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Abstract Actinobacteria of the acI lineage are the most abundant microbes in freshwater systems, but there are so far no pure living cultures of these organisms, possibly because of metabolic dependencies on other microbes. This, in turn, has hampered an in-depth assessment of the genomic basis for their success in the environment. Here we present genomes from 16 axenic cultures of acI Actinobacteria. The isolates were not only of minute cell size, but also among the most streamlined free-living microbes, with extremely small genome sizes (1.2–1.4 Mbp) and low genomic GC content. Genome reduction in these bacteria might have led to auxotrophy for various vitamins, amino acids and reduced sulphur sources, thus creating dependencies to co-occurring organisms (the ‘Black Queen’ hypothesis). Genome analyses, moreover, revealed a surprising degree of inter- and intraspecific diversity in metabolic pathways, especially of carbohydrate transport and metabolism, and mainly encoded in genomic islands. The striking genotype microdiversification of acI Actinobacteria might explain their global success in highly dynamic freshwater environments with complex seasonal patterns of allochthonous and autochthonous carbon sources. We propose a new order within Actinobacteria (‘Candidatus Nanopelagicales’) with two new genera (‘Candidatus Nanopelagicus’ and ‘Candidatus Planktophila’) and nine new species.

Abstract Thaumarchaeota have been detected in several industrial and municipal wastewater treatment plants (WWTPs), despite the fact that ammonia-oxidizing archaea (AOA) are thought to be adapted to low ammonia environments. However, the activity, physiology and metabolism of WWTP-associated AOA remain poorly understood. We report the cultivation and complete genome sequence of Candidatus Nitrosocosmicus exaquare, a novel AOA representative from a municipal WWTP in Guelph, Ontario (Canada). In enrichment culture, Ca. N. exaquare oxidizes ammonia to nitrite stoichiometrically, is mesophilic, and tolerates at least 15 mm of ammonium chloride or sodium nitrite. Microautoradiography (MAR) for enrichment cultures demonstrates that Ca. N. exaquare assimilates bicarbonate in association with ammonia oxidation. However, despite using inorganic carbon, the ammonia-oxidizing activity of Ca. N. exaquare is greatly stimulated in enrichment culture by the addition of organic compounds, especially malate and succinate. Ca. N. exaquare cells are coccoid with a diameter of ~1–2 μm. Phylogenetically, Ca. N. exaquare belongs to the Nitrososphaera sister cluster within the Group I.1b Thaumarchaeota, a lineage which includes most other reported AOA sequences from municipal and industrial WWTPs. The 2.99 Mbp genome of Ca. N. exaquare encodes pathways for ammonia oxidation, bicarbonate fixation, and urea transport and breakdown. In addition, this genome encodes several key genes for dealing with oxidative stress, including peroxidase and catalase. Incubations of WWTP biofilm demonstrate partial inhibition of ammonia-oxidizing activity by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), suggesting that Ca. N. exaquare-like AOA may contribute to nitrification in situ. However, CARD-FISH-MAR showed no incorporation of bicarbonate by detected Thaumarchaeaota, suggesting that detected AOA may incorporate non-bicarbonate carbon sources or rely on an alternative and yet unknown metabolism.

A recently described symbiosis between the metabolically streamlined nitrogen‐fixing cyanobacterium UCYN‐A and a single‐celled eukaryote prymnesiophyte alga is widely distributed throughout tropical and subtropical marine waters, and is thought to contribute significantly to nitrogen fixation in these regions. Several UCYN‐A sublineages have been defined based on UCYN‐A nitrogenase (nifH) sequences. Due to the low abundances of UCYN‐A in the global oceans, currently existing molecular techniques are limited for detecting and quantifying these organisms. A targeted approach is needed to adequately characterize the diversity of this important marine cyanobacterium, and to advance understanding of its ecological importance. We present findings on the distribution of UCYN‐A sublineages based on high throughput sequencing of UCYN‐A nifH PCR amplicons from 78 samples distributed throughout many major oceanic provinces. These UCYN‐A nifH fragments were used to define oligotypes, alternative taxonomic units defined by nucleotide positions with high variability. The data set was dominated by a single oligotype associated with the UCYN‐A1 sublineage, consistent with previous observations of relatively high abundances in tropical and subtropical regions. However, this analysis also revealed for the first time the widespread distribution of the UCYN‐A3 sublineage in oligotrophic waters. Furthermore, distinct assemblages of UCYN‐A oligotypes were found in oligotrophic and coastally influenced waters. This unique data set provides a framework for determining the environmental controls on UCYN‐A distributions and the ecological importance of the different sublineages.

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

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

Candidatus Midichloria mitochondrii is the sheep tick Ixodes ricinus endosymbiont. This unique bacteria can occupy and persist within the mitochondria of animals. I. ricinus is an important vector of human pathogens in natural focal of infections. Candidatus M. mitochondrii found in the intermembrane space of mitochondria and in the cytoplasm of ovarian cells in 100% females of I. ricinus. The bacteria contain flagella in the salivary glands of ticks. Candidatus M. mitochondrii has two groups of unique genes for the members of the order Rickettsiales (cbb3 cytochrome oxidase and flagellin), which allows it to play an important role in embryogenesis of the I. ricinus ticks and cause seroconversion in 58% of patients after ticks bloodsucking. This bacterium formed MALOs group (midichloria and like organisms) with genetically closely related organisms which demonstrated a association with a wide range of host from arthropods to ciliates, amoebae, sponges, fish and various animals and humans. Now there is no data about replication the Candidatus M. mitochondrii in humans and pathogenicity of this microorganism. Although a high percentage of seropositive samples obtained from patients after bloodsucking of I. ricinus in anamnesis, this bacterium cannot yet be regarded as responsible for the pathology as known human pathogenic from order Rickettsi-ales (Rickettsia, Anaplasma and Ehrlichia spp.). Needed to reconsider the attitude to an immune response to the saliva of I. ricinus, taking into account the potential impact of Candidatus M. mitochondrii. It is considered highly possible role of this bacterium in the immune response and immunomodulation in humans with bloodsucking of I. ricinus in anamnesis. DNA of Candidatus M. mitochondrii was the first time detected in I. ricinus ticks from European part of Russia.

Abstract Cel.lu.lo.si.ly.ti.ca.ce'ae. N.L. neut. n. Cellulosilyticum a bacterial genus; N.L. fem. pl. n. Cellulosilyticaceae the Cellulosilyticum family. Slightly curved rod‐shaped cells . Gram‐positive cell‐wall structure but stain Gram‐negative. Terminal spores formed. Mesophilic, optimum growth temperature is approximately 40°C. Obligate anaerobic . No microaerophilic or aerobic growth occurs. Produce acetate, ethanol, and/or formate as the major end products, but not butyric or propionic acid. Hydrolyze cellulose and xylan and ferment cellobiose, but not ferment lactate and pyruvate. The predominant fatty acids are C 16:0 (27.1 %), C 14:0 (9.2 %), and iso‐C 16:0 (6.4%). Phylogenetically distant from the family Lachnospriaceae (Clostridum cluster XIVb), a member of the phylum Firmicutes, with less than 87.7% 16S rRNA similarity. Thus far, only one genus Cellulosilyticum is described. DNA G + C content (mol%) : 33–36. Type genus : Cellulosilyticum Cai and Dong 2010, 848 VP . Taxonomic and Nomenclature Notes According to the List of Prokaryotic names with Standing in Nomenclature (LPSN), the taxonomic status of the family Cellulosilyticaceae is: synonym (last update, February 2025) * . Correct name: Lachnospiraceae LPSN classification: Bacteria / Bacillati / Bacillota / Clostridia / Eubacteriales / Lachnospiraceae The family Cellulosilyticaceae can also be recovered in the Genome Taxonomy Database (GTDB) as f__Cellulosilyticaceae (version v220) ** . GTDB classification: d__Bacteria / p__Bacillota_A / c__Clostridia / o__Lachnospirales / f__Cellulosilyticaceae * 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 The recently isolated strain L21-Fru-ABT represents moderately halophilic, obligately anaerobic and saccharolytic bacteria that thrive in the suboxic transition zones of hypersaline microbial mats. Phylogenetic analyses based on 16S rRNA genes, RpoB proteins and gene content indicated that strain L21-Fru-ABT represents a novel species and genus affiliated with a distinct phylum-level lineage originally designated Verrucomicrobia subdivision 5. A survey of environmental 16S rRNA gene sequences revealed that members of this newly recognized phylum are wide-spread and ecologically important in various anoxic environments ranging from hypersaline sediments to wastewater and the intestine of animals. Characteristic phenotypic traits of the novel strain included the formation of extracellular polymeric substances, a Gram-negative cell wall containing peptidoglycan and the absence of odd-numbered cellular fatty acids. Unusual metabolic features deduced from analysis of the genome sequence were the production of sucrose as osmoprotectant, an atypical glycolytic pathway lacking pyruvate kinase and the synthesis of isoprenoids via mevalonate. On the basis of the analyses of phenotypic, genomic and environmental data, it is proposed that strain L21-Fru-ABT and related bacteria are specifically adapted to the utilization of sulfated glycopolymers produced in microbial mats or biofilms.

Abstract Tenericutes are a unique class of bacteria that lack a cell wall and are typically parasites or commensals of eukaryotic hosts. Environmental 16S rDNA surveys have identified a number of tenericute clades in diverse environments, introducing the possibility that these Tenericutes may represent non-host-associated, free-living microorganisms. Metagenomic sequencing of deep-sea methane seep sediments resulted in the assembly of two genomes from a Tenericutes-affiliated clade currently known as ‘NB1-n’ (SILVA taxonomy) or ‘RF3’ (Greengenes taxonomy). Metabolic reconstruction revealed that, like cultured members of the Mollicutes, these ‘NB1-n’ representatives lack a tricarboxylic acid cycle and instead use anaerobic fermentation of simple sugars for substrate level phosphorylation. Notably, the genomes also contained a number of unique metabolic features including hydrogenases and a simplified electron transport chain containing an RNF complex, cytochrome bd oxidase and complex I. On the basis of the metabolic potential predicted from the annotated genomes, we devised an anaerobic enrichment media that stimulated the growth of these Tenericutes at 10 °C, resulting in a mixed culture where these organisms represented ~60% of the total cells by targeted fluorescence in situ hybridization (FISH). Visual identification by FISH confirmed these organisms were not directly associated with Eukaryotes and electron cryomicroscopy of cells in the enrichment culture confirmed an ultrastructure consistent with the defining phenotypic property of Tenericutes, with a single membrane and no cell wall. On the basis of their unique gene content, phylogenetic placement and ultrastructure, we propose these organisms represent a novel class within the Tenericutes, and suggest the names Candidatus ‘Izimaplasma sp. HR1’ and Candidatus ‘Izimaplasma sp. HR2’ for the two genome representatives.