Stepan V. Toshchakov

Mobilitalea sibirica gen. nov., sp. nov., a halotolerant polysaccharide-degrading bacterium.

A novel strictly anaerobic, halotolerant, organotrophic bacterium, strain P3M-3(T), was isolated from a microbial mat formed under the flow of hot water emerging from a 2775 m-deep well in Tomsk region (western Siberia, Russia). Cells of strain P3M-3(T) were straight and curved rods, 0.2-0.4 µm in width and 1.5-20 µm in length. Strain P3M-3(T) grew optimally at 37 °C, pH 7.0-7.5 and in a NaCl concentration of 15 g l(-1). Under optimum growth conditions, the doubling time was 1 h. The isolate was able to ferment a variety of mono-, di- and polysaccharides, including microcrystalline cellulose. Acetate, ethanol, H2 and CO2 were the main products of glucose fermentation. The DNA G+C content was 33.4 mol%. 16S rRNA gene-based phylogenetic analysis showed that strain P3M-3(T) was a member of family Lachnospiraceae, whose representatives are also found in Clostridium cluster XIVa. 16S rRNA gene sequence similarity with Clostridium jejuense HY-35-12(T), the closest relative, was 93.9%. A novel genus and species, Mobilitalea sibirica gen. nov., sp. nov., are proposed based on phylogenetic analysis and physiological properties of the novel isolate. The type strain of the type species is P3M-3(T) ( = DSM 26468(T) = VKM B-2804(T)).

Tepidisphaera mucosa gen. nov., sp. nov., a moderately thermophilic member of the class Phycisphaerae in the phylum Planctomycetes, and proposal of a new family, Tepidisphaeraceae fam. nov., and a new order, Tepidisphaerales ord. nov.

Three strains of facultatively aerobic, moderately thermophilic bacteria were isolated from terrestrial hot springs in Baikal Lake region and Kamchatka (Russia). Cells of the new isolates were cocci reproducing by binary fission. The temperature range for growth was between 20 and 56 °C and the pH range for growth from pH 4.5 to 8.5, with optimal growth at 47-50 °C and pH 7.0-7.5. The organisms were chemoheterotrophs preferring sugars and polysaccharides as growth substrates. 16S rRNA gene sequences of strains 2842, 2813 and 2918Kr were nearly identical (99.7-100 % similarity) and indicated that the strains belonged to the phylum Planctomycetes. The phylogenetically closest cultivated relatives were Algisphaera agarilytica 06SJR6-2(T) and Phycisphaera mikurensis FYK2301M01(T) with 16S rRNA gene sequence similarity values of 82.4 and 80.3 %, respectively. The novel strains differed from them by higher growth temperature, sensitivity to NaCl concentration above 3.0 % and by their cellular fatty acids profile. On the basis of phylogenetic and physiological data, strains 2842(T), 2813 and 2918Kr represent a novel genus and species for which we propose the name Tepidisphaera mucosa sp. nov. The type strain is 2842(T) ( = VKM B-2832(T) = JCM 19875(T)). We also propose that Tepidisphaera gen. nov. is the type genus of a novel family, Tepidisphaeraceae fam. nov. and a novel order, Tepidisphaerales ord. nov.

Halo(natrono)archaea isolated from hypersaline lakes utilize cellulose and chitin as growth substrates

Until recently, extremely halophilic euryarchaeota were considered mostly as aerobic heterotrophs utilizing simple organic compounds as growth substrates. Almost nothing is known on the ability of these prokaryotes to utilize complex polysaccharides, such as cellulose, xylan, and chitin. Although few haloarchaeal cellulases and chitinases were recently characterized, the analysis of currently available haloarchaeal genomes deciphered numerous genes-encoding glycosidases of various families including endoglucanases and chitinases. However, all these haloarchaea were isolated and cultivated on simple substrates and their ability to grow on polysaccharides in situ or in vitro is unknown. This study examines several halo(natrono)archaeal strains from geographically distant hypersaline lakes for the ability to grow on insoluble polymers as a sole growth substrate in salt-saturated mineral media. Some of them belonged to known taxa, while other represented novel phylogenetic lineages within the class Halobacteria. All isolates produced extracellular extremely salt-tolerant cellulases or chitinases, either cell-free or cell-bound. Obtained results demonstrate a presence of diverse populations of haloarchaeal cellulo/chitinotrophs in hypersaline habitats indicating that euryarchaea participate in aerobic mineralization of recalcitrant organic polymers in salt-saturated environments.

High Diversity of Planctomycetes in Soils of Two Lichen-Dominated Sub-Arctic Ecosystems of Northwestern Siberia

A wide variety of terrestrial ecosystems in tundra have a ground vegetation cover composed of reindeer lichens (genera Cladonia and Cetraria). The microbial communities of two lichen-dominated ecosystems typical of the sub-arctic zone of northwestern Siberia, that is a forested tundra soil and a shallow acidic peatland, were examined in our study. As revealed by molecular analyses, soil and peat layers just beneath the lichen cover were abundantly colonized by bacteria from the phylum Planctomycetes. Highest abundance of planctomycetes detected by fluorescence in situ hybridization was in the range 2.2–2.7 × 107 cells per gram of wet weight. 16S rRNA gene fragments from the Planctomycetes comprised 8–13% of total 16S rRNA gene reads retrieved using Illumina pair-end sequencing from the soil and peat samples. Lichen-associated assemblages of planctomycetes displayed unexpectedly high diversity, with a total of 89,662 reads representing 1723 operational taxonomic units determined at 97% sequence identity. The soil of forested tundra was dominated by uncultivated members of the family Planctomycetaceae (53–71% of total Planctomycetes-like reads), while sequences affiliated with the Phycisphaera-related group WD2101 (recently assigned to the order Tepidisphaerales) were most abundant in peat (28–51% of total reads). Representatives of the Isosphaera–Singulisphaera group (14–28% of total reads) and the lineages defined by the genera Gemmata (1–4%) and Planctopirus–Rubinisphaera (1–3%) were present in both habitats. Two strains of Singulisphaera-like bacteria were isolated from studied soil and peat samples. These planctomycetes displayed good tolerance of low temperatures (4–15°C) and were capable of growth on a number of polysaccharides, including lichenan, a characteristic component of lichen-derived phytomass.

Complete genome sequence of 'Halanaeroarchaeum sulfurireducens' M27-SA2, a sulfur-reducing and acetate-oxidizing haloarchaeon from the deep-sea hypersaline anoxic lake Medee

Strain M27-SA2 was isolated from the deep-sea salt-saturated anoxic lake Medee, which represents one of the most hostile extreme environments on our planet. On the basis of physiological studies and phylogenetic positioning this extremely halophilic euryarchaeon belongs to a novel genus ‘Halanaeroarchaeum’ within the family Halobacteriaceae. All members of this genus cultivated so far are strict anaerobes using acetate as the sole carbon and energy source and elemental sulfur as electron acceptor. Here we report the complete genome sequence of the strain M27-SA2 which is composed of a 2,129,244-bp chromosome and a 124,256-bp plasmid. This is the second complete genome sequence within the genus Halanaeroarchaeum. We demonstrate that genome of ‘Halanaeroarchaeum sulfurireducens’ M27-SA2 harbors complete metabolic pathways for acetate and sulfur catabolism and for de novo biosynthesis of 19 amino acids. The genomic analysis also reveals that ‘Halanaeroarchaeum sulfurireducens’ M27-SA2 harbors two prophage loci and one CRISPR locus, highly similar to that of Kulunda Steppe (Altai, Russia) isolate ‘H. sulfurireducens’ HSR2T. The discovery of sulfur-respiring acetate-utilizing haloarchaeon in deep-sea hypersaline anoxic lakes has certain significance for understanding the biogeochemical functioning of these harsh ecosystems, which are incompatible with life for common organisms. Moreover, isolations of Halanaeroarchaeum members from geographically distant salt-saturated sites of different origin suggest a high degree of evolutionary success in their adaptation to this type of extreme biotopes around the world.

‘ARMAN’ archaea depend on association with euryarchaeal host in culture and in situ

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.In the absence of complete genomes, the metabolic capabilities of uncultured ARMAN-like archaea have been uncertain. Here, Golyshina et al. apply an enrichment culture technique and find that the ungapped genome of the ARMAN-like archaeon Mia14 has lost key metabolic pathways, suggesting dependence on the host archaeon Cuniculiplasma divulgatum.

Thermodesulfobium acidiphilum sp. nov., a new thermoacidophilic sulfate-reducing chemoautotrophic bacterium from a Kamchatkan thermal site.

An obligately anaerobic, sulfate-reducing micro-organism, strain 3127-1T, was isolated from geothermally heated soil (Oil Site, Uzon Caldera, Kamchatka, Russia). The new isolate was a moderately thermoacidophilic anaerobe able to grow with H2 or formate by respiration of sulfate or thiosulfate. The pH range for growth was 3.7-6.5, with an optimum at 4.8-5.0. The temperature range for growth was 37-65 °C, with an optimum at 55 °C. The G+C content of the genomic DNA was 33.7 mol%. The genome of strain 3127-1T contained two almost identical 16S rRNA genes, differing by a single nucleotide substitution. The closest 16S rRNA gene sequence of a validly published species belonged to Thermodesulfobium narugense Na82T (99.5 % similarity). However, the average nucleotide identity of the genomes of strain 3127-1T and T. narugense Na82T and the predicted DNA-DNA hybridization value (GGDC 2.1 blast+, formula 2) were as low as 86 and 32.5±2.5 %, respectively. This, together with phenotypic data, showed the new isolate to belong to a novel species, for which the name Thermodesulfobium acidiphilum sp. nov. is proposed. The type strain is 3127-1T (=DSM 102892T=VKM B-3043T).

Discovery of anaerobic lithoheterotrophic haloarchaea, ubiquitous in hypersaline habitats.

Hypersaline anoxic habitats harbour numerous novel uncultured archaea whose metabolic and ecological roles remain to be elucidated. Until recently, it was believed that energy generation via dissimilatory reduction of sulfur compounds is not functional at salt saturation conditions. Recent discovery of the strictly anaerobic acetotrophic Halanaeroarchaeum compels to change both this assumption and the traditional view on haloarchaea as aerobic heterotrophs. Here we report on isolation and characterization of a novel group of strictly anaerobic lithoheterotrophic haloarchaea, which we propose to classify as a new genus Halodesulfurarchaeum. Members of this previously unknown physiological group are capable of utilising formate or hydrogen as electron donors and elemental sulfur, thiosulfate or dimethylsulfoxide as electron acceptors. Using genome-wide proteomic analysis we have detected the full set of enzymes required for anaerobic respiration and analysed their substrate-specific expression. Such advanced metabolic plasticity and type of respiration, never seen before in haloarchaea, empower the wide distribution of Halodesulfurarchaeum in hypersaline inland lakes, solar salterns, lagoons and deep submarine anoxic brines. The discovery of this novel functional group of sulfur-respiring haloarchaea strengthens the evidence of their possible role in biogeochemical sulfur cycling linked to the terminal anaerobic carbon mineralisation in so far overlooked hypersaline anoxic habitats.

Biology of archaea from a novel family Cuniculiplasmataceae (Thermoplasmata) ubiquitous in hyperacidic environments.

The order Thermoplasmatales (Euryarchaeota) is represented by the most acidophilic organisms known so far that are poorly amenable to cultivation. Earlier culture-independent studies in Iron Mountain (California) pointed at an abundant archaeal group, dubbed ‘G-plasma’. We examined the genomes and physiology of two cultured representatives of a Family Cuniculiplasmataceae, recently isolated from acidic (pH 1–1.5) sites in Spain and UK that are 16S rRNA gene sequence-identical with ‘G-plasma’. Organisms had largest genomes among Thermoplasmatales (1.87–1.94 Mbp), that shared 98.7–98.8% average nucleotide identities between themselves and ‘G-plasma’ and exhibited a high genome conservation even within their genomic islands, despite their remote geographical localisations. Facultatively anaerobic heterotrophs, they possess an ancestral form of A-type terminal oxygen reductase from a distinct parental clade. The lack of complete pathways for biosynthesis of histidine, valine, leucine, isoleucine, lysine and proline pre-determines the reliance on external sources of amino acids and hence the lifestyle of these organisms as scavengers of proteinaceous compounds from surrounding microbial community members. In contrast to earlier metagenomics-based assumptions, isolates were S-layer-deficient, non-motile, non-methylotrophic and devoid of iron-oxidation despite the abundance of methylotrophy substrates and ferrous iron in situ, which underlines the essentiality of experimental validation of bioinformatic predictions.

Calorithrix insularis gen. nov., sp. nov., a novel representative of the phylum Calditrichaeota.

A moderately thermophilic, anaerobic bacterium designated as strain KRT was isolated from a shallow-water submarine hydrothermal vent (Kunashir Island, Southern Kurils, Russia). Cells of strain KRT were thin (0.2-0.3 µm), flexible, motile, Gram-stain-negative rods of variable length. Optimal growth conditions were pH 6.6, 55 °C and 1-3 % (w/v) NaCl. Strain KRT was able to ferment a wide range of proteinaceous substrates, pyruvate, and mono-, di- and polysaccharides. The best growth occurred with proteinaceous compounds. Nitrate significantly stimulated the growth on proteinaceous substrates decreasing H2 formation, ammonium being the main product of nitrate reduction. Strain KRT did not need the presence of a reducing agent in the medium and tolerated the presence of oxygen in the gas phase up to 3 % (v/v). In the presence of nitrate, aerotolerance of isolate KRT was enhanced up to 6-8 % O2 (v/v). Strain KRT was able to grow chemolithoheterotrophically, oxidizing H2 and reducing nitrate to ammonium. Yeast extract (0.05 g l-1) was required for growth. The G+C content of the genomic DNA of strain KRT was 47.3 mol%. 16S rRNA gene sequence analysis placed isolate KRT in the phylum Calditrichaeota where it represented a novel species of a new genus, for which the name Calorithrix insularis gen. nov., sp. nov. is proposed. The type strain of Calorithrix insularis is KRT (=DSM 101605T=VKM B-3022T).