Distribution and Genomic Variation of Thermophilic Cyanobacteria in Diverse Microbial Mats at the Upper Temperature Limits of Photosynthesis.

Thermophilic cyanobacteria have been extensively studied in Yellowstone National Park (YNP) hot springs, particularly during decades of work on the thick laminated mats of Octopus and Mushroom springs. However, focused studies of cyanobacteria outside these two hot springs have been lacking, especially regarding how physical and chemical parameters along with community morphology influence the genomic makeup of these organisms. Here, we used a metagenomic approach to examine cyanobacteria existing at the upper temperature limit of photosynthesis. We examined 15 alkaline hot spring samples across six geographic areas of YNP, all with various physical and chemical parameters and community morphology. We recovered 22 metagenome-assembled genomes (MAGs) belonging to thermophilic cyanobacteria, notably an uncultured Synechococcus-like taxon recovered from a setting at the upper temperature limit of photosynthesis, 73°C, in addition to thermophilic Gloeomargarita. Furthermore, we found that three distinct groups of Synechococcus-like MAGs recovered from different temperature ranges vary in their genomic makeup. MAGs from the uncultured very-high-temperature (up to 73°C) Synechococcus-like taxon lack key nitrogen metabolism genes and have genes implicated in cellular stress responses that diverge from other Synechococcus-like MAGs. Across all parameters measured, temperature was the primary determinant of taxonomic makeup of recovered cyanobacterial MAGs. However, total Fe, community morphology, and biogeography played an additional role in the distribution and abundance of upper-temperature-limit-adapted Synechococcus-like MAGs. These findings expand our understanding of cyanobacterial diversity in YNP and provide a basis for interrogation of understudied thermophilic cyanobacteria. IMPORTANCE Oxygenic photosynthesis arose early in microbial evolution-approximately 2.5 to 3.5 billion years ago-and entirely reshaped the biological makeup of Earth. However, despite the span of time in which photosynthesis has been refined, it is strictly limited to temperatures below 73°C, a barrier that many other biological processes have been able to overcome. Furthermore, photosynthesis at temperatures above 56°C is limited to circumneutral and alkaline pH. Hot springs in Yellowstone National Park (YNP), which have a large diversity in temperatures, pH, and geochemistry, provide a natural laboratory to study thermophilic microbial mats and the cyanobacteria within. While cyanobacteria in YNP microbial mats have been studied for decades, a vast majority of the work has focused on two springs within the same geyser basin, both containing similar community morphologies. Thus, the drivers of cyanobacterial adaptations to the upper limits of photosynthesis across a variety of environmental parameters have been understudied. Our findings provide new insights into the influence of these parameters on both taxonomic diversity and genomic content of cyanobacteria across a range of hot spring samples.

Temperature and Geographic Location Impact the Distribution and Diversity of Photoautotrophic Gene Variants in Alkaline Yellowstone Hot Springs.

Alkaline hot springs in Yellowstone National Park (YNP) provide a framework to study the relationship between photoautotrophs and temperature. Previous work has focused on studying how cyanobacteria (oxygenic phototrophs) vary with temperature, sulfide, and pH, but many questions remain regarding the ecophysiology of anoxygenic photosynthesis due to the taxonomic and metabolic diversity of these taxa. To this end, we examined the distribution of genes involved in phototrophy, carbon fixation, and nitrogen fixation in eight alkaline (pH 7.3-9.4) hot spring sites near the upper temperature limit of photosynthesis (71ºC) in YNP using metagenome sequencing. Based on genes encoding key reaction center proteins, geographic isolation plays a larger role than temperature in selecting for distinct phototrophic Chloroflexi, while genes typically associated with autotrophy in anoxygenic phototrophs, did not have distinct distributions with temperature. Additionally, we recovered Calvin cycle gene variants associated with Chloroflexi, an alternative carbon fixation pathway in anoxygenic photoautotrophs. Lastly, we recovered several abundant nitrogen fixation gene sequences associated with Roseiflexus, providing further evidence that genes involved in nitrogen fixation in Chloroflexi are more common than previously assumed. Together, our results add to the body of work on the distribution and functional potential of phototrophic bacteria in Yellowstone National Park hot springs and support the hypothesis that a combination of abiotic and biotic factors impact the distribution of phototrophic bacteria in hot springs. Future studies of isolates and metagenome assembled genomes (MAGs) from these data and others will further our understanding of the ecology and evolution of hot spring anoxygenic phototrophs. IMPORTANCE Photosynthetic bacteria in hot springs are of great importance to both microbial evolution and ecology. While a large body of work has focused on oxygenic photosynthesis in cyanobacteria in Mushroom and Octopus Springs in Yellowstone National Park, many questions remain regarding the metabolic potential and ecology of hot spring anoxygenic phototrophs. Anoxygenic phototrophs are metabolically and taxonomically diverse, and further investigations into their physiology will lead to a deeper understanding of microbial evolution and ecology of these taxa. Here, we have quantified the distribution of key genes involved in carbon and nitrogen metabolism in both oxygenic and anoxygenic phototrophs. Our results suggest that temperature >68ºC selects for distinct groups of cyanobacteria and that carbon fixation pathways associated with these taxa are likely subject to the same selective pressure. Additionally, our data suggest that phototrophic Chloroflexi genes and carbon fixation genes are largely influenced by local conditions as evidenced by our gene variant analysis. Lastly, we recovered several genes associated with potentially novel phototrophic Chloroflexi. Together, our results add to the body of work on hot springs in Yellowstone National Park and set the stage for future work on metagenome assembled genomes.

Genomics, Exometabolomics, and Metabolic Probing Reveal Conserved Proteolytic Metabolism of Thermoflexus hugenholtzii and Three Candidate Species From China and Japan.

Thermoflexus hugenholtzii JAD2T, the only cultured representative of the Chloroflexota order Thermoflexales, is abundant in Great Boiling Spring (GBS), NV, United States, and close relatives inhabit geothermal systems globally. However, no defined medium exists for T. hugenholtzii JAD2T and no single carbon source is known to support its growth, leaving key knowledge gaps in its metabolism and nutritional needs. Here, we report comparative genomic analysis of the draft genome of T. hugenholtzii JAD2T and eight closely related metagenome-assembled genomes (MAGs) from geothermal sites in China, Japan, and the United States, representing "Candidatus Thermoflexus japonica," "Candidatus Thermoflexus tengchongensis," and "Candidatus Thermoflexus sinensis." Genomics was integrated with targeted exometabolomics and 13C metabolic probing of T. hugenholtzii. The Thermoflexus genomes each code for complete central carbon metabolic pathways and an unusually high abundance and diversity of peptidases, particularly Metallo- and Serine peptidase families, along with ABC transporters for peptides and some amino acids. The T. hugenholtzii JAD2T exometabolome provided evidence of extracellular proteolytic activity based on the accumulation of free amino acids. However, several neutral and polar amino acids appear not to be utilized, based on their accumulation in the medium and the lack of annotated transporters. Adenine and adenosine were scavenged, and thymine and nicotinic acid were released, suggesting interdependency with other organisms in situ. Metabolic probing of T. hugenholtzii JAD2T using 13C-labeled compounds provided evidence of oxidation of glucose, pyruvate, cysteine, and citrate, and functioning glycolytic, tricarboxylic acid (TCA), and oxidative pentose-phosphate pathways (PPPs). However, differential use of position-specific 13C-labeled compounds showed that glycolysis and the TCA cycle were uncoupled. Thus, despite the high abundance of Thermoflexus in sediments of some geothermal systems, they appear to be highly focused on chemoorganotrophy, particularly protein degradation, and may interact extensively with other microorganisms in situ.

Temperature impacts community structure and function of phototrophic Chloroflexi and Cyanobacteria in two alkaline hot springs in Yellowstone National Park.

Photosynthetic bacteria are abundant in alkaline, terrestrial hot springs and there is a long history of research on phototrophs in Yellowstone National Park (YNP). Hot springs provide a framework to examine the ecophysiology of phototrophs in situ because they provide natural gradients of geochemistry, pH and temperature. Phototrophs within the Cyanobacteria and Chloroflexi groups are frequently observed in alkaline hot springs. Decades of research has determined that temperature constrains Cyanobacteria in alkaline hot springs, but factors that constrain the distribution of phototrophic Chloroflexi remain unresolved. Using a combination of 16S rRNA gene sequencing and photoassimilation microcosms, we tested the hypothesis that temperature would constrain the activity and composition of phototrophic Cyanobacteria and Chloroflexi. We expected diversity and rates of photoassimilation to decrease with increasing temperature. We report 16S rRNA amplicon sequencing along with carbon isotope signatures and photoassimilation from 45 to 72°C in two alkaline hot springs. We find that Roseiflexus, Chloroflexus (Chloroflexi) and Leptococcus (Cyanobacteria) operational taxonomic units (OTUs) have distinct distributions with temperature. This distribution suggests that, like phototrophic Cyanobacteria, temperature selects for specific phototrophic Chloroflexi taxa. The richness of phototrophic Cyanobacteria decreased with increasing temperature along with a decrease in oxygenic photosynthesis, whereas Chloroflexi richness and rates of anoxygenic photosynthesis did not decrease with increasing temperature, even at temperatures approaching the upper limit of photosynthesis (~72-73°C). Our carbon isotopic data suggest an increasing prevalence of the 3-hydroxypropionate pathway with decreasing temperature coincident with photoautotrophic Chloroflexi. Together these results indicate temperature plays a role in defining the niche space of phototrophic Chloroflexi (as has been observed for Cyanobacteria), but other factors such as morphology, geochemistry, or metabolic diversity of Chloroflexi, in addition to temperature, could determine the niche space of this highly versatile group.

Diverse respiratory capacity among Thermus strains from US Great Basin hot springs.

Thermus species are thermophilic heterotrophs, with most capable of using a variety of organic and inorganic electron donors for respiration. Here, a combined cultivation-independent and -dependent approach was used to explore the diversity of Thermus in Great Boiling Spring (GBS) and Little Hot Creek (LHC) in the US Great Basin. A cultivation-independent 16S rRNA gene survey of ten LHC sites showed that Thermus made up 0-3.5% of sequences and were predominately Thermus thermophilus. 189 Thermus isolates from GBS and LHC were affiliated with T. aquaticus (73.0%), T. oshimai (25.4%), T. sediminis (1.1%), and T. thermophilus (0.5%), with T. aquaticus and T. oshimai forming biogeographic clusters. 22 strains were selected for characterization, including chemolithotrophic oxidation of thiosulfate and arsenite, and reduction of ferric iron, polysulfide, and nitrate, revealing phenotypic diversity and broad respiratory capability within each species. PCR demonstrated the wide distribution of aerobic arsenite oxidase genes. A GBS sediment metaproteome contained sulfite oxidase and Fe3+ ABC transporter permease peptides, suggesting sulfur and iron transformations in situ. This study expands our knowledge of the physiological diversity of Thermus, suggesting widespread chemolithotrophic and anaerobic respiration phenotypes, and providing a foundation for better understanding the ecology of this genus in thermal ecosystems.

Anoxygenic Phototrophs Span Geochemical Gradients and Diverse Morphologies in Terrestrial Geothermal Springs.

Extant anoxygenic phototrophs are taxonomically, physiologically, and metabolically diverse and include examples from all seven bacterial phyla with characterized phototrophic members. pH, temperature, and sulfide are known to constrain phototrophs, but how these factors dictate the distribution and activity of specific taxa of anoxygenic phototrophs has not been reported. Here, we hypothesized that within the known limits of pH, temperature, and sulfide, the distribution, abundance, and activity of specific anoxygenic phototrophic taxa would vary due to key differences in the physiology of these organisms. To test this hypothesis, we examined the distribution, abundance, and potential activity of anoxygenic phototrophs in filaments, microbial mats, and sediments across geochemical gradients in geothermal features of Yellowstone National Park, which ranged in pH from 2.2 to 9.4 and in temperature from 31.5°C to 71.0°C. Indeed, our data indicate putative aerobic anoxygenic phototrophs within the Proteobacteria are more abundant at lower pH and lower temperature, while phototrophic Chloroflexi are prevalent in circumneutral to alkaline springs. In contrast to previous studies, our data suggest sulfide is not a key determinant of anoxygenic phototrophic taxa. Finally, our data underscore a role for photoheterotrophy (or photomixotrophy) across geochemical gradients in terrestrial geothermal ecosystems.IMPORTANCE There is a long and rich history of literature on phototrophs in terrestrial geothermal springs. These studies have revealed sulfide, pH, and temperature are the main constraints on phototrophy. However, the taxonomic and physiological diversity of anoxygenic phototrophs suggests that, within these constraints, specific geochemical parameters determine the distribution and activity of individual anoxygenic phototrophic taxa. Here, we report the recovery of sequences affiliated with characterized anoxygenic phototrophs in sites that range in pH from 2 to 9 and in temperature from 31°C to 71°C. Transcript abundance indicates anoxygenic phototrophs are active across this temperature and pH range. Our data suggest sulfide is not a key determinant of anoxygenic phototrophic taxa and underscore a role for photoheterotrophy in terrestrial geothermal ecosystems. These data provide the framework for high-resolution sequencing and in situ activity approaches to characterize the physiology of specific anoxygenic phototrophic taxa across a broad range of temperatures and pH.

Position-Specific Metabolic Probing and Metagenomics of Microbial Communities Reveal Conserved Central Carbon Metabolic Network Activities at High Temperatures.

Temperature is a primary driver of microbial community composition and taxonomic diversity; however, it is unclear to what extent temperature affects characteristics of central carbon metabolic pathways (CCMPs) at the community level. In this study, 16S rRNA gene amplicon and metagenome sequencing were combined with 13C-labeled metabolite probing of the CCMPs to assess community carbon metabolism along a temperature gradient (60-95°C) in Great Boiling Spring, NV. 16S rRNA gene amplicon diversity was inversely proportional to temperature, and Archaea were dominant at higher temperatures. KO richness and diversity were also inversely proportional to temperature, yet CCMP genes were similarly represented across the temperature gradient and many individual metagenome-assembled genomes had complete pathways. In contrast, genes encoding cellulosomes and many genes involved in plant matter degradation and photosynthesis were absent at higher temperatures. In situ 13C-CO2 production from labeled isotopomer pairs of glucose, pyruvate, and acetate suggested lower relative oxidative pentose phosphate pathway activity and/or fermentation at 60°C, and a stable or decreased maintenance energy demand at higher temperatures. Catabolism of 13C-labeled citrate, succinate, L-alanine, L-serine, and L-cysteine was observed at 85°C, demonstrating broad heterotrophic activity and confirming functioning of the TCA cycle. Together, these results suggest that temperature-driven losses in biodiversity and gene content in geothermal systems may not alter CCMP function or maintenance energy demands at a community level.

Thermus sediminis sp. nov., a thiosulfate-oxidizing and arsenate-reducing organism isolated from Little Hot Creek in the Long Valley Caldera, California.

Thermus species are widespread in natural and artificial thermal environments. Two new yellow-pigmented strains, L198T and L423, isolated from Little Hot Creek, a geothermal spring in eastern California, were identified as novel organisms belonging to the genus Thermus. Cells are Gram-negative, rod-shaped, and non-motile. Growth was observed at temperatures from 45 to 75 °C and at salinities of 0-2.0% added NaCl. Both strains grow heterotrophically or chemolithotrophically by oxidation of thiosulfate to sulfate. L198T and L423 grow by aerobic respiration or anaerobic respiration with arsenate as the terminal electron acceptor. Values for 16S rRNA gene identity (≤ 97.01%), digital DNA-DNA hybridization (≤ 32.7%), OrthoANI (≤ 87.5%), and genome-to-genome distance (0.13) values to all Thermus genomes were less than established criteria for microbial species. The predominant respiratory quinone was menaquinone-8 and the major cellular fatty acids were iso-C15:0, iso-C17:0 and anteiso-C15:0. One unidentified phospholipid (PL1) and one unidentified glycolipid (GL1) dominated the polar lipid pattern. The new strains could be differentiated from related taxa by ß-galactosidase and ß-glucosidase activity and the presence of hydroxy fatty acids. Based on phylogenetic, genomic, phenotypic, and chemotaxonomic evidence, the novel species Thermus sediminis sp. nov. is proposed, with the type strain L198T (= CGMCC 1.13590T = KCTC XXX).

Effect of the Synthetic Bile Salt Analog CamSA on the Hamster Model of Clostridium difficile Infection.

Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea and has gained worldwide notoriety due to emerging hypervirulent strains and the high incidence of recurrence. We previously reported protection of mice from CDI using the antigerminant bile salt analog CamSA. Here we describe the effects of CamSA in the hamster model of CDI. CamSA treatment of hamsters showed no toxicity and did not affect the richness or diversity of gut microbiota; however, minor changes in community composition were observed. Treatment of C. difficile-challenged hamsters with CamSA doubled the mean time to death, compared to control hamsters. However, CamSA alone was insufficient to prevent CDI in hamsters. CamSA in conjunction with suboptimal concentrations of vancomycin led to complete protection from CDI in 70% of animals. Protected animals remained disease-free at least 30 days postchallenge and showed no signs of colonic tissue damage. In a delayed-treatment model of hamster CDI, CamSA was unable to prevent infection signs and death. These data support a putative model in which CamSA reduces the number of germinating C. difficile spores but does not keep all of the spores from germinating. Vancomycin halts division of any vegetative cells that are able to grow from spores that escape CamSA.

Localized high abundance of Marine Group II archaea in the subtropical Pearl River Estuary: implications for their niche adaptation.

Marine Group II archaea are widely distributed in global oceans and dominate the total archaeal community within the upper euphotic zone of temperate waters. However, factors controlling the distribution of MGII are poorly delineated and the physiology and ecological functions of these still-uncultured organisms remain elusive. In this study, we investigated the planktonic MGII associated with particles and in free-living forms in the Pearl River Estuary (PRE) over a 10-month period. We detected high abundance of particle-associated MGII in PRE (up to ∼108 16S rRNA gene copies/l), which was around 10-fold higher than the free-living MGII in the same region, and an order of magnitude higher than previously reported in other marine environments. 10‰ salinity appeared to be a threshold value for these MGII because MGII abundance decreased sharply below it. Above 10‰ salinity, the abundance of MGII on the particles was positively correlated with phototrophs and MGII in the surface water was negatively correlated with irradiance. However, the abundances of those free-living MGII showed positive correlations with salinity and temperature, suggesting the different physiological characteristics between particle-attached and free-living MGIIs. A nearly completely assembled metagenome, MGIIa_P, was recovered using metagenome binning methods. Compared with the other two MGII genomes from surface ocean, MGIIa_P contained higher proportions of glycoside hydrolases, indicating the ability of MGIIa_P to hydrolyse glycosidic bonds in complex sugars in PRE. MGIIa_P is the first assembled MGII metagenome containing a catalase gene, which might be involved in scavenging reactive oxygen species generated by the abundant phototrophs in the eutrophic PRE. Our study presented the widespread and high abundance of MGII in the water columns of PRE, and characterized the determinant abiotic factors affecting their distribution. Their association with heterotrophs, preference for particles and resourceful metabolic traits indicate MGII might play a significant role in metabolising organic matters in the PRE and other temperate estuarine systems.

Genomic Comparison of Two Family-Level Groups of the Uncultivated NAG1 Archaeal Lineage from Chemically and Geographically Disparate Hot Springs.

Recent progress based on single-cell genomics and metagenomic investigations of archaea in a variety of extreme environments has led to significant advances in our understanding of the diversity, evolution, and metabolic potential of archaea, yet the vast majority of archaeal diversity remains undersampled. In this work, we coordinated single-cell genomics with metagenomics in order to construct a near-complete genome from a deeply branching uncultivated archaeal lineage sampled from Great Boiling Spring (GBS) in the U.S. Great Basin, Nevada. This taxon is distantly related (distinct families) to an archaeal genome, designated "Novel Archaeal Group 1" (NAG1), which was extracted from a metagenome recovered from an acidic iron spring in Yellowstone National Park (YNP). We compared the metabolic predictions of the NAG1 lineage to better understand how these archaea could inhabit such chemically distinct environments. Similar to the NAG1 population previously studied in YNP, the NAG1 population from GBS is predicted to utilize proteins as a primary carbon source, ferment simple carbon sources, and use oxygen as a terminal electron acceptor under oxic conditions. However, GBS NAG1 populations contained distinct genes involved in central carbon metabolism and electron transfer, including nitrite reductase, which could confer the ability to reduce nitrite under anaerobic conditions. Despite inhabiting chemically distinct environments with large variations in pH, GBS NAG1 populations shared many core genomic and metabolic features with the archaeon identified from YNP, yet were able to carve out a distinct niche at GBS.

High-Quality Draft Genome Sequence of Thermocrinis jamiesonii GBS1T Isolated from Great Boiling Spring, Nevada.

The draft genome of Thermocrinis jamiesonii GBS1T is 1,315,625 bp in 10 contigs and encodes 1,463 predicted genes. The presence of sox genes and various glycoside hydrolases and the absence of uptake NiFe hydrogenases (hyaB) are consistent with a requirement for thiosulfate and suggest the ability to use carbohydrate polymers.

High-Quality Draft Genomes from Thermus caliditerrae YIM 77777 and T. tengchongensis YIM 77401, Isolates from Tengchong, China.

The draft genomes of Thermus  tengchongensis YIM 77401 and T. caliditerrae YIM 77777 are 2,562,314 and 2,218,114 bp and encode 2,726 and 2,305 predicted genes, respectively. Gene content and growth experiments demonstrate broad metabolic capacity, including starch hydrolysis, thiosulfate oxidation, arsenite oxidation, incomplete denitrification, and polysulfide reduction.

High-quality draft genome sequence of the Thermus amyloliquefaciens type strain YIM 77409(T) with an incomplete denitrification pathway.

Thermus amyloliquefaciens type strain YIM 77409(T) is a thermophilic, Gram-negative, non-motile and rod-shaped bacterium isolated from Niujie Hot Spring in Eryuan County, Yunnan Province, southwest China. In the present study we describe the features of strain YIM 77409(T) together with its genome sequence and annotation. The genome is 2,160,855 bp long and consists of 6 scaffolds with 67.4 % average GC content. A total of 2,313 genes were predicted, comprising 2,257 protein-coding and 56 RNA genes. The genome is predicted to encode a complete glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle. Additionally, a large number of transporters and enzymes for heterotrophy highlight the broad heterotrophic lifestyle of this organism. A denitrification gene cluster included genes predicted to encode enzymes for the sequential reduction of nitrate to nitrous oxide, consistent with the incomplete denitrification phenotype of this strain.

Single-Cell-Genomics-Facilitated Read Binning of Candidate Phylum EM19 Genomes from Geothermal Spring Metagenomes.

The vast majority of microbial life remains uncatalogued due to the inability to cultivate these organisms in the laboratory. This "microbial dark matter" represents a substantial portion of the tree of life and of the populations that contribute to chemical cycling in many ecosystems. In this work, we leveraged an existing single-cell genomic data set representing the candidate bacterial phylum "Calescamantes" (EM19) to calibrate machine learning algorithms and define metagenomic bins directly from pyrosequencing reads derived from Great Boiling Spring in the U.S. Great Basin. Compared to other assembly-based methods, taxonomic binning with a read-based machine learning approach yielded final assemblies with the highest predicted genome completeness of any method tested. Read-first binning subsequently was used to extract Calescamantes bins from all metagenomes with abundant Calescamantes populations, including metagenomes from Octopus Spring and Bison Pool in Yellowstone National Park and Gongxiaoshe Spring in Yunnan Province, China. Metabolic reconstruction suggests that Calescamantes are heterotrophic, facultative anaerobes, which can utilize oxidized nitrogen sources as terminal electron acceptors for respiration in the absence of oxygen and use proteins as their primary carbon source. Despite their phylogenetic divergence, the geographically separate Calescamantes populations were highly similar in their predicted metabolic capabilities and core gene content, respiring O2, or oxidized nitrogen species for energy conservation in distant but chemically similar hot springs.

Phylogeny and physiology of candidate phylum 'Atribacteria' (OP9/JS1) inferred from cultivation-independent genomics.

The 'Atribacteria' is a candidate phylum in the Bacteria recently proposed to include members of the OP9 and JS1 lineages. OP9 and JS1 are globally distributed, and in some cases abundant, in anaerobic marine sediments, geothermal environments, anaerobic digesters and reactors and petroleum reservoirs. However, the monophyly of OP9 and JS1 has been questioned and their physiology and ecology remain largely enigmatic due to a lack of cultivated representatives. Here cultivation-independent genomic approaches were used to provide a first comprehensive view of the phylogeny, conserved genomic features and metabolic potential of members of this ubiquitous candidate phylum. Previously available and heretofore unpublished OP9 and JS1 single-cell genomic data sets were used as recruitment platforms for the reconstruction of atribacterial metagenome bins from a terephthalate-degrading reactor biofilm and from the monimolimnion of meromictic Sakinaw Lake. The single-cell genomes and metagenome bins together comprise six species- to genus-level groups that represent most major lineages within OP9 and JS1. Phylogenomic analyses of these combined data sets confirmed the monophyly of the 'Atribacteria' inclusive of OP9 and JS1. Additional conserved features within the 'Atribacteria' were identified, including a gene cluster encoding putative bacterial microcompartments that may be involved in aldehyde and sugar metabolism, energy conservation and carbon storage. Comparative analysis of the metabolic potential inferred from these data sets revealed that members of the 'Atribacteria' are likely to be heterotrophic anaerobes that lack respiratory capacity, with some lineages predicted to specialize in either primary fermentation of carbohydrates or secondary fermentation of organic acids, such as propionate.

High-Quality Draft Genome Sequence of Kallotenue papyrolyticum JKG1T Reveals Broad Heterotrophic Capacity Focused on Carbohydrate and Amino Acid Metabolism.

The draft genome of Kallotenue papyrolyticum JKG1(T), a member of the order Kallotenuales, class Chloroflexia, consists of 4,475,263 bp in 4 contigs and encodes 4,010 predicted genes, 49 tRNA-encoding genes, and 3 rRNA operons. The genome is consistent with a heterotrophic lifestyle including catabolism of polysaccharides and amino acids.

Exploring microbial dark matter to resolve the deep archaeal ancestry of eukaryotes.

The origin of eukaryotes represents an enigmatic puzzle, which is still lacking a number of essential pieces. Whereas it is currently accepted that the process of eukaryogenesis involved an interplay between a host cell and an alphaproteobacterial endosymbiont, we currently lack detailed information regarding the identity and nature of these players. A number of studies have provided increasing support for the emergence of the eukaryotic host cell from within the archaeal domain of life, displaying a specific affiliation with the archaeal TACK superphylum. Recent studies have shown that genomic exploration of yet-uncultivated archaea, the so-called archaeal 'dark matter', is able to provide unprecedented insights into the process of eukaryogenesis. Here, we provide an overview of state-of-the-art cultivation-independent approaches, and demonstrate how these methods were used to obtain draft genome sequences of several novel members of the TACK superphylum, including Lokiarchaeum, two representatives of the Miscellaneous Crenarchaeotal Group (Bathyarchaeota), and a Korarchaeum-related lineage. The maturation of cultivation-independent genomics approaches, as well as future developments in next-generation sequencing technologies, will revolutionize our current view of microbial evolution and diversity, and provide profound new insights into the early evolution of life, including the enigmatic origin of the eukaryotic cell.

Uncultivated thermophiles: current status and spotlight on 'Aigarchaeota'.

Meta-analysis of cultivation-independent sequence data shows that geothermal systems host an abundance of novel organisms, representing a vast unexplored phylogenetic and functional diversity among yet-uncultivated thermophiles. A number of thermophiles have recently been interrogated using metagenomic and/or single-cell genomic approaches, including members of taxonomic groups that inhabit both thermal and non-thermal environments, such as 'Acetothermia' (OP1) and 'Atribacteria' (OP9/JS1), as well as the exclusively thermophilic lineages 'Korarchaeota', 'Calescamantes' (EM19), 'Fervidibacteria' (OctSpA1-106), and 'Aigarchaeota' (HWCG-I). The 'Aigarchaeota', a sister lineage to the Thaumarchaeota, likely includes both hyperthermophiles and moderate thermophiles. They inhabit terrestrial, marine, and subsurface thermal environments and comprise at least nine genus-level lineages, several of which are globally distributed.

Impact of single-cell genomics and metagenomics on the emerging view of extremophile "microbial dark matter".

Despite >130 years of microbial cultivation studies, many microorganisms remain resistant to traditional cultivation approaches, including numerous candidate phyla of bacteria and archaea. Unraveling the mysteries of these candidate phyla is a grand challenge in microbiology and is especially important in habitats where they are abundant, including some extreme environments and low-energy ecosystems. Over the past decade, parallel advances in DNA amplification, DNA sequencing and computing have enabled rapid progress on this problem, particularly through metagenomics and single-cell genomics. Although each approach suffers limitations, metagenomics and single-cell genomics are particularly powerful when combined synergistically. Studies focused on extreme environments have revealed the first substantial genomic information for several candidate phyla, encompassing putative acidophiles (Parvarchaeota), halophiles (Nanohaloarchaeota), thermophiles (Acetothermia, Aigarchaeota, Atribacteria, Calescamantes, Korarchaeota, and Fervidibacteria), and piezophiles (Gracilibacteria). These data have enabled insights into the biology of these organisms, including catabolic and anabolic potential, molecular adaptations to life in extreme environments, unique genomic features such as stop codon reassignments, and predictions about cell ultrastructure. In addition, the rapid expansion of genomic coverage enabled by these studies continues to yield insights into the early diversification of microbial lineages and the relationships within and between the phyla of Bacteria and Archaea. In the next 5 years, the genomic foliage within the tree of life will continue to grow and the study of yet-uncultivated candidate phyla will firmly transition into the post-genomic era.