Metagenomic sequencing of a Patescibacteria-containing enrichment from Zodletone spring in Oklahoma, USA.

An enrichment of sulfidic sediments from Zodletone spring was sequenced as a metagenome. Draft genomes representing Cloacimonadota, Deltabacterota, Firmicutes, and Patescibacteria were binned and annotated and will aid functional genomics and cultivation efforts.

Anaerobic fungi in the tortoise alimentary tract illuminate early stages of host-fungal symbiosis and Neocallimastigomycota evolution.

Anaerobic gut fungi (AGF, Neocallimastigomycota) reside in the alimentary tract of herbivores. While their presence in mammals is well documented, evidence for their occurrence in non-mammalian hosts is currently sparse. Culture-independent surveys of AGF in tortoises identified a unique community, with three novel deep-branching genera representing >90% of sequences in most samples. Representatives of all genera were successfully isolated under strict anaerobic conditions. Transcriptomics-enabled phylogenomic and molecular dating analyses indicated an ancient, deep-branching position in the AGF tree for these genera, with an evolutionary divergence time estimate of 104-112 million years ago (Mya). Such estimates push the establishment of animal-Neocallimastigomycota symbiosis from the late to the early Cretaceous. Further, tortoise-associated isolates (T-AGF) exhibited limited capacity for plant polysaccharides metabolism and lacked genes encoding several carbohydrate-active enzyme (CAZyme) families. Finally, we demonstrate that the observed curtailed degradation capacities and reduced CAZyme repertoire is driven by the paucity of horizontal gene transfer (HGT) in T-AGF genomes, compared to their mammalian counterparts. This reduced capacity was reflected in an altered cellulosomal production capacity in T-AGF. Our findings provide insights into the phylogenetic diversity, ecological distribution, evolutionary history, evolution of fungal-host nutritional symbiosis, and dynamics of genes acquisition in Neocallimastigomycota.

Diversity and community structure of anaerobic gut fungi in the rumen of wild and domesticated herbivores.

The rumen houses a diverse community that plays a major role in the digestion process in ruminants. Anaerobic gut fungi (AGF) are key contributors to plant digestion in the rumen. Here, we present a global amplicon-based survey of the rumen AGF mycobiome by examining 206 samples from 15 animal species, 15 countries, and 6 continents. The rumen AGF mycobiome was highly diverse, with 81 out of 88 currently recognized AGF genera or candidate genera identified. However, only six genera (Neocallimastix, Orpinomyces, Caecomyces, Cyllamyces, NY9, and Piromyces) were present at >4% relative abundance. AGF diversity was higher in members of the families Antilocapridae and Cervidae compared to Bovidae. Community structure analysis identified a pattern of phylosymbiosis, where host family (10% of total variance) and species (13.5%) partially explained the rumen mycobiome composition. As well, diet composition (9%-19%), domestication (11.14%), and biogeography (14.1%) also partially explained AGF community structure; although sampling limitation, geographic range restrictions, and direct association between different factors hindered accurate elucidation of the relative contribution of each factor. Pairwise comparison of rumen and fecal samples obtained from the same subject (n = 13) demonstrated greater diversity and inter-sample variability in rumen versus fecal samples. The genera Neocallimastix and Orpinomyces were present in higher abundance in rumen samples, while Cyllamyces and Caecomyces were enriched in fecal samples. Comparative analysis of global rumen and feces data sets revealed a similar pattern. Our results provide a global view of AGF community in the rumen and identify patterns of AGF variability between rumen and feces in herbivores Gastrointestinal (GI) tract.IMPORTANCERuminants are highly successful and economically important mammalian suborder. Ruminants are herbivores that digest plant material with the aid of microorganisms residing in their GI tract. In ruminants, the rumen compartment represents the most important location where microbially mediated plant digestion occurs, and is known to house a bewildering array of microbial diversity. An important component of the rumen microbiome is the anaerobic gut fungi (AGF), members of the phylum Neocallimastigomycota. So far, studies examining AGF diversity have mostly employed fecal samples, and little is currently known regarding the identity of AGF residing in the rumen compartment, factors that impact the observed patterns of diversity and community structure of AGF in the rumen, and how AGF communities in the rumen compare to AGF communities in feces. Here, we examined the rumen AGF diversity using an amplicon-based survey targeting a wide range of wild and domesticated ruminants (n = 206, 15 different animal species) obtained from 15 different countries. Our results demonstrate that while highly diverse, no new AGF genera were identified in the rumen mycobiome samples examined. Our analysis also indicate that animal host phylogeny, diet, biogeography, and domestication status could play a role in shaping AGF community structure. Finally, we demonstrate that a greater level of diversity and higher inter-sample variability was observed in rumen compared to fecal samples, with two genera (Neocallimastix and Orpinomyces) present in higher abundance in rumen samples, and two others (Cyllamyces and Caecomyces) enriched in fecal samples. Our results provide a global view of the identity, diversity, and community structure of AGF in ruminants, elucidate factors impacting diversity and community structure of the rumen mycobiome, and identify patterns of AGF community variability between the rumen and feces in the herbivorous GI tract.

Anaerobic gut fungal communities in marsupial hosts.

The anaerobic gut fungi (AGF) inhabit the alimentary tracts of herbivores. In contrast to placental mammals, information regarding the identity, diversity, and community structure of AGF in marsupials is extremely sparse. Here, we characterized AGF communities in 61 fecal samples from 10 marsupial species belonging to four families in the order Diprotodontia: Vombatidae (wombats), Phascolarctidae (koalas), Phalangeridae (possums), and Macropodidae (kangaroos, wallabies, and pademelons). An amplicon-based diversity survey using the D2 region of the large ribosomal subunit as a phylogenetic marker indicated that marsupial AGF communities were dominated by eight genera commonly encountered in placental herbivores (Neocallimastix, Caecomyces, Cyllamyces, Anaeromyces, Orpinomyces, Piromyces, Pecoramyces, and Khoyollomyces). Community structure analysis revealed a high level of stochasticity, and ordination approaches did not reveal a significant role for the animal host, gut type, dietary preferences, or lifestyle in structuring marsupial AGF communities. Marsupial foregut and hindgut communities displayed diversity and community structure patterns comparable to AGF communities typically encountered in placental foregut hosts while exhibiting a higher level of diversity and a distinct community structure compared to placental hindgut communities. Quantification of AGF load using quantitative PCR indicated a significantly smaller load in marsupial hosts compared to their placental counterparts. Isolation efforts were only successful from a single red kangaroo fecal sample and yielded a Khoyollomyces ramosus isolate closely related to strains previously isolated from placental hosts. Our results suggest that AGF communities in marsupials are in low abundance and show little signs of selection based on ecological and evolutionary factors.IMPORTANCEThe AGF are integral part of the microbiome of herbivores. They play a crucial role in breaking down plant biomass in hindgut and foregut fermenters. The majority of research has been conducted on the AGF community in placental mammalian hosts. However, it is important to note that many marsupial mammals are also herbivores and employ a hindgut or foregut fermentation strategy for breaking down plant biomass. So far, very little is known regarding the AGF diversity and community structure in marsupial mammals. To fill this knowledge gap, we conducted an amplicon-based diversity survey targeting AGF in 61 fecal samples from 10 marsupial species. We hypothesize that, given the distinct evolutionary history and alimentary tract architecture, novel and unique AGF communities would be encountered in marsupials. Our results indicate that marsupial AGF communities are highly stochastic, present in relatively low loads, and display community structure patterns comparable to AGF communities typically encountered in placental foregut hosts. Our results indicate that marsupial hosts harbor AGF communities; however, in contrast to the strong pattern of phylosymbiosis typically observed between AGF and placental herbivores, the identity and gut architecture appear to play a minor role in structuring AGF communities in marsupials.

Temporal progression of anaerobic fungal communities in dairy calves from birth to maturity.

Establishment of microbial communities in neonatal calves is vital for their growth and overall health. While this process has received considerable attention for bacteria, our knowledge on temporal progression of anaerobic gut fungi (AGF) in calves is lacking. Here, we examined AGF communities in faecal samples from six dairy cattle collected at 24 different time points during the pre-weaning (days 1-48), weaning (days 48-60), and post-weaning (days 60-360) phases. Quantitative polymerase chain reaction indicated that AGF colonisation occurs within 24 h after birth, with loads slowly increasing during pre-weaning and weaning, then drastically increasing post-weaning. Culture-independent amplicon surveys identified higher alpha diversity during pre-weaning/weaning, compared to post-weaning. AGF community structure underwent a drastic shift post-weaning, from a community enriched in genera commonly encountered in hindgut fermenters to one enriched in genera commonly encountered in adult ruminants. Comparison of AGF community between calves day 1 post-birth and their mothers suggest a major role for maternal transmission, with additional input from cohabitating subjects. This distinct pattern of AGF progression could best be understood in-light of their narrower niche preferences, metabolic specialisation, and physiological optima compared to bacteria, hence eliciting a unique response to changes in feeding pattern and associated structural GIT development during maturation.

Patterns and determinants of the global herbivorous mycobiome.

Despite their role in host nutrition, the anaerobic gut fungal (AGF) component of the herbivorous gut microbiome remains poorly characterized. Here, to examine global patterns and determinants of AGF diversity, we generate and analyze an amplicon dataset from 661 fecal samples from 34 mammalian species, 9 families, and 6 continents. We identify 56 novel genera, greatly expanding AGF diversity beyond current estimates (31 genera and candidate genera). Community structure analysis indicates that host phylogenetic affiliation, not domestication status and biogeography, shapes the community rather than. Fungal-host associations are stronger and more specific in hindgut fermenters than in foregut fermenters. Transcriptomics-enabled phylogenomic and molecular clock analyses of 52 strains from 14 genera indicate that most genera with preferences for hindgut hosts evolved earlier (44-58 Mya) than those with preferences for foregut hosts (22-32 Mya). Our results greatly expand the documented scope of AGF diversity and provide an ecologically and evolutionary-grounded model to explain the observed patterns of AGF diversity in extant animal hosts.

Comparative transcriptomics and genomics from continuous axenic media growth identifies Coxiella burnetii intracellular survival strategies.

Coxiella burnetii (Cb) is an obligate intracellular pathogen in nature and the causative agent of acute Q fever as well as chronic diseases. In an effort to identify genes and proteins crucial to their normal intracellular growth lifestyle, we applied a 'reverse evolution' approach where the avirulent Nine Mile Phase II strain of Cb was grown for 67 passages in chemically defined ACCM-D media and gene expression patterns and genome integrity from various passages was compared to passage number one following intracellular growth. Transcriptomic analysis identified a marked downregulation of the structural components of the type 4B secretion system (T4BSS), the general secretory (Sec) pathway, as well as 14 out of 118 previously identified genes encoding effector proteins. Additional downregulated pathogenicity determinants genes included several chaperones, LPS, and peptidoglycan biosynthesis. A general marked downregulation of central metabolic pathways was also observed, which was balanced by a marked upregulation of genes encoding transporters. This pattern reflected the richness of the media and diminishing anabolic, and ATP-generation needs. Finally, genomic sequencing and comparative genomic analysis demonstrated an extremely low level of mutation across passages, despite the observed Cb gene expression changes following acclimation to axenic media.

Testudinimyces gracilis gen. nov, sp. nov. and Astrotestudinimyces divisus gen. nov, sp. nov., two novel, deep-branching anaerobic gut fungal genera from tortoise faeces.

The anaerobic gut fungi (AGF, Neocallimastigomycota) represent a basal zoosporic phylum within the kingdom Fungi. Twenty genera are currently described, all of which were isolated from the digestive tracts of mammalian herbivores. Here, we report on the isolation and characterization of novel AGF taxa from faecal samples of tortoises. Twenty-nine fungal isolates were obtained from seven different tortoise species. Phylogenetic analysis using the D1/D2 region of the LSU rRNA gene, ribosomal internal transcribed spacer 1, and RNA polymerase II large subunit grouped all isolates into two distinct, deep-branching clades (clades T and B), with a high level of sequence divergence to their closest cultured relative (Khoyollomyces ramosus). Average amino acid identity values calculated using predicted peptides from the isolates' transcriptomes ranged between 60.80-66.21  % (clade T), and 61.24-64.83  % (clade B) when compared to all other AGF taxa; values that are significantly below recently recommended thresholds for genus (85%) and family (75%) delineation in the Neocallimastigomycota. Both clades displayed a broader temperature growth range (20-45 °C, optimal 30 °C for clade T, and 30-42 °C, optimal 39 °C for clade B) compared to all other AGF taxa. Microscopic analysis demonstrated that strains from both clades produced filamentous hyphae, polycentric rhizoidal growth patterns, and monoflagellated zoospores. Isolates in clade T were characterized by the production of unbranched, predominantly narrow hyphae, and small zoospores, while isolates in clade B were characterized by the production of multiple sporangiophores and sporangia originating from a single central swelling resulting in large multi-sporangiated structures. Based on the unique phylogenetic positions, AAI values, and phenotypic characteristics, we propose to accommodate these isolates into two novel genera (Testudinimyces and Astrotestudinimyces), and species (T. gracilis and A. divisus) within the order Neocallimastigales. The type species are strains T130AT (T. gracilis) and B1.1T (A. divisus).

Complete Genome Sequence of Desulfomicrobium sp. Strain ZS1 from Zodletone Spring in Oklahoma, USA.

Desulfomicrobium sp. strain ZS1 is an obligate anaerobic, sulfate-reducing member of the Desulfobacterota from Zodletone Spring, an anoxic sulfide-rich spring in southwestern Oklahoma. Its complete genome was sequenced using a combination of Illumina and Oxford Nanopore platforms and encodes 3,364 proteins and 81 RNAs on a single chromosome.

Comparative Transcriptomics and Genomics from Continuous Axenic Media Growth Identifies Coxiella burnetii Intracellular Survival Strategies.

Coxiella burnetii (Cb) is an obligate intracellular pathogen in nature and the causative agent of acute Q fever as well as chronic diseases. In an effort to identify genes and proteins crucial to their normal intracellular growth lifestyle, we applied a "Reverse evolution" approach where the avirulent Nine Mile Phase II strain of Cb was grown for 67 passages in chemically defined ACCM-D media and gene expression patterns and genome integrity from various passages was compared to passage number one following intracellular growth. Transcriptomic analysis identified a marked downregulation of the structural components of the type 4B secretion system (T4BSS), the general secretory (sec) pathway, as well as 14 out of 118 previously identified genes encoding effector proteins. Additional downregulated pathogenicity determinants genes included several chaperones, LPS, and peptidoglycan biosynthesis. A general marked downregulation of central metabolic pathways was also observed, which was balanced by a marked upregulation of genes encoding transporters. This pattern reflected the richness of the media and diminishing anabolic and ATP-generation needs. Finally, genomic sequencing and comparative genomic analysis demonstrated an extremely low level of mutation across passages, despite the observed Cb gene expression changes following acclimation to axenic media.

Phylogenomic analysis of the Neocallimastigomycota: proposal of Caecomycetaceae fam. nov., Piromycetaceae fam. nov., and emended description of the families Neocallimastigaceae and Anaeromycetaceae.

The anaerobic gut fungi (AGF) represent a coherent phylogenetic clade within the Mycota. Twenty genera have been described so far. Currently, the phylogenetic and evolutionary relationships between AGF genera remain poorly understood. Here, we utilized 52 transcriptomic datasets from 14 genera to resolve AGF inter-genus relationships using phylogenomics, and to provide a quantitative estimate (amino acid identity, AAI) for intermediate rank assignments. We identify four distinct supra-genus clades, encompassing all genera producing polyflagellated zoospores, bulbous rhizoids, the broadly circumscribed genus Piromyces, and the Anaeromyces and affiliated genera. We also identify the genus Khoyollomyces as the earliest evolving AGF genus. Concordance between phylogenomic outputs and RPB1 and D1/D2 LSU, but not RPB2, MCM7, EF1α or ITS1, phylogenies was observed. We combine phylogenomic analysis and AAI outputs with informative phenotypic traits to propose accommodating 14/20 AGF genera into four families: Caecomycetaceae fam. nov. (encompassing the genera Caecomyces and Cyllamyces), Piromycetaceae fam. nov. (encompassing the genus Piromyces), emend the description of the family Neocallimastigaceae to encompass the genera Neocallimastix, Orpinomyces, Pecoramyces, Feramyces, Ghazallomyces, Aestipascuomyces and Paucimyces, as well as the family Anaeromycetaceae to include the genera Oontomyces, Liebetanzomyces and Capellomyces in addition to Anaeromyces. We refrain from proposing families for the deeply branching genus Khoyollomyces and for genera with uncertain position (Buwchfawromyces, Joblinomyces, Tahromyces, Agriosomyces and Aklioshbomyces) pending availability of additional isolates and sequence data; and these genera are designated as 'genera incertae sedis' in the order Neocallimastigales. Our results establish an evolutionary-grounded Linnaean taxonomic framework for the AGF, provide quantitative estimates for rank assignments, and demonstrate the utility of RPB1 as an additional informative marker in Neocallimastigomycota taxonomy.

Simultaneous Metabarcoding and Quantification of Neocallimastigomycetes from Environmental Samples: Insights into Community Composition and Novel Lineages.

Anaerobic fungi from the herbivore digestive tract (Neocallimastigomycetes) are primary lignocellulose modifiers and hold promise for biotechnological applications. Their molecular detection is currently difficult due to the non-specificity of published primer pairs, which impairs evolutionary and ecological research with environmental samples. We developed and validated a Neocallimastigomycetes-specific PCR primer pair targeting the D2 region of the ribosomal large subunit suitable for screening, quantifying, and sequencing. We evaluated this primer pair in silico on sequences from all known genera, in vitro with pure cultures covering 16 of the 20 known genera, and on environmental samples with highly diverse microbiomes. The amplified region allowed phylogenetic differentiation of all known genera and most species. The amplicon is about 350 bp long, suitable for short-read high-throughput sequencing as well as qPCR assays. Sequencing of herbivore fecal samples verified the specificity of the primer pair and recovered highly diverse and so far unknown anaerobic gut fungal taxa. As the chosen barcoding region can be easily aligned and is taxonomically informative, the sequences can be used for classification and phylogenetic inferences. Several new Neocallimastigomycetes clades were obtained, some of which represent putative novel lineages such as a clade from feces of the rodent Dolichotis patagonum (mara).

Insoluble dietary fibers: structure, metabolism, interactions with human microbiome, and role in gut homeostasis.

Consumption of food rich in dietary fibers (DFs) has been long recognized to exert an overall beneficial effect on human health. This review aims to provide a holistic overview on how IDFs impact human gut health either directly, or through modulation of the gut microbiome. Several databases were searched for collecting papers such as PubMed, Google Scholar, Web of Science, Scopus and Reaxys from 2000 till 2022. Firstly, an overview of the chemical structure of the various IDFs and the pathways employed by gut microbiota for their degradation is provided. The impact of IDFs on microbial community structure and pathogens colonization inside the human gut was discussed. Finally, the impact of IDFs on gut homeostasis and systemic effects at the cellular level, as well as the overall immunological benefits of IDFs consumption were analyzed. IDFs viz., cellulose, hemicellulose, resistant starch, and lignin found enriched in food are discussed for these effects. IDFs were found to induce gut immunity, improve intestinal integrity and mucosal proliferation, and favor adhesion of probiotics and hence improve human health. Also, IDFs were concluded to improve the bioavailability of plant polyphenols and improve their health-related functional roles. Ultimately, dietary fibers processing by modification shows potential to enhance fibers-based functional food production, in addition to increase the economic value and usage of food-rich fibers and their by-products.

Taxonomy of the anaerobic gut fungi (Neocallimastigomycota): a review of classification criteria and description of current taxa.

Members of the anaerobic gut fungi (Neocallimastigomycota) reside in the rumen and alimentary tract of larger mammalian and some reptilian, marsupial and avian herbivores. The recent decade has witnessed a significant expansion in the number of described Neocallimastigomycota genera and species. However, the difficulties associated with the isolation and maintenance of Neocallimastigomycota strains has greatly complicated comparative studies to resolve inter- and intra-genus relationships. Here, we provide an updated outline of Neocallimastigomycota taxonomy. We critically evaluate various morphological, microscopic and phylogenetic traits previously and currently utilized in Neocallimastigomycota taxonomy, and provide an updated key for quick characterization of all genera. We then synthesize data from taxa description manuscripts, prior comparative efforts and molecular sequence data to present an updated list of Neocallimastigomycota genera and species, with an emphasis on resolving relationships and identifying synonymy between recent and historic strains. We supplement data from published manuscripts with information and illustrations from strains in the authors' collections. Twenty genera and 36 species are recognized, but the status of 10 species in the genera Caecomyces, Piromyces, Anaeromyces and Cyllamyces remains uncertain due to the unavailability of culture and conferre (cf.) strains, lack of sequence data, and/or inadequacy of available microscopic and phenotypic data. Six cases of synonymy are identified in the genera Neocallimastix and Caecomyces, and two names in the genus Piromyces are rejected based on apparent misclassification.

Influence of tick sex and geographic region on the microbiome of Dermacentor variabilis collected from dogs and cats across the United States.

As tick-borne diseases continue to increase across North America, current research strives to understand how the tick microbiome may affect pathogen acquisition, maintenance, and transmission. Prior high throughput amplicon-based microbial diversity surveys of the widespread tick Dermacentor variabilis have suggested that life stage, sex, and geographic region may influence the composition of the tick microbiome. Here, adult D. variabilis ticks (n = 145) were collected from dogs and cats from 32 states with specimens originating from all four regions of the United States (West, Midwest, South, and Northeast), and the tick microbiome was examined via V4-16S rRNA gene amplification and Illumina sequencing. A total of 481,246 bacterial sequences were obtained (median 2924 per sample, range 399-11,990). Fifty genera represented the majority (>80%) of the sequences detected, with the genera Allofrancisella and Francisella being the most abundant. Further, 97%, 23%, and 5.5% of the ticks contained sequences belonging to Francisella spp., Rickettsia spp., and Coxiella spp., respectively. No Ehrlichia spp. or Anaplasma spp. were identified. Co-occurrence analysis, by way of correlation coefficients, between the top 50 most abundant genera demonstrated five strong positive and no strong negative correlation relationships. Geographic region had a consistent effect on species richness with ticks from the Northeast having a significantly greater level of richness. Alpha diversity patterns were dependent on tick sex, with males exhibiting higher levels of diversity, and geographical region, with higher level of diversity observed in ticks obtained from the Northeast, but not on tick host. Community structure, or beta diversity, of tick microbiome was impacted by tick sex and geographic location, with microbiomes of ticks from the western US exhibiting a distinct community structure when compared to those from the other three regions (Northeast, South, and Midwest). In total, LEfSe (Linear discriminant analysis Effect Size) identified 18 specific genera driving these observed patterns of diversity and community structure. Collectively, these findings highlight the differences in bacterial diversity of D. variabilis across the US and supports the interpretation that tick sex and geographic region affects microbiome composition across a broad sampling distribution.

Characterization and rank assignment criteria for the anaerobic fungi (Neocallimastigomycota).

Establishing a solid taxonomic framework is crucial for enabling discovery and documentation efforts. This ensures effective communication between scientists as well as reproducibility of results between laboratories, and facilitates the exchange and preservation of biological material. Such framework can only be achieved by establishing clear criteria for taxa characterization and rank assignment. Within the anaerobic fungi (phylum Neocallimastigomycota), the need for such criteria is especially vital. Difficulties associated with their isolation, maintenance and long-term storage often result in limited availability and loss of previously described taxa. To this end, we provide here a list of morphological, microscopic, phylogenetic and phenotypic criteria for assessment and documentation when characterizing newly obtained Neocallimastigomycota isolates. We also recommend a polyphasic rank-assignment scheme for novel genus-, species- and strain-level designations for newly obtained Neocallimastigomycota isolates.

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.

Genomes of Novel Myxococcota Reveal Severely Curtailed Machineries for Predation and Cellular Differentiation.

Cultured Myxococcota are predominantly aerobic soil inhabitants, characterized by their highly coordinated predation and cellular differentiation capacities. Little is currently known regarding yet-uncultured Myxococcota from anaerobic, nonsoil habitats. We analyzed genomes representing one novel order (o__JAFGXQ01) and one novel family (f__JAFGIB01) in the Myxococcota from an anoxic freshwater spring (Zodletone Spring) in Oklahoma, USA. Compared to their soil counterparts, anaerobic Myxococcota possess smaller genomes and a smaller number of genes encoding biosynthetic gene clusters (BGCs), peptidases, one- and two-component signal transduction systems, and transcriptional regulators. Detailed analysis of 13 distinct pathways/processes crucial to predation and cellular differentiation revealed severely curtailed machineries, with the notable absence of homologs for key transcription factors (e.g., FruA and MrpC), outer membrane exchange receptor (TraA), and the majority of sporulation-specific and A-motility-specific genes. Further, machine learning approaches based on a set of 634 genes informative of social lifestyle predicted a nonsocial behavior for Zodletone Myxococcota. Metabolically, Zodletone Myxococcota genomes lacked aerobic respiratory capacities but carried genes suggestive of fermentation, dissimilatory nitrite reduction, and dissimilatory sulfate-reduction (in f_JAFGIB01) for energy acquisition. We propose that predation and cellular differentiation represent a niche adaptation strategy that evolved circa 500 million years ago (Mya) in response to the rise of soil as a distinct habitat on Earth. IMPORTANCE The phylum Myxococcota is a phylogenetically coherent bacterial lineage that exhibits unique social traits. Cultured Myxococcota are predominantly aerobic soil-dwelling microorganisms that are capable of predation and fruiting body formation. However, multiple yet-uncultured lineages within the Myxococcota have been encountered in a wide range of nonsoil, predominantly anaerobic habitats, and the metabolic capabilities, physiological preferences, and capacity of social behavior of such lineages remain unclear. Here, we analyzed genomes recovered from a metagenomic analysis of an anoxic freshwater spring in Oklahoma, USA, that represent novel, yet-uncultured, orders and families in the Myxococcota. The genomes appear to lack the characteristic hallmarks for social behavior encountered in Myxococcota genomes and displayed a significantly smaller genome size and a smaller number of genes encoding biosynthetic gene clusters, peptidases, signal transduction systems, and transcriptional regulators. Such perceived lack of social capacity was confirmed through detailed comparative genomic analysis of 13 pathways associated with Myxococcota social behavior, as well as the implementation of machine learning approaches to predict social behavior based on genome composition. Metabolically, these novel Myxococcota are predicted to be strict anaerobes, utilizing fermentation, nitrate reduction, and dissimilarity sulfate reduction for energy acquisition. Our results highlight the broad patterns of metabolic diversity within the yet-uncultured Myxococcota and suggest that the evolution of predation and fruiting body formation in the Myxococcota has occurred in response to soil formation as a distinct habitat on Earth.

Five Metagenome-Assembled Genomes of the Rare Phylum CSSED10-310 from Zodletone Spring (Oklahoma, USA).

We analyzed five metagenome-assembled genomes (MAGs) belonging to the rare, yet-uncultured phylum CSSED10-310 recovered from the anoxic sediments of Zodletone Spring (Oklahoma). Our analysis suggests their potential involvement in sulfite respiration.

Genomic Analysis of Family UBA6911 (Group 18 Acidobacteria) Expands the Metabolic Capacities of the Phylum and Highlights Adaptations to Terrestrial Habitats.

Approaches for recovering and analyzing genomes belonging to novel, hitherto-unexplored bacterial lineages have provided invaluable insights into the metabolic capabilities and ecological roles of yet-uncultured taxa. The phylum Acidobacteria is one of the most prevalent and ecologically successful lineages on Earth, yet currently, multiple lineages within this phylum remain unexplored. Here, we utilize genomes recovered from Zodletone Spring, an anaerobic sulfide and sulfur-rich spring in southwestern Oklahoma, as well as from multiple disparate soil and nonsoil habitats, to examine the metabolic capabilities and ecological role of members of family UBA6911 (group 18) Acidobacteria. The analyzed genomes clustered into five distinct genera, with genera Gp18_AA60 and QHZH01 recovered from soils, genus Ga0209509 from anaerobic digestors, and genera Ga0212092 and UBA6911 from freshwater habitats. All genomes analyzed suggested that members of Acidobacteria group 18 are metabolically versatile heterotrophs capable of utilizing a wide range of proteins, amino acids, and sugars as carbon sources, possess respiratory and fermentative capacities, and display few auxotrophies. Soil-dwelling genera were characterized by larger genome sizes, higher numbers of CRISPR loci, an expanded carbohydrate active enzyme (CAZyme) machinery enabling debranching of specific sugars from polymers, possession of a C1 (methanol and methylamine) degradation machinery, and a sole dependence on aerobic respiration. In contrast, nonsoil genomes encoded a more versatile respiratory capacity for oxygen, nitrite, sulfate, and trimethylamine N-oxide (TMAO) respiration, as well as the potential for utilizing the Wood-Ljungdahl (WL) pathway as an electron sink during heterotrophic growth. Our results not only expand our knowledge of the metabolism of a yet-uncultured bacterial lineage but also provide interesting clues on how terrestrialization and niche adaptation drive metabolic specialization within the Acidobacteria. IMPORTANCE Members of the Acidobacteria are important players in global biogeochemical cycles, especially in soils. A wide range of acidobacterial lineages remain currently unexplored. We present a detailed genomic characterization of genomes belonging to family UBA6911 (also known as group 18) within the phylum Acidobacteria. The genomes belong to different genera and were obtained from soil (genera Gp18_AA60 and QHZH01), freshwater habitats (genera Ga0212092 and UBA6911), and an anaerobic digestor (genus Ga0209509). While all members of the family shared common metabolic features, e.g., heterotrophic respiratory abilities, broad substrate utilization capacities, and few auxotrophies, distinct differences between soil and nonsoil genera were observed. Soil genera were characterized by expanded genomes, higher numbers of CRISPR loci, a larger carbohydrate active enzyme (CAZyme) repertoire enabling monomer extractions from polymer side chains, and methylotrophic (methanol and methylamine) degradation capacities. In contrast, nonsoil genera encoded more versatile respiratory capacities for utilizing nitrite, sulfate, TMAO, and the WL pathway, in addition to oxygen as electron acceptors. Our results not only broaden our understanding of the metabolic capacities within the Acidobacteria but also provide interesting clues on how terrestrialization shaped Acidobacteria evolution and niche adaptation.