reference genomes. Groundwater-associated Elusimicrobia are predicted to be capable of heterotrophic or autotrophic lifestyles, reliant on oxygen or nitrate/nitrite-dependent respiration, or a variety of organic compounds and Rhodobacter nitrogen fixation (Rnf) complex-dependent acetogenesis with hydrogen and carbon dioxide as the substrates. Genomes from two clades of groundwater-associated Elusimicrobia often encode a new group of nitrogenase paralogs that co-occur with an extensive suite of radical S-Adenosylmethionine (SAM) proteins. We identified similar genomic loci in genomes of bacteria from the Gracilibacteria phylum and the Myxococcales order and predict that the gene clusters reduce a tetrapyrrole, possibly to form a novel cofactor. The animal-associated Elusimicrobia clades nest phylogenetically within two free-living-associated clades. Thus, we propose an evolutionary trajectory in which some Elusimicrobia adapted to animal-associated lifestyles from free-living species via genome reduction.

Metabolic potentials of archaeal lineages resolved from metagenomes of deep Costa Rica sediments.

Numerous archaeal lineages are known to inhabit marine subsurface sediments, although their distributions, metabolic capacities, and interspecies interactions are still not well understood. Abundant and diverse archaea were recently reported in Costa Rica (CR) margin subseafloor sediments recovered during IODP Expedition 334. Here, we recover metagenome-assembled genomes (MAGs) of archaea from the CR margin and compare them to their relatives from shallower settings. We describe 31 MAGs of six different archaeal lineages (Lokiarchaeota, Thorarchaeota, Heimdallarchaeota, Bathyarcheota, Thermoplasmatales, and Hadesarchaea) and thoroughly analyze representative MAGs from the phyla Lokiarchaeota and Bathyarchaeota. Our analysis suggests the potential capability of Lokiarchaeota members to anaerobically degrade aliphatic and aromatic hydrocarbons. We show it is genetically possible and energetically feasible for Lokiarchaeota to degrade benzoate if they associate with organisms using nitrate, nitrite, and sulfite as electron acceptors, which suggests a possibility of syntrophic relationships between Lokiarchaeota and nitrite and sulfite reducing bacteria. The novel Bathyarchaeota lineage possesses an incomplete methanogenesis pathway lacking the methyl coenzyme M reductase complex and encodes a noncanonical acetogenic pathway potentially coupling methylotrophy to acetogenesis via the methyl branch of Wood-Ljungdahl pathway. These metabolic characteristics suggest the potential of this Bathyarchaeota lineage to be a transition between methanogenic and acetogenic Bathyarchaeota lineages. This work expands our knowledge about the metabolic functional repertoire of marine benthic archaea.

Clades of huge phages from across Earth's ecosystems.

Bacteriophages typically have small genomes1 and depend on their bacterial hosts for replication2. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is-to our knowledge-the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR-Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR-Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR-Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth's ecosystems.

Candidatus Mcinerneyibacterium aminivorans gen. nov., sp. nov., the first representative of the candidate phylum Mcinerneyibacteriota phyl. nov. recovered from a high temperature, high salinity tertiary oil reservoir in north central Oklahoma, USA.

We report on the characterization of a novel genomic assembly (ARYD3) recovered from formation water (17.6% salinity) and crude oil enrichment amended by isolated soy proteins (0.2%), and incubated for 100 days under anaerobic conditions at 50°C. Phylogenetic and phylogenomic analysis demonstrated that the ARYD3 is unaffiliated with all currently described bacterial phyla and candidate phyla, as evident by the low AAI (34.7%), shared gene content (19.4%), and 78.9% 16S rRNA gene sequence similarity to Halothiobacillus neapolitanus, its closest cultured relative. Genomic characterization predicts a slow-growing, non-spore forming, and non-motile Gram-negative rod. Adaptation to high salinity is potentially mediated by the production of the compatible solutes cyclic 2,3-diphosphoglycerate (cDPG), α-glucosylglycerate, as well as the uptake of glycine betaine. Metabolically, the genome encodes primarily aminolytic capabilities for a wide range of amino acids and peptides. Interestingly, evidence of propionate degradation to succinate via methyl-malonyl CoA was identified, suggesting possible capability for syntrophic propionate degradation. Analysis of ARYD3 global distribution patterns identified its occurrence in a very small fraction of Earth Microbiome Project datasets examined (318/27,068), where it consistently represented an extremely rare fraction (maximum 0.28%, average 0.004%) of the overall community. We propose the Candidatus name Mcinerneyibacterium aminivorans gen. nov, sp. nov. for ARYD3T, with the genome serving as the type material for the novel family Mcinerneyibacteriaceae fam. nov., order Mcinerneyibacteriales ord. nov., class Mcinerneyibacteria class nov., and phylum Mcinerneyibacteriota phyl. nov. The type material genome assembly is deposited in GenBank under accession number VSIX00000000.

Horizontal Gene Transfer as an Indispensable Driver for Evolution of Neocallimastigomycota into a Distinct Gut-Dwelling Fungal Lineage.

Survival and growth of the anaerobic gut fungi (AGF; Neocallimastigomycota) in the herbivorous gut necessitate the possession of multiple abilities absent in other fungal lineages. We hypothesized that horizontal gene transfer (HGT) was instrumental in forging the evolution of AGF into a phylogenetically distinct gut-dwelling fungal lineage. The patterns of HGT were evaluated in the transcriptomes of 27 AGF strains, 22 of which were isolated and sequenced in this study, and 4 AGF genomes broadly covering the breadth of AGF diversity. We identified 277 distinct incidents of HGT in AGF transcriptomes, with subsequent gene duplication resulting in an HGT frequency of 2 to 3.5% in AGF genomes. The majority of HGT events were AGF specific (91.7%) and wide (70.8%), indicating their occurrence at early stages of AGF evolution. The acquired genes allowed AGF to expand their substrate utilization range, provided new venues for electron disposal, augmented their biosynthetic capabilities, and facilitated their adaptation to anaerobiosis. The majority of donors were anaerobic fermentative bacteria prevalent in the herbivorous gut. This study strongly indicates that HGT indispensably forged the evolution of AGF as a distinct fungal phylum and provides a unique example of the role of HGT in shaping the evolution of a high-rank taxonomic eukaryotic lineage.IMPORTANCE The anaerobic gut fungi (AGF) represent a distinct basal phylum lineage (Neocallimastigomycota) commonly encountered in the rumen and alimentary tracts of herbivores. Survival and growth of anaerobic gut fungi in these anaerobic, eutrophic, and prokaryote-dominated habitats necessitates the acquisition of several traits absent in other fungal lineages. We assess here the role of horizontal gene transfer as a relatively fast mechanism for trait acquisition by the Neocallimastigomycota postsequestration in the herbivorous gut. Analysis of 27 transcriptomes that represent the broad diversity of Neocallimastigomycota identified 277 distinct HGT events, with subsequent gene duplication resulting in an HGT frequency of 2 to 3.5% in AGF genomes. These HGT events have allowed AGF to survive in the herbivorous gut by expanding their substrate utilization range, augmenting their biosynthetic pathway, providing new routes for electron disposal by expanding fermentative capacities, and facilitating their adaptation to anaerobiosis. HGT in the AGF is also shown to be mainly a cross-kingdom affair, with the majority of donors belonging to the bacteria. This study represents a unique example of the role of HGT in shaping the evolution of a high-rank taxonomic eukaryotic lineage.

The Wood-Ljungdahl pathway as a key component of metabolic versatility in candidate phylum Bipolaricaulota (Acetothermia, OP1).

The Wood-Ljungdahl (WL) pathway is an important component of the metabolic machinery in multiple anaerobic prokaryotes, including numerous yet-uncultured bacterial phyla. The pathway can operate in the reductive and oxidative directions, enabling a wide range of metabolic processes. Here, we present a detailed analysis of 14 newly acquired, previously analysed, and publicly available genomic assemblies belonging to the candidate phylum Bipolaricaulota (candidate division OP1, and candidatus Acetothermia), where the occurrence of WL pathway appears to be universal. In silico analysis of predicted metabolic capabilities indicates that the pathway enables homoacetogenic fermentation of sugars and amino acids in all three Bipolaricaulota orders (RBG-16-55-9, UBA7950 and Bipolaricaulales). In addition, members of RBG-16-55-9 appear to possess the additional capacity for syntrophic acetate oxidation using the WL pathway; as well as for respiratory growth using oxygen or nitrate. Anabolically, all UBA7950, and the majority of the Bipolaricaulales genomes possess the capacity for autotrophic growth using the WL pathway. Our results highlight the WL-enabled metabolic versatility in the Bipolaricaulota, emphasize the need for examining the WL pathway in context of the overall metabolic circuitry in uncultured taxa, and demonstrate the value of comparative genomic analysis for providing a detailed overview of metabolic potential in a target microbial lineage and its potential functional niche in an ecosystem.

Genomic Characterization of Candidate Division LCP-89 Reveals an Atypical Cell Wall Structure, Microcompartment Production, and Dual Respiratory and Fermentative Capacities.

Recent experimental and bioinformatic advances enable the recovery of genomes belonging to yet-uncultured microbial lineages directly from environmental samples. Here, we report on the recovery and characterization of single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) representing candidate phylum LCP-89, previously defined based on 16S rRNA gene sequences. Analysis of LCP-89 genomes recovered from Zodletone Spring, an anoxic spring in Oklahoma, predicts slow-growing, rod-shaped organisms. LCP-89 genomes contain genes for cell wall lipopolysaccharide (LPS) production but lack the entire machinery for peptidoglycan biosynthesis, suggesting an atypical cell wall structure. The genomes, however, encode S-layer homology domain-containing proteins, as well as machinery for the biosynthesis of CMP-legionaminate, inferring the possession of an S-layer glycoprotein. A nearly complete chemotaxis machinery coupled to the absence of flagellar synthesis and assembly genes argues for the utilization of alternative types of motility. A strict anaerobic lifestyle is predicted, with dual respiratory (nitrite ammonification) and fermentative capacities. Predicted substrates include a wide range of sugars and sugar alcohols and a few amino acids. The capability of rhamnose metabolism is confirmed by the identification of bacterial microcompartment genes to sequester the toxic intermediates generated. Comparative genomic analysis identified differences in oxygen sensitivities, respiratory capabilities, substrate utilization preferences, and fermentation end products between LCP-89 genomes and those belonging to its four sister phyla (Calditrichota, SM32-31, AABM5-125-24, and KSB1) within the broader FCB (Fibrobacteres-Chlorobi-Bacteroidetes) superphylum. Our results provide a detailed characterization of members of the candidate division LCP-89 and highlight the importance of reconciling 16S rRNA-based and genome-based phylogenies.IMPORTANCE Our understanding of the metabolic capacities, physiological preferences, and ecological roles of yet-uncultured microbial phyla is expanding rapidly. Two distinct approaches are currently being utilized for characterizing microbial communities in nature: amplicon-based 16S rRNA gene surveys for community characterization and metagenomics/single-cell genomics for detailed metabolic reconstruction. The occurrence of multiple yet-uncultured bacterial phyla has been documented using 16S rRNA surveys, and obtaining genome representatives of these yet-uncultured lineages is critical to our understanding of the role of yet-uncultured organisms in nature. This study provides a genomics-based analysis highlighting the structural features and metabolic capacities of a yet-uncultured bacterial phylum (LCP-89) previously identified in 16S rRNA surveys for which no prior genomes have been described. Our analysis identifies several interesting structural features for members of this phylum, e.g., lack of peptidoglycan biosynthetic machinery and the ability to form bacterial microcompartments. Predicted metabolic capabilities include degradation of a wide range of sugars, anaerobic respiratory capacity, and fermentative capacities. In addition to the detailed structural and metabolic analysis provided for candidate division LCP-89, this effort represents an additional step toward a unified scheme for microbial taxonomy by reconciling 16S rRNA gene-based and genomics-based taxonomic outlines.

Candidatus Krumholzibacterium zodletonense gen. nov., sp nov, the first representative of the candidate phylum Krumholzibacteriota phyl. nov. recovered from an anoxic sulfidic spring using genome resolved metagenomics.

The accumulation of genomes of uncultured organisms has highlighted the need for devising a taxonomic and nomenclature scheme to validate names and prevent redundancies. We here report on the recovery and analysis of four phylogenetically related genomes recovered from an anoxic sulfide and sulfur-rich spring (Zodletone spring) in southwestern Oklahoma. Phylogenetic analysis based on 120 single copy markers attested to their position as a novel distinct bacterial phylum. Genomic analysis suggests Gram-negative flagellated organisms that possess type IV pili. The organisms are predicted to be rod-shaped, slow-growers, with an anoxic, heterotrophic, and fermentative lifestyle. Predicted substrate utilization pattern includes multiple amino acids, dipeptides, tripeptides, and oligpopeptides; as well as few sugars. Predicted auxotrophies include proline, vitamin B6, lipoic acid, biotin, and vitamin B12. Assessment of the putative global distribution pattern of this novel lineage suggests its preference to anoxic marine, terrestrial, hydrocarbon-impacted, and freshwater habitats. We propose the candidatus name Krumholzibacterium zodletonense gen. nov, sp. nov. for Zgenome0171T, with the genome serving as the type material for the novel family Krumholzibacteriaceae fam. nov., order Krumholzibacteriales ord. nov., class Krumholzibacteria class nov., and phylum Krumholzibacteriota phyl. nov. The type material genome assembly is deposited in GenBank under accession number QTKG01000000.

Global Distribution Patterns and Pangenomic Diversity of the Candidate Phylum "Latescibacteria" (WS3).

We investigated the global distribution patterns and pangenomic diversity of the candidate phylum "Latescibacteria" (WS3) in 16S rRNA gene as well as metagenomic data sets. We document distinct distribution patterns for various "Latescibacteria" orders in 16S rRNA gene data sets, with prevalence of orders sediment_1 in terrestrial, PBSIII_9 in groundwater and temperate freshwater, and GN03 in pelagic marine, saline-hypersaline, and wastewater habitats. Using a fragment recruitment approach, we identified 68.9 Mb of "Latescibacteria"-affiliated contigs in publicly available metagenomic data sets comprising 73,079 proteins. Metabolic reconstruction suggests a prevalent saprophytic lifestyle in all "Latescibacteria" orders, with marked capacities for the degradation of proteins, lipids, and polysaccharides predominant in plant, bacterial, fungal/crustacean, and eukaryotic algal cell walls. As well, extensive transport and central metabolic pathways for the metabolism of imported monomers were identified. Interestingly, genes and domains suggestive of the production of a cellulosome-e.g., protein-coding genes harboring dockerin I domains attached to a glycosyl hydrolase and scaffoldin-encoding genes harboring cohesin I and CBM37 domains-were identified in order PBSIII_9, GN03, and MSB-4E2 fragments recovered from four anoxic aquatic habitats; hence extending the cellulosomal production capabilities in Bacteria beyond the Gram-positive Firmicutes In addition to fermentative pathways, a complete electron transport chain with terminal cytochrome c oxidases Caa3 (for operation under high oxygen tension) and Cbb3 (for operation under low oxygen tension) were identified in PBSIII_9 and GN03 fragments recovered from oxygenated and partially/seasonally oxygenated aquatic habitats. Our metagenomic recruitment effort hence represents a comprehensive pangenomic view of this yet-uncultured phylum and provides insights broader than and complementary to those gained from genome recovery initiatives focusing on a single or few sampled environments.IMPORTANCE Our understanding of the phylogenetic diversity, metabolic capabilities, and ecological roles of yet-uncultured microorganisms is rapidly expanding. However, recent efforts mainly have been focused on recovering genomes of novel microbial lineages from a specific sampling site, rather than from a wide range of environmental habitats. To comprehensively evaluate the genomic landscape, putative metabolic capabilities, and ecological roles of yet-uncultured candidate phyla, efforts that focus on the recovery of genomic fragments from a wide range of habitats and that adequately sample the intraphylum diversity within a specific target lineage are needed. Here, we investigated the global distribution patterns and pangenomic diversity of the candidate phylum "Latescibacteria" Our results document the preference of specific "Latescibacteria" orders to specific habitats, the prevalence of plant polysaccharide degradation abilities within all "Latescibacteria" orders, the occurrence of all genes/domains necessary for the production of cellulosomes within three "Latescibacteria" orders (GN03, PBSIII_9, and MSB-4E2) in data sets recovered from anaerobic locations, and the identification of the components of an aerobic respiratory chain, as well as occurrence of multiple O2-dependent metabolic reactions in "Latescibacteria" orders GN03 and PBSIII_9 recovered from oxygenated habitats. The results demonstrate the value of phylocentric pangenomic surveys for understanding the global ecological distribution and panmetabolic abilities of yet-uncultured microbial lineages since they provide broader and more complementary insights than those gained from single-cell genomic and/or metagenomic-enabled genome recovery efforts focusing on a single sampling site.

In Silico Analysis of the Metabolic Potential and Niche Specialization of Candidate Phylum "Latescibacteria" (WS3).

The "Latescibacteria" (formerly WS3), member of the Fibrobacteres-Chlorobi-Bacteroidetes (FCB) superphylum, represents a ubiquitous candidate phylum found in terrestrial, aquatic, and marine ecosystems. Recently, single-cell amplified genomes (SAGs) representing the "Latescibacteria" were obtained from the anoxic monimolimnion layers of Sakinaw Lake (British Columbia, Canada), and anoxic sediments of a coastal lagoon (Etoliko lagoon, Western Greece). Here, we present a detailed in-silico analysis of the four SAGs to gain some insights on their metabolic potential and apparent ecological roles. Metabolic reconstruction suggests an anaerobic fermentative mode of metabolism, as well as the capability to degrade multiple polysaccharides and glycoproteins that represent integral components of green (Charophyta and Chlorophyta) and brown (Phaeophycaea) algae cell walls (pectin, alginate, ulvan, fucan, hydroxyproline-rich glycoproteins), storage molecules (starch and trehalose), and extracellular polymeric substances (EPSs). The analyzed SAGs also encode dedicated transporters for the uptake of produced sugars and amino acids/oligopeptides, as well as an extensive machinery for the catabolism of all transported sugars, including the production of a bacterial microcompartment (BMC) to sequester propionaldehyde, a toxic intermediate produced during fucose and rhamnose metabolism. Finally, genes for the formation of gas vesicles, flagella, type IV pili, and oxidative stress response were found, features that could aid in cellular association with algal detritus. Collectively, these results indicate that the analyzed "Latescibacteria" mediate the turnover of multiple complex organic polymers of algal origin that reach deeper anoxic/microoxic habitats in lakes and lagoons. The implications of such process on our understanding of niche specialization in microbial communities mediating organic carbon turnover in stratified water bodies are discussed.

Global patterns of abundance, diversity and community structure of the Aminicenantes (candidate phylum OP8).

We investigated the global patterns of abundance, diversity, and community structure of members of the Aminicenantes (candidate phylum OP8). Our aim was to identify the putative ecological role(s) played by members of this poorly characterized bacterial lineages in various ecosystems. Analysis of near full-length 16S rRNA genes identified four classes and eight orders within the Aminicenantes. Within 3,134 datasets comprising ∼1.8 billion high throughput-generated partial 16S rRNA genes, 47,351 Aminicenantes-affiliated sequences were identified in 913 datasets. The Aminicenantes exhibited the highest relative abundance in hydrocarbon-impacted environments, followed by marine habitats (especially hydrothermal vents and coral-associated microbiome samples), and aquatic, non-marine habitats (especially in terrestrial springs and groundwater samples). While the overall abundance of the Aminicenantes was higher in low oxygen tension as well as non-saline and low salinity habitats, it was encountered in a wide range of oxygen tension, salinities, and temperatures. Analysis of the community structure of the Aminicenantes showed distinct patterns across various datasets that appear to be, mostly, driven by habitat variations rather than prevalent environmental parameters. We argue that the detection of the Aminicenantes across environmental extremes and the observed distinct community structure patterns reflect a high level of intraphylum metabolic diversity and adaptive capabilities that enable its survival and growth in a wide range of habitats and environmental conditions.