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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.

Abstract Members of the candidate phylum Hyd24-12 are globally distributed, but no genomic information or knowledge about their morphology, physiology or ecology is available. In this study, members of the Hyd24-12 lineage were shown to be present and abundant in full-scale mesophilic anaerobic digesters at Danish wastewater treatment facilities. In some samples, a member of the Hyd24-12 lineage was one of the most abundant genus-level bacterial taxa, accounting for up to 8% of the bacterial biomass. Three closely related and near-complete genomes were retrieved using metagenome sequencing of full-scale anaerobic digesters. Genome annotation and metabolic reconstruction showed that they are Gram-negative bacteria likely involved in acidogenesis, producing acetate and hydrogen from fermentation of sugars, and may play a role in the cycling of sulphur in the digesters. Fluorescence in situ hybridization revealed single rod-shaped cells dispersed within the flocs. The genomic information forms a foundation for a more detailed understanding of their role in anaerobic digestion and provides the first insight into a hitherto undescribed branch in the tree of life.

Abstract New and emerging environmental pathogens pose some of the greatest threats to modern aquaculture, a critical source of food protein globally. As with other intensive farming practices, increasing our understanding of the biology of infections is important to improve animal welfare and husbandry. The gill infection epitheliocystis is increasingly problematic in gilthead seabream (Sparus aurata), a major Mediterranean aquaculture species. Epitheliocystis is generally associated with chlamydial bacteria, yet we were not able to localise chlamydial targets within the major gilthead seabream lesions. Two previously unidentified species within a novel β-proteobacterial genus were instead identified. These co-infecting intracellular bacteria have been characterised using high-resolution imaging and genomics, presenting the most comprehensive study on epitheliocystis agents to date. Draft genomes of the two uncultured species, Ca. Ichthyocystis hellenicum and Ca. Ichthyocystis sparus, have been de novo sequenced and annotated from preserved material. Analysis of the genomes shows a compact core indicating a metabolic dependency on the host, and an accessory genome with an unprecedented number of tandemly arrayed gene families. This study represents a critical insight into novel, emerging fish pathogens and will be used to underpin future investigations into the bacterial origins, and to develop diagnostic and treatment strategies.

Abstract Microorganisms in biological wastewater treatment plants require adaptive strategies to deal with rapidly fluctuating environmental conditions. At the population level, the filamentous bacterium Candidatus Microthrix parvicella (Ca. M. parvicella) has been found to fine-tune its gene expression for optimized substrate assimilation. Here we investigated in situ substrate assimilation by single cells of Ca. M. parvicella using nano-scale secondary-ion mass spectrometry (nanoSIMS). NanoSIMS imaging highlighted phenotypic heterogeneity among Ca. M. parvicella cells of the same filament, whereby 13C-oleic acid and 13C-glycerol-3-phosphate assimilation occurred in ≈21–55% of cells, despite non-assimilating cells being intact and alive. In response to alternating aerobic–anoxic regimes, 13C-oleic acid assimilation occurred among subpopulations of Ca. M. parvicella cells (≈3–28% of cells). Furthermore, Ca. M. parvicella cells exhibited two temperature optima for 13C-oleic acid assimilation and associated growth rates. These results suggest that phenotypic heterogeneity among Ca. M. parvicella cells allows the population to adapt rapidly to fluctuating environmental conditions facilitating its widespread occurrence in biological wastewater treatment plants.

Abstract The candidate archaeal phylum ‘Aigarchaeota’ contains microorganisms from terrestrial and subsurface geothermal ecosystems. The phylogeny and metabolic potential of Aigarchaeota has been deduced from several recent single-cell amplified genomes; however, a detailed description of their metabolic potential and in situ transcriptional activity is absent. Here, we report a comprehensive metatranscriptome-based reconstruction of the in situ metabolism of Aigarchaeota in an oxic, hot spring filamentous ‘streamer’ community. Fluorescence in situ hybridization showed that these newly discovered Aigarchaeota are filamentous, which is consistent with the presence and transcription of an actin-encoding gene. Aigarchaeota filaments are intricately associated with other community members, which include both bacteria (for example, filamentous Thermocrinis spp.) and archaea. Metabolic reconstruction of genomic and metatranscriptomic data suggests that this aigarchaeon is an aerobic, chemoorganoheterotroph with autotrophic potential. A heme copper oxidase complex was identified in the environmental genome assembly and highly transcribed in situ. Potential electron donors include acetate, fatty acids, amino acids, sugars and aromatic compounds, which may originate from extracellular polymeric substances produced by other microorganisms shown to exist in close proximity and/or autochthonous dissolved organic carbon (OC). Transcripts related to genes specific to each of these potential electron donors were identified, indicating that this aigarchaeon likely utilizes several OC substrates. Characterized members of this lineage cannot synthesize heme, and other cofactors and vitamins de novo, which suggests auxotrophy. We propose the name Candidatus ‘Calditenuis aerorheumensis’ for this aigarchaeon, which describes its filamentous morphology and its primary electron acceptor, oxygen.

A Gram-stain-negative, non-motile, non-spore-forming, long rod-shaped bacterium, designated strain GYP-15T, was isolated from the culture broth of a marine microalga,Picochlorumasp. 122. Phylogenetic analyses revealed that strain GYP-15Tshared 90.6 % 16S rRNA gene sequence similarity with its closest relative,Kangiella aquimarinaKCTC 12183T, and represents a distinct phylogenetic lineage in a robust clade consisting of GYP-15Tand members of the generaKangiellaandPleioneain the orderOceanospirillales. Chemotaxonomic and physiological characteristics, including major cellular fatty acids, NaCl tolerance and pattern of carbon source utilization, could also readily distinguish strain GYP-15Tfrom all established genera and species. Thus, it is concluded that strain GYP-15Trepresents a novel species of a new genus, for which the nameAliikangiella marinagen. nov., sp. nov. is proposed. The type strain ofAliikangiella marinais GYP-15T( = MCCC 1K01163T = KCTC 42667T). Based on phylogenetic results, 16S rRNA gene signature nucleotide pattern and some physiological characteristics, the three generaKangiella,PleioneaandAliikangiellaare proposed to make up a novel family,Kangiellaceaefam. nov., in the orderOceanospirillales.

Abstract The bacterial family Rickettsiaceae includes a group of well-known etiological agents of many human and vertebrate diseases, including epidemic typhus-causing pathogen Rickettsia prowazekii. Owing to their medical relevance, rickettsiae have attracted a great deal of attention and their host-pathogen interactions have been thoroughly investigated. All known members display obligate intracellular lifestyles, and the best-studied genera, Rickettsia and Orientia, include species that are hosted by terrestrial arthropods. Their obligate intracellular lifestyle and host adaptation is reflected in the small size of their genomes, a general feature shared with all other families of the Rickettsiales. Yet, despite that the Rickettsiaceae and other Rickettsiales families have been extensively studied for decades, many details of the origin and evolution of their obligate host-association remain elusive. Here we report the discovery and single-cell sequencing of ‘Candidatus Arcanobacter lacustris’, a rare environmental alphaproteobacterium that was sampled from Damariscotta Lake that represents a deeply rooting sister lineage of the Rickettsiaceae. Intriguingly, phylogenomic and comparative analysis of the partial ‘Candidatus Arcanobacter lacustris’ genome revealed the presence chemotaxis genes and vertically inherited flagellar genes, a novelty in sequenced Rickettsiaceae, as well as several host-associated features. This finding suggests that the ancestor of the Rickettsiaceae might have had a facultative intracellular lifestyle. Our study underlines the efficacy of single-cell genomics for studying microbial diversity and evolution in general, and for rare microbial cells in particular.

Abstract Methylotrophic planktonic bacteria fulfill a particular role in the carbon cycle of lakes via the turnover of single-carbon compounds. We studied two planktonic freshwater lineages (LD28 and PRD01a001B) affiliated with Methylophilaceae (Betaproteobacteria) in Lake Zurich, Switzerland, by a combination of molecular and cultivation-based approaches. Their spatio-temporal distribution was monitored at high resolution (n=992 samples) for 4 consecutive years. LD28 methylotrophs constituted up to 11 × 107 cells l−1 with pronounced peaks in spring and autumn–winter, concomitant with blooms of primary producers. They were rare in the warm water layers during summer but abundant in the cold hypolimnion, hinting at psychrophilic growth. Members of the PRD01a001B lineage were generally less abundant but also had maxima in spring. More than 120 axenic strains from these so far uncultivated lineages were isolated from the pelagic zone by dilution to extinction. Phylogenetic analysis separated isolates into two distinct genotypes. Isolates grew slowly (μmax=0.4 d−1), were of conspicuously small size, and were indeed psychrophilic, with higher growth yield at low temperatures. Growth was enhanced upon addition of methanol and methylamine to sterile lake water. Genomic analyses of two strains confirmed a methylotrophic lifestyle with a reduced set of genes involved in C1 metabolism. The very small and streamlined genomes (1.36 and 1.75 Mb) shared several pathways with the marine OM43 lineage. As the closest described taxa (Methylotenera sp.) are only distantly related to either set of isolates, we propose a new genus with two species, that is, ‘Candidatus Methylopumilus planktonicus’ (LD28) and ‘Candidatus Methylopumilus turicensis’ (PRD01a001B).

Abstract Fri'tsche.a. N.L. fem. n. Fritschea named after Thomas R. Fritsche, an American physician and parasitologist, in honor of his contributions to our current knowledge on chlamydial diversity. Coccoid to rod‐shaped, nonmotile, obligately intracellular bacteria, up to 2.5 μm in length . Cells show a developmental cycle with morphologically distinct stages similar to those of the Chlamydiaceae . Organisms were found in whitefly and scale insects, but have not yet been obtained in cell culture. The genus is based on the description of “ Candidatus Fritschea bemisiae” and “ Candidatus Fritschea eriococci” (Everett et al., 2005; Thao et al., 2003) (Figure 1). Type species : “ Candidatus Fritschea bemisiae ” Everett, Thao, Horn, Dyszynski and Baumann 2005, 1585. Taxonomic and Nomenclature Notes According to the List of Prokaryotic names with Standing in Nomenclature (LPSN), the taxonomic status of the genus Candidatus Fritschea is: preferred name (not correct name) (last update, February 2025) * . LPSN classification: Bacteria / Pseudomonadati / Chlamydiota / Chlamydiia / Chlamydiales / Simkaniaceae / Candidatus Fritschea Candidatus Fritschea 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

Abstract Me.tha.no.bre.vi.bac'ter. M.L. neut. n. methanum methane; L. masc. adj. brevis short; M.L. masc. n. bacter equivalent of Gr. neut. n. bakterion rod, staff; M.L. masc. n. Methanobrevibacter short methane (‐producing) rod. Oval rods or cocci to short rods , usually occurring in pairs or chains; about 0.5–0.7 µm in width and 0.8–1.4 µm in length. Rarely, filaments are formed. Nonsporing, Gram‐positive. Cell walls are composed of pseudomurein . Nonmotile. Strict anaerobes. Optimum temperature, 37–40°C; maximum, ~45°C; minimum, ~30°C. The mol % G + C of the DNA is : 27.5–31.6. Type species : Methanobrevibacter ruminantium (Smith and Hungate 1958) Balch and Wolfe 1981, 216 (Effective publication: Balch and Wolfe in Balch, Fox, Magrum, Woese and Wolfe, 1979, 284) ( Methanobacterium ruminantium Smith and Hungate 1958, 717). Taxonomic and Nomenclature Notes According to the List of Prokaryotic names with Standing in Nomenclature (LPSN), the taxonomic status of the genus Methanobrevibacter is: correct name (last update, February 2025) * . LPSN classification: Archaea / Methanobacteriati / Methanobacteriota / Methanobacteria / Methanobacteriales / Methanobacteriaceae / Methanobrevibacter The genus Methanobrevibacter can also be recovered in the Genome Taxonomy Database (GTDB) as g__Methanobrevibacter (version v220) ** . GTDB classification: d__Archaea / p__Methanobacteriota / c__Methanobacteria / o__Methanobacteriales / f__Methanobacteriaceae / g__Methanobrevibacter * 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