Hedlund, Brian P.

Recent advances in sequencing technology promoted the blowout discovery of super tiny microbes in the Diapherotrites , Parvarchaeota , Aenigmarchaeota , Nanoarchaeota , and Nanohaloarchaeota (DPANN) superphylum. However, the unculturable properties of the majority of microbes impeded our investigation of their behavior and symbiotic lifestyle in the corresponding community.

“Candidatus Nitrosocaldaceae” are globally distributed in neutral or slightly alkaline hot springs and geothermally heated soils. Despite their essential role in the nitrogen cycle in high-temperature ecosystems, they remain poorly understood because they have never been isolated in pure culture, and very few genomes are available. In the present study, a metagenomics approach was employed to obtain “Ca. Nitrosocaldaceae” metagenomic-assembled genomes (MAGs) from hot spring samples collected from India and China. Phylogenomic analysis placed these MAGs within “Ca. Nitrosocaldaceae.” Average nucleotide identity and average amino acid identity analysis suggested the new MAGs represent two novel species of “Candidatus Nitrosocaldus” and a novel genus, herein proposed as “Candidatus Nitrosothermus.” Key genes responsible for chemolithotrophic ammonia oxidation and a thaumarchaeal 3HP/4HB cycle were detected in all MAGs. Furthermore, genes coding for urea degradation were only present in “Ca. Nitrosocaldus,” while biosynthesis of the vitamins, biotin, cobalamin, and riboflavin were detected in almost all MAGs. Comparison of “Ca. Nitrosocaldales/Nitrosocaldaceae” with other AOA revealed 526 specific orthogroups. This included genes related to thermal adaptation (cyclic 2,3-diphosphoglycerate, and S-adenosylmethionine decarboxylase), indicating their importance for life at high temperature. In addition, these MAGs acquired genes from members from archaea (Crenarchaeota) and bacteria (Firmicutes), mainly involved in metabolism and stress responses, which might play a role to allow this group to adapt to thermal habitats.

In prokaryotic taxonomy, a set of criteria is commonly used to delineate species. These criteria are generally based on cohesion at the phylogenetic, phenotypic and genomic levels. One such criterion shown to have promise in the genomic era is average nucleotide identity (ANI), which provides an average measure of similarity across homologous regions shared by a pair of genomes. However, despite the popularity and relative ease of using this metric, ANI has undergone numerous refinements, with variations in genome fragmentation, homologue detection parameters and search algorithms. To test the robustness of a 95–96 % species cut-off range across all the commonly used ANI approaches, seven different methods were used to calculate ANI values for intra- and interspecies datasets representing three classes in the Proteobacteria . As a reference point, these methods were all compared to the widely used blast-based ANI (i.e. ANIb as implemented in JSpecies), and regression analyses were performed to investigate the correlation of these methods to ANIb with more than 130000 individual data points. From these analyses, it was clear that ANI methods did not provide consistent results regarding the conspecificity of isolates. Most of the methods investigated did not correlate perfectly with ANIb, particularly between 90 and 100% identity, which includes the proposed species boundary. There was also a difference in the correlation of methods for the different taxon sets. Our study thus suggests that the specific approach employed needs to be considered when ANI is used to delineate prokaryotic species. We furthermore suggest that one would first need to determine an appropriate cut-off value for a specific taxon set, based on the intraspecific diversity of that group, before conclusions on conspecificity of isolates can be made, and that the resulting species hypotheses be confirmed with analyses based on evolutionary history as part of the polyphasic approach to taxonomy.

Abstract Background: ‘Ca. Aenigmarchaeota’ represents an evolutionary branch within the DPANN superphylum. However, their ecological roles and potential host-symbiont interactions are poorly understood.Results: Here, we analyze eight metagenomic-assembled genomes from hot spring habitats and reveal their functional potentials. Although they have limited metabolic capacities, they harbor substantial carbohydrate metabolizing abilities. Further investigation suggests that horizontal gene transfer might be the main driver that endows these abilities to ‘Ca. Aenigmarchaeota’, including enzymes involved in glycolysis. Additionally, members from the TACK superphylum and Euryarchaeota contribute substantially to the niche expansion of ‘Ca. Aenigmarchaeota’, especially genes related to carbohydrate metabolism and stress responses. Based on co-occurrence network analysis, we conjecture that ‘Ca. Aenigmarchaeota’ may be symbionts associated with TACK archaea and Euryarchaeota, though host-specificity might be wide and variable across different ‘Ca. Aenigmarchaeota’ genomes. Conclusion: This study provides significant insights into possible host-symbiont interactions and ecological roles of ‘Ca. Aenigmarchaeota’.

AbstractSeveral recent studies have shown the presence of genes for the key enzyme associated with archaeal methane/alkane metabolism, methyl-coenzyme M reductase (Mcr), in metagenome-assembled genomes (MAGs) divergent to existing archaeal lineages. Here, we study the mcr-containing archaeal MAGs from several hot springs, which reveal further expansion in the diversity of archaeal organisms performing methane/alkane metabolism. Significantly, an MAG basal to organisms from the phylum Thaumarchaeota that contains mcr genes, but not those for ammonia oxidation or aerobic metabolism, is identified. Together, our phylogenetic analyses and ancestral state reconstructions suggest a mostly vertical evolution of mcrABG genes among methanogens and methanotrophs, along with frequent horizontal gene transfer of mcr genes between alkanotrophs. Analysis of all mcr-containing archaeal MAGs/genomes suggests a hydrothermal origin for these microorganisms based on optimal growth temperature predictions. These results also suggest methane/alkane oxidation or methanogenesis at high temperature likely existed in a common archaeal ancestor.

Abstract Ni.tro.so.cal'dus. N.L. adj. nitrosus nitrous; L. masc. adj. caldus hot; N.L. masc. n. Nitrosocaldus a nitrite producer at thermophilic growth temperatures. Thermophilic, neutrophilic, autotrophic, and aerobic ammonia‐oxidizing archaea. Urea can be used as a source of energy for growth. Cells fix CO 2 via a modified 3‐hydroxypropionate/4‐hydroxybutyrate carbon fixation pathway. Cells are irregular cocci between 0.65 and 0.75 microns in diameter. Cells reproduce by binary fission. Cell membrane lipids consist mainly of acyclic and cyclized glycerol trialkyl glycerol tetraethers (GTGTs) and glycerol dibiphytanyl glycerol tetraethers (GDGTs), with acyclic GTGT and crenarchaeol as the dominant components. Quinone systems include menaquinones with fully saturated and monounsaturated side chains that comprised six isoprenoid units. The members of the genus Ca . Nitrosocaldus can be found in neutral or slightly alkaline terrestrial geothermal environments at temperatures ranging from 65 to 85°C. Phylogenetic analysis on the basis of both 16S rRNA and amoA genes supports the monophyletic genus Ca . Nitrosocaldus within the family Ca . Nitrosocaldaceae. DNA G + C content (mol%) : 37.1. Type species : Ca . Nitrosocaldus yellowstonensis (Nitrosocaldus yellowstonii) de la Torre, Walker, Ingalls, Könneke and Stahl 2008, 815. Taxonomic and Nomenclature Notes According to the List of Prokaryotic names with Standing in Nomenclature (LPSN), the taxonomic status of the genus Candidatus Nitrosocaldus is: preferred name (not correct name) (last update, February 2025) * . LPSN classification: Archaea / Thermoproteati / Thermoproteota / incertae sedis / Candidatus Nitrosocaldales / Candidatus Nitrosocaldaceae / Candidatus Nitrosocaldus The genus Candidatus Nitrosocaldus can also be recovered in the Genome Taxonomy Database (GTDB) as g__Nitrosocaldus (version v220) ** . GTDB classification: d__Archaea / p__Thermoproteota / c__Nitrososphaeria / o__Nitrososphaerales / f__Nitrosocaldaceae / g__Nitrosocaldus * Meier‐Kolthoff et al. ( 2022 ). Nucleic Acids Res , 50 , D801 – D807 ; DOI: 10.1093/nar/gkab902 ** Parks et al. ( 2022 ). Nucleic Acids Res , 50 , D785 – D794 ; DOI: 10.1093/nar/gkab776

Abstract Ni.tro.so.cal.da'les. N.L. masc. n. Nitrosocaldus type genus of the order; ‐ ales ending to denote order; N.L. fem. pl. n. Nitrosocaldales the Nitrosocaldus order. Coccoid cells reproduce by binary fission. Thermophilic and neutrophilic. Obligately aerobic. Chemolithoautotrophic growth by ammonia oxidation to nitrite using CO 2 as carbon source. Some members can also use urea as an alternative source of energy for growth. Cell membrane is composed of isoprenoid tetraether lipids, including large amounts of acyclic and cyclized glycerol trialkyl glycerol tetraethers (GTGTs) and crenarchaeol (a unique glycerol dibiphytanyl glycerol tetraether (GDGT) containing one cyclohexane ring and four cyclopentane rings). Fully saturated and monounsaturated menaquinones with six isoprenoid units are present. They are globally distributed in various geothermal environments. Phylogenetic analyses of 16S rRNA and amoA gene sequences place the monophyletic order Ca . Nitrosocaldales as a basal lineage within the phylum Thaumarchaeota . This order can be distinguished from the closest order Nitrososphaerales by a lower mol% G + C content, a higher growth temperature (>60°C), and a higher relative abundance of GTGT. The order Ca . Nitrosocaldales is currently represented by a single family Ca . Nitrosocaldaceae. Type genus : Ca . Nitrosocaldus de la Torre, Walker, Ingalls, Könneke and Stahl 2008, 815. Taxonomic and Nomenclature Notes According to the List of Prokaryotic names with Standing in Nomenclature (LPSN), the taxonomic status of the order Candidatus Nitrosocaldales is: preferred name (not correct name) (last update, February 2025) * . LPSN classification: Archaea / Thermoproteati / Thermoproteota / incertae sedis / Candidatus Nitrosocaldales The order Candidatus Nitrosocaldales can also be recovered in the Genome Taxonomy Database (GTDB) as o__Nitrososphaerales (version v220) ** . GTDB classification: d__Archaea / p__Thermoproteota / c__Nitrososphaeria / o__Nitrososphaerales * Meier‐Kolthoff et al. ( 2022 ). Nucleic Acids Res , 50 , D801 – D807 ; DOI: 10.1093/nar/gkab902 ** Parks et al. ( 2022 ). Nucleic Acids Res , 50 , D785 – D794 ; DOI: 10.1093/nar/gkab776

Abstract Ni.tro.so.cal.da.ce'ae. N.L. masc. n. Nitrosocaldus type genus of the family; ‐ aceae ending to denote family; N.L. fem. pl. n. Nitrosocaldaceae the Nitrosocaldus family. Cells are irregular cocci and divide by binary fission. Strictly aerobic. Thermophilic, neutrophilic, and lithoautotrophic archaea that are able to gain energy via ammonia oxidation to nitrite. Membrane lipids contain glycerol dibiphytanyl glycerol tetraethers (GDGTs) bound to monoglycosidic, diglycosidic, phosphohexose, and hexose‐phosphohexose headgroups. Like other described Thaumarchaeota , members of this family contain crenarchaeol as a major membrane core lipid. However, their membrane lipids are characterized by the presence of the high abundances of acyclic and cyclized glycerol trialkyl glycerol tetraethers (GTGTs). Respiratory lipoquinones are saturated and monounsaturated menaquinones with six isoprenoid units. Cells and molecular markers have been found in neutral or slightly alkaline geothermal environments, such as hot springs and subsurface gold mines. At present, the family Ca . Nitrosocaldaceae only harbors a single genus, which is so far represented by only one species. Therefore, future phylogenetic analysis of additional cultivated representatives may reveal further subdivisions within the family. DNA G+C content (mol%):36.8–41.7%. Type genus: Candidatus Nitrosocaldus de la Torre, Walker, Ingalls, Könneke and Stahl 2008, 815. Taxonomic and Nomenclature Notes According to the List of Prokaryotic names with Standing in Nomenclature (LPSN), the taxonomic status of the family Candidatus Nitrosocaldaceae is: preferred name (not correct name) (last update, February 2025) * . LPSN classification: Archaea / Thermoproteati / Thermoproteota / incertae sedis / Candidatus Nitrosocaldales / Candidatus Nitrosocaldaceae The family Candidatus Nitrosocaldaceae can also be recovered in the Genome Taxonomy Database (GTDB) as f__Nitrosocaldaceae (version v220) ** . GTDB classification: d__Archaea / p__Thermoproteota / c__Nitrososphaeria / o__Nitrososphaerales / f__Nitrosocaldaceae * Meier‐Kolthoff et al. ( 2022 ). Nucleic Acids Res , 50 , D801 – D807 ; DOI: 10.1093/nar/gkab902 ** Parks et al. ( 2022 ). Nucleic Acids Res , 50 , D785 – D794 ; DOI: 10.1093/nar/gkab776

AbstractAnalysis of the increasing wealth of metagenomic data collected from diverse environments can lead to the discovery of novel branches on the tree of life. Here we analyse 5.2 Tb of metagenomic data collected globally to discover a novel bacterial phylum (‘Candidatus Kryptonia’) found exclusively in high-temperature pH-neutral geothermal springs. This lineage had remained hidden as a taxonomic ‘blind spot’ because of mismatches in the primers commonly used for ribosomal gene surveys. Genome reconstruction from metagenomic data combined with single-cell genomics results in several high-quality genomes representing four genera from the new phylum. Metabolic reconstruction indicates a heterotrophic lifestyle with conspicuous nutritional deficiencies, suggesting the need for metabolic complementarity with other microbes. Co-occurrence patterns identifies a number of putative partners, including an uncultured Armatimonadetes lineage. The discovery of Kryptonia within previously studied geothermal springs underscores the importance of globally sampled metagenomic data in detection of microbial novelty, and highlights the extraordinary diversity of microbial life still awaiting discovery.

Abstract Xi.phi.ne.ma.to.bac'ter. Gr. neut. n. Xiphinema, ‐atos the genus name of the host organism; N.L. masc. n. bacter the equivalent of Gr. neut. n. baktron a rod; N.L. masc. n. Xiphinematobacter the rod‐shaped microbe associated with Xiphinema . Full‐grown cells are rod‐shaped with rounded ends, 0.7–1.0 × 2.1–3.2 μm ; however, cells in the J 1 (first juvenile) stage of nematode development have a wrinkled, pleomorphic shape. The longer entities usually consist of a mother cell from which a daughter cell is budding, giving rise to serial pairs typical of this bacterial genus. In thin sections cells have two or three membranes consisting of, from inside to outside, a cytoplasmic membrane, an electron‐dense outer membrane, and, in many individuals, a vacuolar membrane which is probably derived from the host cell membrane and which often shows discontinuities. No peptidoglycan layer is evident ; however, a periplasmic hexagonally arrayed monolayer of 10 nm protein units is sometimes present. Gram‐stain‐negative, nonmotile, and nonsporulating. DNA is often condensed at cell poles . Bacteria live as obligate cytoplasmic symbionts with maternal transmission in nematodes of the Xiphinema americanum group ( Nematoda, Longidoridae ), in which they are presumed to induce thelytokous ( mother‐to‐daughter ) parthenogenesis . DNA G + C content ( mol %): not determined. Type species : “ Candidatus Xiphinematobacter brevicolli ” Vandekerckhove, Willems, Gillis and Coomans 2000, 2203. Taxonomic and Nomenclature Notes According to the List of Prokaryotic names with Standing in Nomenclature (LPSN), the taxonomic status of the genus Candidatus Xiphinematobacter is: preferred name (not correct name) (last update, February 2025) * . LPSN classification: Bacteria / Pseudomonadati / Verrucomicrobiota / Terrimicrobiia / Terrimicrobiales / Chthoniobacteraceae / Candidatus Xiphinematobacter Candidatus Xiphinematobacter could not be recovered in GTDB ** . * Meier‐Kolthoff et al. ( 2022 ). Nucleic Acids Res , 50 , D801 – D807 ; DOI: 10.1093/nar/gkab902 ** Parks et al. ( 2022 ). Nucleic Acids Res , 50 , D785 – D794 ; DOI: 10.1093/nar/gkab776