Ecological and genomic analyses of candidate phylum WPS-2 bacteria in an unvegetated soil.

Members of the bacterial candidate phylum WPS-2 (or Eremiobacterota) are abundant in several dry, bare soil environments. In a bare soil deposited by an extinct iron-sulfur spring, we found that WPS-2 comprised up to 24% of the bacterial community and up to 108 cells per g of soil based on 16S rRNA gene sequencing and quantification. A single genus-level cluster (Ca. Rubrimentiphilum) predominated in bare soils but was less abundant in adjacent forest. Nearly complete genomes of Ca. Rubrimentiphilum were recovered as single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs). Surprisingly, given the abundance of WPS-2 in bare soils, the genomes did not indicate any capacity for autotrophy, phototrophy, or trace gas metabolism. Instead, they suggest a predominantly aerobic organoheterotrophic lifestyle, perhaps based on scavenging amino acids, nucleotides, and complex oligopeptides, along with lithotrophic capacity on thiosulfate. Network analyses of the entire community showed that some species of Chloroflexi, Actinobacteria, and candidate phylum AD3 (or Dormibacterota) co-occurred with Ca. Rubrimentiphilum and may represent ecological or metabolic partners. We propose that Ca. Rubrimentiphilum act as efficient heterotrophic scavengers. Combined with previous studies, these data suggest that the phylum WPS-2 includes bacteria with diverse metabolic capabilities.

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.

Microbiome Responses to Oral Fecal Microbiota Transplantation in a Cohort of Domestic Dogs.

Fecal microbiota transplants (FMTs) have been successful at treating digestive and skin conditions in dogs. The degree to which the microbiome is impacted by FMT in a cohort of dogs has not been thoroughly investigated. Using 16S rRNA gene sequencing, we document the changes in the microbiome of fifty-four dogs that took capsules of lyophilized fecal material for their chronic diarrhea, vomiting, or constipation. We found that the relative abundances of five bacterial genera (Butyricicoccus, Faecalibacterium, Fusobacterium, Megamonas, and Sutterella) were higher after FMT than before FMT. Fecal microbiome alpha- and beta-diversity were correlated with kibble and raw food consumption, and prior antibiotic use. On average, 18% of the stool donor's bacterial amplicon sequence variants (ASVs) engrafted in the FMT recipient, with certain bacterial taxa like Bacteroides spp., Fusobacterium spp., and Lachnoclostridium spp. engrafting more frequently than others. Lastly, analyses indicated that the degree of overlap between the donor bacteria and the community of microbes already established in the FMT recipient likely impacts engraftment. Collectively, our work provides further insight into the microbiome and engraftment dynamics of dogs before and after taking oral FMTs.

Characterization and Description of the Fecal Microbiomes of Pet Domestic Ferrets (Mustela putorius furo) Living in Homes.

The domestic ferret (Mustela putorius furo) is a popular companion pet in the United States, with an estimated population of 500,000. Despite being obligate carnivores with a fast digestive system, little is known about their gut microbiomes. This study aims to compare the fecal microbiomes of healthy domestic ferrets and cats, which are both obligate carnivores. We collected and analyzed stool samples from 36 healthy ferrets and 36 healthy cats, sequencing the V4 region of the 16S rRNA gene. Using QIIME 2, we assessed the alpha and beta diversities and identified the taxa differences. Compared to cats, ferrets exhibited a higher representation of Firmicutes and Proteobacteria, while Bacteroidota and Actinomycetota were more prevalent in cats. The ferrets' microbiomes displayed lower alpha diversities. The highly present bacterial genera in the gut microbiomes of ferrets included Clostridium sensu stricto, Streptococcus, Romboutsia, Paeniclostridium, Lactobacillus, Enterococcus, and Lactococcus. Notably, the ferrets' microbiomes significantly differed from those of cats. This research highlights the potential differences in gastrointestinal care for ferrets, emphasizing the need for tailored approaches. Future studies should explore microbiome variations in ferrets with gastrointestinal issues and their responses to dietary and medical interventions.

Microbiome Responses to Fecal Microbiota Transplantation in Cats with Chronic Digestive Issues.

There is growing interest in the application of fecal microbiota transplants (FMTs) in small animal medicine, but there are few published studies that have tested their effects in the domestic cat (Felis catus). Here we use 16S rRNA gene sequencing to examine fecal microbiome changes in 46 domestic cats with chronic digestive issues that received FMTs using lyophilized stool that was delivered in oral capsules. Fecal samples were collected from FMT recipients before and two weeks after the end of the full course of 50 capsules, as well as from their stool donors (N = 10), and other healthy cats (N = 113). The fecal microbiomes of FMT recipients varied with host clinical signs and dry kibble consumption, and shifts in the relative abundances of Clostridium, Collinsella, Megamonas, Desulfovibrio and Escherichia were observed after FMT. Overall, donors shared 13% of their bacterial amplicon sequence variants (ASVs) with FMT recipients and the most commonly shared ASVs were classified as Prevotella 9, Peptoclostridium, Bacteroides, and Collinsella. Lastly, the fecal microbiomes of cats with diarrhea became more similar to the microbiomes of age-matched and diet-matched healthy cats compared to cats with constipation. Overall, our results suggest that microbiome responses to FMT may be modulated by the FMT recipient's initial presenting clinical signs, diet, and their donor's microbiome.

A predicted CRISPR-mediated symbiosis between uncultivated archaea.

CRISPR-Cas systems defend prokaryotic cells from invasive DNA of viruses, plasmids and other mobile genetic elements. Here, we show using metagenomics, metatranscriptomics and single-cell genomics that CRISPR systems of widespread, uncultivated archaea can also target chromosomal DNA of archaeal episymbionts of the DPANN superphylum. Using meta-omics datasets from Crystal Geyser and Horonobe Underground Research Laboratory, we find that CRISPR spacers of the hosts Candidatus Altiarchaeum crystalense and Ca. A. horonobense, respectively, match putative essential genes in their episymbionts' genomes of the genus Ca. Huberiarchaeum and that some of these spacers are expressed in situ. Metabolic interaction modelling also reveals complementation between host-episymbiont systems, on the basis of which we propose that episymbionts are either parasitic or mutualistic depending on the genotype of the host. By expanding our analysis to 7,012 archaeal genomes, we suggest that CRISPR-Cas targeting of genomes associated with symbiotic archaea evolved independently in various archaeal lineages.

The Kitty Microbiome Project: Defining the Healthy Fecal "Core Microbiome" in Pet Domestic Cats.

Here, we present a taxonomically defined fecal microbiome dataset for healthy domestic cats (Felis catus) fed a range of commercial diets. We used this healthy reference dataset to explore how age, diet, and living environment correlate with fecal microbiome composition. Thirty core bacterial genera were identified. Prevotella, Bacteroides, Collinsella, Blautia, and Megasphaera were the most abundant, and Bacteroides, Blautia, Lachnoclostridium, Sutterella, and Ruminococcus gnavus were the most prevalent. While community composition remained relatively stable across different age classes, the number of core taxa present decreased significantly with age. Fecal microbiome composition varied with host diet type. Cats fed kibble had a slightly, but significantly greater number of core taxa compared to cats not fed any kibble. The core microbiomes of cats fed some raw food contained taxa not as highly prevalent or abundant as cats fed diets that included kibble. Living environment also had a large effect on fecal microbiome composition. Cats living in homes differed significantly from those in shelters and had a greater portion of their microbiomes represented by core taxa. Collectively our work reinforces the findings that age, diet, and living environment are important factors to consider when defining a core microbiome in a population.

Best Practices for Microbiome Study Design in Companion Animal Research.

The gut microbiome is a community of microorganisms that inhabits an animal host's gastrointestinal tract, with important effects on animal health that are shaped by multiple environmental, dietary, and host-associated factors. Clinical and dietary trials in companion animals are increasingly including assessment of the microbiome, but interpretation of these results is often hampered by suboptimal choices in study design. Here, we review best practices for conducting feeding trials or clinical trials that intend to study the effects of an intervention on the microbiota. Choices for experimental design, including a review of basic designs, controls, and comparison groups, are discussed in the context of special considerations necessary for microbiome studies. Diet is one of the strongest influences on the composition of gut microbiota, so applications specific to nutritional interventions are discussed in detail. Lastly, we provide specific advice for successful recruitment of colony animals and household pets into an intervention study. This review is intended to serve as a resource to academic and industry researchers, clinicians, and veterinarians alike, for studies that test many different types of interventions.

GAL08, an Uncultivated Group of Acidobacteria, Is a Dominant Bacterial Clade in a Neutral Hot Spring.

GAL08 are bacteria belonging to an uncultivated phylogenetic cluster within the phylum Acidobacteria. We detected a natural population of the GAL08 clade in sediment from a pH-neutral hot spring located in British Columbia, Canada. To shed light on the abundance and genomic potential of this clade, we collected and analyzed hot spring sediment samples over a temperature range of 24.2-79.8°C. Illumina sequencing of 16S rRNA gene amplicons and qPCR using a primer set developed specifically to detect the GAL08 16S rRNA gene revealed that absolute and relative abundances of GAL08 peaked at 65°C along three temperature gradients. Analysis of sediment collected over multiple years and locations revealed that the GAL08 group was consistently a dominant clade, comprising up to 29.2% of the microbial community based on relative read abundance and up to 4.7 × 105 16S rRNA gene copy numbers per gram of sediment based on qPCR. Using a medium quality threshold, 25 single amplified genomes (SAGs) representing these bacteria were generated from samples taken at 65 and 77°C, and seven metagenome-assembled genomes (MAGs) were reconstructed from samples collected at 45-77°C. Based on average nucleotide identity (ANI), these SAGs and MAGs represented three separate species, with an estimated average genome size of 3.17 Mb and GC content of 62.8%. Phylogenetic trees constructed from 16S rRNA gene sequences and a set of 56 concatenated phylogenetic marker genes both placed the three GAL08 bacteria as a distinct subgroup of the phylum Acidobacteria, representing a candidate order (Ca. Frugalibacteriales) within the class Blastocatellia. Metabolic reconstructions from genome data predicted a heterotrophic metabolism, with potential capability for aerobic respiration, as well as incomplete denitrification and fermentation. In laboratory cultivation efforts, GAL08 counts based on qPCR declined rapidly under atmospheric levels of oxygen but increased slightly at 1% (v/v) O2, suggesting a microaerophilic lifestyle.

Trait-Based Comparison of Coral and Sponge Microbiomes.

Corals and sponges harbor diverse microbial communities that are integral to the functioning of the host. While the taxonomic diversity of their microbiomes has been well-established for corals and sponges, their functional roles are less well-understood. It is unclear if the similarities of symbiosis in an invertebrate host would result in functionally similar microbiomes, or if differences in host phylogeny and environmentally driven microhabitats within each host would shape functionally distinct communities. Here we addressed this question, using metatranscriptomic and 16S rRNA gene profiling techniques to compare the microbiomes of two host organisms from different phyla. Our results indicate functional similarity in carbon, nitrogen, and sulfur assimilation, and aerobic nitrogen cycling. Additionally, there were few statistical differences in pathway coverage or abundance between the two hosts. For example, we observed higher coverage of phosphonate and siderophore metabolic pathways in the star coral, Montastraea cavernosa, while there was higher coverage of chloroalkane metabolism in the giant barrel sponge, Xestospongia muta. Higher abundance of genes associated with carbon fixation pathways was also observed in M. cavernosa, while in X. muta there was higher abundance of fatty acid metabolic pathways. Metagenomic predictions based on 16S rRNA gene profiling analysis were similar, and there was high correlation between the metatranscriptome and metagenome predictions for both hosts. Our results highlight several metabolic pathways that exhibit functional similarity in these coral and sponge microbiomes despite the taxonomic differences between the two microbiomes, as well as potential specialization of some microbially based metabolism within each host.

Hiding in Plain Sight: The Globally Distributed Bacterial Candidate Phylum PAUC34f.

Bacterial candidate phylum PAUC34f was originally discovered in marine sponges and is widely considered to be composed of sponge symbionts. Here, we report 21 single amplified genomes (SAGs) of PAUC34f from a variety of environments, including the dark ocean, lake sediments, and a terrestrial aquifer. The diverse origins of the SAGs and the results of metagenome fragment recruitment suggest that some PAUC34f lineages represent relatively abundant, free-living cells in environments other than sponge microbiomes, including the deep ocean. Both phylogenetic and biogeographic patterns, as well as genome content analyses suggest that PAUC34f associations with hosts evolved independently multiple times, while free-living lineages of PAUC34f are distinct and relatively abundant in a wide range of environments.

Insights into the dynamics between viruses and their hosts in a hot spring microbial mat.

Our current knowledge of host-virus interactions in biofilms is limited to computational predictions based on laboratory experiments with a small number of cultured bacteria. However, natural biofilms are diverse and chiefly composed of uncultured bacteria and archaea with no viral infection patterns and lifestyle predictions described to date. Herein, we predict the first DNA sequence-based host-virus interactions in a natural biofilm. Using single-cell genomics and metagenomics applied to a hot spring mat of the Cone Pool in Mono County, California, we provide insights into virus-host range, lifestyle and distribution across different mat layers. Thirty-four out of 130 single cells contained at least one viral contig (26%), which, together with the metagenome-assembled genomes, resulted in detection of 59 viruses linked to 34 host species. Analysis of single-cell amplification kinetics revealed a lack of active viral replication on the single-cell level. These findings were further supported by mapping metagenomic reads from different mat layers to the obtained host-virus pairs, which indicated a low copy number of viral genomes compared to their hosts. Lastly, the metagenomic data revealed high layer specificity of viruses, suggesting limited diffusion to other mat layers. Taken together, these observations indicate that in low mobility environments with high microbial abundance, lysogeny is the predominant viral lifestyle, in line with the previously proposed "Piggyback-the-Winner" theory.

Ancestral Absence of Electron Transport Chains in Patescibacteria and DPANN.

Recent discoveries suggest that the candidate superphyla Patescibacteria and DPANN constitute a large fraction of the phylogenetic diversity of Bacteria and Archaea. Their small genomes and limited coding potential have been hypothesized to be ancestral adaptations to obligate symbiotic lifestyles. To test this hypothesis, we performed cell-cell association, genomic, and phylogenetic analyses on 4,829 individual cells of Bacteria and Archaea from 46 globally distributed surface and subsurface field samples. This confirmed the ubiquity and abundance of Patescibacteria and DPANN in subsurface environments, the small size of their genomes and cells, and the divergence of their gene content from other Bacteria and Archaea. Our analyses suggest that most Patescibacteria and DPANN in the studied subsurface environments do not form specific physical associations with other microorganisms. These data also suggest that their unusual genomic features and prevalent auxotrophies may be a result of ancestral, minimal cellular energy transduction mechanisms that lack respiration, thus relying solely on fermentation for energy conservation.

Four Draft Single-Cell Genome Sequences of Novel, Nearly Identical Kiritimatiellaeota Strains Isolated from the Continental Deep Subsurface.

The recently proposed bacterial phylum Kiritimatiellaeota represents a globally distributed monophyletic clade distinct from other members of the Planctomycetes, Verrucomicrobia, and Chlamydiae (PVC) superphylum. Here, we present four phylogenetically distinct single-cell genome sequences from within the Kiritimatiellaeota lineage sampled from deep continental subsurface aquifer fluids of the Death Valley Regional Flow System in the United States.

Diets with and without edible cricket support a similar level of diversity in the gut microbiome of dogs.

The gut microbiome plays an important role in the health of dogs. Both beneficial microbes and overall diversity can be modulated by diet. Fermentable sources of fiber in particular often increase the abundance of beneficial microbes. Banded crickets (Gryllodes sigillatus) contain the fermentable polysaccharides chitin and chitosan. In addition, crickets are an environmentally sustainable protein source. Considering crickets as a potential source of both novel protein and novel fiber for dogs, four diets ranging from 0% to 24% cricket content were fed to determine their effects on healthy dogs' (n = 32) gut microbiomes. Fecal samples were collected serially at 0, 14, and 29 days, and processed using high-throughput sequencing of 16S rRNA gene PCR amplicons. Microbiomes were generally very similar across all diets at both the phylum and genus level, and alpha and beta diversities did not differ between the various diets at 29 days. A total of 12 ASVs (amplicon sequence variants) from nine genera significantly changed in abundance following the addition of cricket, often in a dose-response fashion with increasing amounts of cricket. A net increase was observed in Catenibacterium, Lachnospiraceae [Ruminococcus], and Faecalitalea, whereas Bacteroides, Faecalibacterium, Lachnospiracaeae NK4A136 group and others decreased in abundance. Similar changes in Catenibacterium and Bacteroides have been associated with gut health benefits in other studies. However, the total magnitude of all changes was small and only a few specific taxa changed in abundance. Overall, we found that diets containing cricket supported the same level of gut microbiome diversity as a standard healthy balanced diet. These results support crickets as a potential healthy, novel food ingredient for dogs.

Single-cell genomics of co-sorted Nanoarchaeota suggests novel putative host associations and diversification of proteins involved in symbiosis.

BACKGROUND: Nanoarchaeota are obligate symbionts of other Archaea first discovered 16 years ago, yet little is known about this largely uncultivated taxon. While Nanoarchaeota diversity has been detected in a variety of habitats using 16S rRNA gene surveys, genome sequences have been available for only three Nanoarchaeota and their hosts. The host range and adaptation of Nanoarchaeota to a wide range of environmental conditions has thus largely remained elusive. Single-cell genomics is an ideal approach to address these questions as Nanoarchaeota can be isolated while still attached to putative hosts, enabling the exploration of cell-cell interactions and fine-scale genomic diversity. RESULTS: From 22 single amplified genomes (SAGs) from three hot springs in Yellowstone National Park, we derived a genome-based phylogeny of the phylum Nanoarchaeota, linking it to global 16S rRNA gene diversity. By exploiting sequencing of co-sorted tightly attached cells, we associated Nanoarchaeota with 6 novel putative hosts, 2 of which were found in multiple SAGs, and showed that the same host species may associate with multiple species of Nanoarchaeota. Comparison of single nucleotide polymorphisms (SNPs) within a population of Nanoarchaeota SAGs indicated that Nanoarchaeota attached to a single host cell in situ are likely clonal. In addition to an overall pattern of purifying selection, we found significantly higher densities of non-synonymous SNPs in hypothetical cell surface proteins, as compared to other functional categories. Genes implicated in interactions in other obligate microbe-microbe symbioses, including those encoding a cytochrome bd-I ubiquinol oxidase and a FlaJ/TadC homologue possibly involved in type IV pili production, also had relatively high densities of non-synonymous SNPs. CONCLUSIONS: This population genetics study of Nanoarchaeota greatly expands the known potential host range of the phylum and hints at what genes may be involved in adaptation to diverse environments or different hosts. We provide the first evidence that Nanoarchaeota cells attached to the same host cell are clonal and propose a hypothesis for how clonality may occur despite diverse symbiont populations.

Differential depth distribution of microbial function and putative symbionts through sediment-hosted aquifers in the deep terrestrial subsurface.

An enormous diversity of previously unknown bacteria and archaea has been discovered recently, yet their functional capacities and distributions in the terrestrial subsurface remain uncertain. Here, we continually sampled a CO2-driven geyser (Colorado Plateau, Utah, USA) over its 5-day eruption cycle to test the hypothesis that stratified, sandstone-hosted aquifers sampled over three phases of the eruption cycle have microbial communities that differ both in membership and function. Genome-resolved metagenomics, single-cell genomics and geochemical analyses confirmed this hypothesis and linked microorganisms to groundwater compositions from different depths. Autotrophic Candidatus "Altiarchaeum sp." and phylogenetically deep-branching nanoarchaea dominate the deepest groundwater. A nanoarchaeon with limited metabolic capacity is inferred to be a potential symbiont of the Ca. "Altiarchaeum". Candidate Phyla Radiation bacteria are also present in the deepest groundwater and they are relatively abundant in water from intermediate depths. During the recovery phase of the geyser, microaerophilic Fe- and S-oxidizers have high in situ genome replication rates. Autotrophic Sulfurimonas sustained by aerobic sulfide oxidation and with the capacity for N2 fixation dominate the shallow aquifer. Overall, 104 different phylum-level lineages are present in water from these subsurface environments, with uncultivated archaea and bacteria partitioned to the deeper subsurface.

Comparative Genomics of Color Morphs In the Coral Montastraea cavernosa.

Montastraea cavernosa is a common coral in the Caribbean basin found in several color morphs. To investigate the causes for brown and orange morphs we undertook a genomics approach on corals collected at the same time and depth in the Bahamas. The coral holobiont includes the host, symbiotic dinoflagellates (Symbiodinium spp.), and a diverse microbiome. While the coral host showed significant genetic differentiation between color morphs both the composition of the Symbiodinium spp. communities and the prokaryotic communities did not. Both targeted and global gene expression differences in the transcriptome of the host show no difference in fluorescent proteins while the metatranscriptome of the microbiome shows that pigments such as phycoerythrin and orange carotenoid protein of cyanobacterial origin are significantly greater in orange morphs, which is also consistent with the significantly greater number of cyanobacteria quantified by 16S rRNA reads and flow cytometry. The microbiome of orange color morphs expressed significantly more nitrogenase (nifH) transcripts consistent with their known ability to fix nitrogen. Both coral and Symbiodinium spp. transcriptomes from orange morphs had significantly increased expression of genes related to immune response and apoptosis, which may potentially be involved in maintaining and regulating the unique symbiont population in orange morphs.

Correction to: Towards a balanced view of the bacterial tree of life.

CORRECTION: Following publication of the original article [1], the authors pointed out that the figure shown as figure S1 is actually figure S2 and vice versa. Figure S1 should show the barcharts, and figure S2 should shows the heatmaps. Figures captions are in the correct order. 1) "...SILVA-only OTUs (37,066 97% OTUs and 1266 85% clusters) (Additional file 3: Figure S2 )..." - > ...SILVA-only OTUs (37,066 97% OTUs and 1266 85% clusters) (Additional file 3: Figure S1 )..." 2) "...in groundwater and soil (Fig. 2, Additional file 2: Figure S1 )." - > "in groundwater and soil (Fig. 2, Additional file 2: Figure S2 )." 3) "...from genomes as training data (Additional file 3: Figure S2 , ..." - > "...from genomes as training data (Additional file 3: Figure S1 , ..." 4) "...were predicted to be chimeric (Additional file 3: Figure S2 )" - > "...were predicted to be chimeric (Additional file 3: Figure S1 )".

Towards a balanced view of the bacterial tree of life.

The bacterial tree of life has recently undergone significant expansion, chiefly from candidate phyla retrieved through genome-resolved metagenomics. Bypassing the need for genome availability, we present a snapshot of bacterial phylogenetic diversity based on the recovery of high-quality SSU rRNA gene sequences extracted from nearly 7000 metagenomes and all available reference genomes. We illuminate taxonomic richness within established bacterial phyla together with environmental distribution patterns, providing a revised framework for future phylogeny-driven sequencing efforts.