Summary: A novel moderately thermophilic, facultatively anaerobic chemoorganotrophic bacterium strain P3M ‐2 ^T was isolated from a microbial mat developing on the wooden surface of a chute under the flow of hot water (46° C ) coming out of a 2775‐m‐deep oil exploration well ( T omsk region, R ussia). Strain P3M ‐2 ^T is a moderate thermophile and facultative anaerobe growing on mono‐, di‐ or polysaccharides by aerobic respiration, fermentation or by reducing diverse electron acceptors [nitrite, F e( III ), As ( V )]. Its closest cultivated relative (90.8% rRNA gene sequence identity) is I gnavibacterium album , the only chemoorganotrophic member of the phylum C hlorobi . New genus and species M elioribacter roseus are proposed for isolate P3M ‐2 ^T . Together with I . album , the new organism represents the class I gnavibacteria assigned to the phylum Chlorobi . The revealed group includes a variety of uncultured environmental clones, the 16S rRNA gene sequences of some of which have been previously attributed to the candidate division ZB1 . Phylogenetic analysis of M . roseus and I . album based on their 23S rRNA and RecA sequences confirmed that these two organisms could represent an even deeper, phylum‐level lineage. Hence, we propose a new phylum Ignavibacteriae within the Bacteroidetes – C hlorobi group with a sole class I gnavibacteria , two families I gnavibacteriaceae and M elioribacteraceae and two species I . album and M . roseus . This proposal correlates with chemotaxonomic data and phenotypic differences of both organisms from other cultured representatives of C hlorobi . The most essential differences, supported by the analyses of complete genomes of both organisms, are motility, facultatively anaerobic and obligately organotrophic mode of life, the absence of chlorosomes and the apparent inability to grow phototrophically.

A novel obligately anaerobic, mesophilic, organotrophic bacterium, strain P3M-1^T, was isolated from a microbial mat formed in a wooden bath filled with hot water emerging from a 2775 m-deep well in the Tomsk region of western Siberia, Russia. Cells of strain P3M-1^Twere rod-shaped, 0.3–0.7 µm in width and formed multicellullar filaments that reached up to 400 µm in length. Strain P3M-1^Tgrew optimally at 42–45 °C, pH 7.5–8.0, and with 0.1% (w/v) NaCl. Under optimal conditions, the doubling time was 6 h. The isolate was able to ferment a variety of proteinaceous substrates and sugars, including microcrystalline cellulose. Acetate, ethanol and H₂were the main products of glucose fermentation. The genomic DNA G+C content was 55 mol%. 16S rRNA gene sequence-based phylogenetic analyses showed that strain P3M-1^Twas a member of the class Anaerolinea , with 92.8 % sequence similarity to Levilinea saccharolytica KIBI-1^T. Based on phylogenetic analysis and physiological properties, strain P3M-1^Trepresents a novel species in a new genus, for which the nameOrnatilinea apprimagen. nov., sp. nov. is proposed; the type strain ofO. apprimais P3M-1^T( = DSM 23815^T = VKM B-2669^T).

Intriguing, yet uncultured ‘ARMAN’-like archaea are metabolically dependent on other members of the microbial community. It remains uncertain though which hosts they rely upon, and, because of the lack of complete genomes, to what extent. Here, we report the co-culturing of ARMAN-2-related organism, Mia14, with Cuniculiplasma divulgatum PM4 during the isolation of this strain from acidic streamer in Parys Mountain (Isle of Anglesey, UK). Mia14 is highly enriched in the binary culture (ca. 10% genomic reads) and its ungapped 0.95 Mbp genome points at severe voids in central metabolic pathways, indicating dependence on the host, C. divulgatum PM4. Analysis of C. divulgatum isolates from different sites and shotgun sequence data of Parys Mountain samples suggests an extensive genetic exchange between Mia14 and hosts in situ. Within the subset of organisms with high-quality genomic assemblies representing the ‘DPANN’ superphylum, the Mia14 lineage has had the largest gene flux, with dozens of genes gained that are implicated in the host interaction.

Two novel cell wall-lacking, acidophilic, mesophilic, organotrophic and facultatively anaerobic archaeal strains have been isolated from acidic streamers formed on the surfaces of copper- ore-containing sulfidic deposits in the southwest Spain and North Wales, UK. Cells of strains varied from 0.1 m to 2 m in size and were of pleomorphic shape with tendency to form filamentous structures. The optimal pH and temperature for growth for both strains were 1.0-1.2 and 37- 40 °C, respectively, with the optimal substrates for growth being beef extract (3 g/L) for strain S5 and beef extract with tryptone (3 g/L and 1 g/L, respectively) for strain PM4. The lipid composition was dominated by intact polar lipids consisting of a glycerol dibiphytanyl glycerol tetraether (GDGT) core attached to predominantly glycosidic polar headgroups. In addition, free GDGT and low relative amounts of intact and core diether lipids were present. Strains S5 and PM4 possessed mainly menaquinones with minor fractions of thermoplasmaquinones. G+C content of DNA was 37.3 mol % in strain S5 and 37.16 mol % for strain PM4. Similarity matrix of 16S rRNA gene sequences (identical for both strains) showed their affiliation to the order Thermoplasmatales with 73.9-86.3% identity with sequences from its validly published members. A novel family, Cuniculiplasmataceae fam. nov., genus Cuniculiplasma gen. nov. and species Cuniculiplasma divulgatum sp. nov. were proposed based on the phylogenetic, chemotaxonomic analyses and physiological properties of the two isolates, S5T (=JCM 30642; =VKM B-2941) and PM4 (=JCM 30641;=VKM B-2940).

The current view suggests that in low-temperature acidic environments, archaea are significantly less abundant than bacteria. Thus, this study of the microbiome of Parys Mountain (Anglesey, UK) sheds light on the generality of this current assumption. Parys Mountain is a historically important copper mine and its acid mine drainage (AMD) water streams are characterised by constant moderate temperatures (8–18 °C), extremely low pH (1.7) and high concentrations of soluble iron and other metal cations. Metagenomic and SSU rRNA amplicon sequencing of DNA from Parys Mountain revealed a significant proportion of archaea affiliated with Euryarchaeota, which accounted for ca. 67% of the community. Within this phylum, potentially new clades of Thermoplasmata were overrepresented (58%), with the most predominant group being “E-plasma”, alongside low-abundant Cuniculiplasmataceae, ‘Ca. Micrarchaeota’ and ‘Terrestrial Miscellaneous Euryarchaeal Group’ (TMEG) archaea, which were phylogenetically close to Methanomassilicoccales and clustered with counterparts from acidic/moderately acidic settings. In the sediment, archaea and Thermoplasmata contributed the highest numbers in V3-V4 amplicon reads, in contrast with the water body community, where Proteobacteria, Nitrospirae, Acidobacteria and Actinobacteria outnumbered archaea. Cultivation efforts revealed the abundance of archaeal sequences closely related to Cuniculiplasma divulgatum in an enrichment culture established from the filterable fraction of the water sample. Enrichment cultures with unfiltered samples showed the presence of Ferrimicrobium acidiphilum, C. divulgatum, ‘Ca. Mancarchaeum acidiphilum Mia14’, ‘Ca. Micrarchaeota’-related and diverse minor (< 2%) bacterial metagenomic reads. Contrary to expectation, our study showed a high abundance of archaea in this extremely acidic mine-impacted environment. Further, archaeal populations were dominated by one particular group, suggesting that they are functionally important. The prevalence of archaea over bacteria in these microbiomes and their spatial distribution patterns represents a novel and important advance in our understanding of acidophile ecology. We also demonstrated a procedure for the specific enrichment of cell wall-deficient members of the archaeal component of this community, although the large fraction of archaeal taxa remained unculturable. Lastly, we identified a separate clustering of globally occurring acidophilic members of TMEG that collectively belong to a distinct order within Thermoplasmata with yet unclear functional roles in the ecosystem.

Samples of water from the hot springs of Uzon Caldera with temperatures from 68 to 87°C and pHs of 4.1 to 7.0, supplemented with proteinaceous (albumin, casein, or α- or β-keratin) or carbohydrate (cellulose, carboxymethyl cellulose, chitin, or agarose) biological polymers, were filled with thermal water and incubated at the same sites, with the contents of the tubes freely accessible to the hydrothermal fluid. As a result, several enrichment cultures growing in situ on different polymeric substrates were obtained. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments obtained after PCR withBacteria-specific primers showed that the bacterial communities developing on carbohydrates included the generaCaldicellulosiruptorandDictyoglomusand that those developing on proteins contained members of theThermotogalesorder. DGGE analysis performed after PCR withArchaea- andCrenarchaeota-specific primers showed that archaea related to uncultured environmental clones, particularly those of theCrenarchaeotaphylum, were present in both carbohydrate- and protein-degrading communities. Five isolates obtained from in situ enrichments or corresponding natural samples of water and sediments represented the bacterial generaDictyoglomusandCaldanaerobacteras well as new archaea of theCrenarchaeotaphylum. Thus, in situ enrichment and consequent isolation showed the diversity of thermophilic prokaryotes competing for biopolymers in microbial communities of terrestrial hot springs.