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The class Limnochordia harbors a single cultivated member, the mesophilic Limnochorda pilosa, which was isolated from a meromictic lake. Despite numerous molecular signatures reported in various ecosystems, the ecophysiological versatility of this deeply branched lineage of Firmicutes (Bacillota) remains poorly understood. The objective of this study was to use targeted cultivation, based on metagenome-assembled genomes from a deep terrestrial aquifer in Western Siberia, to isolate two new thermophilic members of the class. These isolates, described as Geochorda subterranea gen. nov. sp. nov. and Carboxydochorda subterranea gen. nov. sp. nov. within the Geochordaceae fam. nov., were capable of both anaerobic and aerobic respiration using fumarate and O2, respectively, with simple sugars as electron donors. The cultivated Geochordaceae have demonstrated fermentative growth and degradation of various polymers, including starch, maltose, maltodextrin, xylan, and chitin. The carboxydotrophic C. subterranea sp. nov. exhibited autotrophic growth via the Calvin–Benson–Bassham cycle, using CO, H2, and formate as electron donors and O2 as an electron acceptor, adding metabolic flexibility to the bacterium in the nutrient-depleted “deep biosphere” and supporting the possibility of aerobic metabolism in the deep subsurface. The broad physiological potential deciphered from physiological experiments and comparative genomic data explains the widespread distribution of uncultivated members of the class Limnochordia in various ecosystems, where they can oxidize complex organic substrates through both aerobic and anaerobic respiration, as well as pursue a chemolithotrophic lifestyle through the oxidation of H2 or CO.

Abstract Wastewater treatment plants (WWTPs) are considered to be hotspots for the spread of antibiotic resistance genes (ARGs). We performed a metagenomic analysis of the raw wastewater, activated sludge and treated wastewater from two large WWTPs responsible for the treatment of urban wastewater in Moscow, Russia. In untreated wastewater, several hundred ARGs that could confer resistance to most commonly used classes of antibiotics were found. WWTPs employed a nitrification/denitrification or an anaerobic/anoxic/oxic process and enabled efficient removal of organic matter, nitrogen and phosphorus, as well as fecal microbiota. The resistome constituted about 0.05% of the whole metagenome, and after water treatment its share decreased by 3–4 times. The resistomes were dominated by ARGs encoding resistance to beta-lactams, macrolides, aminoglycosides, tetracyclines, quaternary ammonium compounds, and sulfonamides. ARGs for macrolides and tetracyclines were removed more efficiently than beta-lactamases, especially ampC, the most abundant ARG in the treated effluent. The removal efficiency of particular ARGs was impacted by the treatment technology. Metagenome-assembled genomes of multidrug-resistant strains were assembled both for the influent and the treated effluent. Ccomparison of resistomes from WWTPs in Moscow and around the world suggested that the abundance and content of ARGs depend on social, economic, medical, and environmental factors.

The phylum Verrucomicrobiota is one of the main groups of soil prokaryotes, which remains poorly represented by cultivated organisms. The major recognized role of Verrucomicrobiota in soils is the degradation of plant-derived organic matter. These bacteria are particularly abundant in peatlands, where xylan-type hemicelluloses represent one of the most actively decomposed peat constituents. The aim of this work was to characterize the microorganisms capable of hydrolyzing xylan under the anoxic conditions typical of peatland soils. The laboratory incubation of peat samples with xylan resulted in the pronounced enrichment of several phylotypes affiliated with the Verrucomicrobiota, Firmicutes, and Alphaproteobacteria. Sequencing of the metagenome of the enrichment culture allowed us to recover high-quality metagenome-assembled genomes (MAGs) assigned to the genera Caproiciproducens, Clostridium, Bacillus (Firmicutes), and Rhizomicrobium (Alphaproteobacteria), Cellulomonas (Actinobacteriota) and the uncultured genus-level lineage of the family Chthoniobacteraceae (Verrucomicrobiota). The latter bacterium, designated “Candidatus Chthoniomicrobium xylanophilum” SH-KS-3, dominated in the metagenome and its MAG was assembled as a complete closed chromosome. An analysis of the SH-KS-3 genome revealed potential endo-1,4-beta-xylanases, as well as xylan beta-1,4-xylosidases and other enzymes involved in xylan utilization. A genome analysis revealed the absence of aerobic respiration and predicted chemoheterotrophic metabolism with the capacity to utilize various carbohydrates, including cellulose, and to perform fermentation or nitrate reduction. An analysis of other MAGs suggested that Clostridium and Rhizomicrobium could play the role of primary xylan degraders while other community members probably took advantage of the availability of xylo-oligosaccharides and xylose or utilized low molecular weight organics.

The search for the microorganisms responsible for sulfide formation and corrosion of steel equipment in the oil fields of Tatarstan (Russia) resulted in the isolation of a new halotolerant strictly anaerobic sulfate-reducing bacterium, strain 5S69T. The cells were motile curved Gram-negative rods. Optimal growth was observed in the presence of 2.0–4.0% (w/v) NaCl, at pH 6.5, and at 23–28 °C under sulfate-reducing conditions. The isolate was capable of chemoorganotrophic growth with sulfate and other sulfoxides as electron acceptors, resulting in sulfide formation; and of pyruvate fermentation resulting in formation of H2 and acetate. The strain utilized lactate, pyruvate, ethanol, methanol, fumarate, and fructose, as well as H2/CO2/acetate for sulfate reduction. The genome size of the type strain 5S69T was 4.16 Mb with a G + C content of 63.0 mol%. On the basis of unique physiological properties and results of the 16S rRNA gene-based phylogenetic analysis, phylogenomic analysis of the 120 conserved single copy proteins and genomic indexes (ANI, AAI, and dDDH), assigning the type strain 5S69T ((VKM B-3653T = KCTC 25499T) to a new species within the genus Pseudodesulfovibrio, is suggested, with the proposed name Pseudodesulfovibrio methanolicus sp. nov. Genome analysis of the new isolate showed several genes involved in sulfate reduction and its sulfide-producing potential in oil fields with high saline formation water.

Burning coal seams, characterized by massive carbon monoxide (CO) emissions, the presence of secondary sulfates, and high temperatures, represent suitable environments for thermophilic sulfate reduction. The diversity and activity of dissimilatory sulfate reducers in these environments remain unexplored. In this study, using metagenomic approaches, in situ activity measurements with a radioactive tracer, and cultivation we have shown that members of the genus Desulfofundulus are responsible for the extremely high sulfate reduction rate (SRR) in burning lignite seams in the Altai Mountains. The maximum SRR reached 564 ± 21.9 nmol S cm−3 day−1 at 60°C and was of the same order of magnitude for both thermophilic (60°C) and mesophilic (23°C) incubations. The 16S rRNA profiles and the search for dsr gene sequences in the metagenome revealed members of the genus Desulfofundulus as the main sulfate reducers. The thermophilic Desulfofundulus sp. strain Al36 isolated in pure culture, did not grow at temperatures below 50°C, but produced spores that germinated into metabolically active cells at 20 and 15°C. Vegetative cells germinating from spores produced up to 0.738 ± 0.026 mM H2S at 20°C and up to 0.629 ± 0.007 mM H2S at 15°C when CO was used as the sole electron donor. The Al36 strain maintains significant production of H2S from sulfate over a wide temperature range from 15°C to 65°C, which is important in variable temperature biotopes such as lignite burning seams. Burning coal seams producing CO are ubiquitous throughout the world, and biogenic H2S may represent an overlooked significant flux to the atmosphere. The thermophilic spore outgrowth and their metabolic activity at temperatures below the growth minimum may be important for other spore-forming bacteria of environmental, industrial and clinical importance.

Currently, the phylogeny of the genus Thiothrix is based on comparative whole genome analysis because of the high homology of the 16S ribosomal RNA gene sequences within the genus. We analyzed the possibility of using various conservative genes as phylogenetic markers for the genus Thiothrix. We found that the levels of similarity of the nucleotide sequences of the tRNA(Ile)-lysidine synthase (tilS) and the β subunit of RNA polymerase (rpoB) genes are in good agreement with the average nucleotide identity (ANI) values between the genomes of various representatives of the genus Thiothrix. The genomes of Thiothrix strains MK1, WS, DNT52, DNT53, and H33 were sequenced. Taxonomic analysis using both whole genomes and the tilS gene consistently showed that MK1 and WS belong to Thiothrix lacustris, while DNT52, DNT53, and H33 belong to Thiothrix subterranea. The tilS gene fragments were subjected to high-throughput sequencing to profile the Thiothrix mat of a sulfidic spring, which revealed the presence of known species of Thiothrix and new species-level phylotypes. Thus, the use of tilS and rpoB as phylogenetic markers will allow for rapid analyses of pure cultures and natural communities for the purpose of phylogenetic identification of representatives of the genus Thiothrix.

The reserves of light conditional oil in reservoirs with low-salinity formation water are decreasing worldwide, necessitating the extraction of heavy oil from petroleum reservoirs with high-salinity formation water. As the first stage of defining the microbial-enhanced oil recovery (MEOR) strategies for depleted petroleum reservoirs, microbial community composition was studied for petroleum reservoirs with high-salinity formation water located in Tatarstan (Russia) using metagenomic and culture-based approaches. Bacteria of the phyla Desulfobacterota, Halanaerobiaeota, Sinergistota, Pseudomonadota, and Bacillota were revealed using 16S rRNA-based high-throughput sequencing in halophilic microbial communities. Sulfidogenic bacteria predominated in the studied oil fields. The 75 metagenome-assembled genomes (MAGs) of prokaryotes reconstructed from water samples were assigned to 16 bacterial phyla, including Desulfobacterota, Bacillota, Pseudomonadota, Thermotogota, Actinobacteriota, Spirochaetota, and Patescibacteria, and to archaea of the phylum Halobacteriota (genus Methanohalophilus). Results of metagenomic analyses were supported by the isolation of 20 pure cultures of the genera Desulfoplanes, Halanaerobium, Geotoga, Sphaerochaeta, Tangfeifania, and Bacillus. The isolated halophilic fermentative bacteria produced oil-displacing metabolites (lower fatty acids, alcohols, and gases) from sugar-containing and proteinaceous substrates, which testify their potential for MEOR. However, organic substrates stimulated the growth of sulfidogenic bacteria, in addition to fermenters. Methods for enhanced oil recovery should therefore be developed, combining the production of oil-displacing compounds with fermentative bacteria and the suppression of sulfidogenesis.

Abstract Microbial communities in wastewater treatment plants (WWTPs) play a key role in water purification. Microbial communities of activated sludge (AS) vary extensively based on plant operating technology, influent characteristics and WWTP capacity. In this study we performed 16S rRNA gene profiling of AS at nine large-scale WWTPs responsible for the treatment of municipal sewage from the city of Moscow, Russia. Two plants employed conventional aerobic process, one plant—nitrification/denitrification technology, and six plants were operated with the University of Cape Town (UCT) anaerobic/anoxic/oxic process. Microbial communities were impacted by the technology and dominated by the Proteobacteria, Bacteroidota and Actinobacteriota. WWTPs employing the UCT process enabled efficient removal of not only organic matter, but also nitrogen and phosphorus, consistently with the high content of ammonia-oxidizing Nitrosomonas sp. and phosphate-accumulating bacteria. The latter group was represented by Candidatus Accumulibacter, Tetrasphaera sp. and denitrifiers. Co-occurrence network analysis provided information on key hub microorganisms in AS, which may be targeted for manipulating the AS stability and performance. Comparison of AS communities from WWTPs in Moscow and worldwide revealed that Moscow samples clustered together indicating that influent characteristics, related to social, cultural and environmental factors, could be more important than a plant operating technology.

Uncultured bacteria of the candidate phylum MBNT15, distantly related to Desulfobacterota, have been identified in a broad range of mostly organic-rich aquatic environments. We assembled a near-complete genome of a member of MBNT15 from a boreal peatland metagenome and used genomic data to analyze the metabolic pathways of this bacterium and its ecological role. This bacterium, designated SHF-111, was predicted to be rod shaped, it lacks flagellar machinery but twitching motility is encoded. Genome-based phylogenetic analysis supported the phylum-level classification of the MBNT15 lineage. Genome annotation and metabolic reconstruction revealed the presence of the Embden–Meyerhof, Entner–Doudoroff and pentose phosphate pathways, as well as the complete tricarboxylic acid (TCA) cycle, and suggested a facultatively anaerobic chemoheterotrophic lifestyle with the ability to ferment peptides, amino acids, fatty acids and simple sugars, and completely oxidize these substrates through aerobic and anaerobic respiration. The SHF-111 genome encodes multiple multiheme c-type cytochromes that probably enable dissimilatory iron reduction. Consistently, the relative abundance of MBNT15 in peatlands positively correlated with iron concentration. Apparently, in the wetland ecosystem, MBNT15 representatives play the role of scavengers, carrying out the complete mineralization of low molecular weight organic substances formed as a result of microbial degradation of complex polymeric substrates. Comparative genome analysis of the MBNT15 phylum revealed that vast majority of its members are capable of aerobic respiration and dissimilatory iron reduction and some species also can reduce sulfur and nitrogen compounds, but not sulfate. Based on phylogenetic and genomic analyses, the novel bacterium is proposed to be classified as Candidatus Deferrimicrobium borealis, within a candidate phylum Deferrimicrobiota.

Abstract There is still a lack of understanding of H2S formation in agricultural waste, which leads to poor odour prevention and control. Microbial sulfate reduction is a major process contributing to sulfide formation in natural and technogenic environments with high sulfate and low oxygen concentration. Agricultural waste can be considered a low-sulfate system with no obvious input of oxidised sulfur compounds. The purpose of this study was to characterise a microbial community participating in H2S production and estimate the microbial sulfate reduction rate (SRR) in manure slurry from a large-scale swine finishing facility in Western Siberia. In a series of manure slurry microcosms, we identified bacterial consortia by 16S rRNA gene profiling and metagenomic analysis and revealed that sulfate-reducing Desulfovibrio were key players responsible for H2S production. The SRR measured with radioactive sulfate in manure slurry was high and comprised 7.25 nmol S cm−3 day−1. Gypsum may be used as a solid-phase electron acceptor for sulfate reduction. Another plausible source of sulfate is a swine diet, which often contains supplements in the form of sulfates, including lysine sulfate. Low-sulfur diet, manure treatment with iron salts, and avoiding gypsum bedding are possible ways to mitigate H2S emissions from swine manure.

The strain WBS, an anaerobic, psychro- and halotolerant bacterium belonging to the genus Fusibacter, was isolated from the littoral bottom sediments of the White Sea, Arctic, Russia. Fusibacter bizertensis WBS grew at temperatures between 8 and 32 °C (optimum growth at 18–20 °C), pH between 5.2 and 8.3 (optimum growth at pH 7.2), and at NaCl concentrations between 0 and 70 g L−1 (optimum growth at 32 g L−1). It reduced sulfate, thiosulfate, and elemental sulfur into sulfide, and, probably, the strain is able to disproportionate thiosulfate. The strain also utilized a wide range of substrates as it is a chemoorganotrophic bacterium. Analysis of the sequenced genome revealed genes for all enzymes involved in the Embden–Meyerhof glycolytic pathway as well as genes for the non-oxidative stage of the pentose phosphate pathway. The presence of genes encoding aldehyde dehydrogenases and alcohol dehydrogenases also suggests that, in addition to acetate, alcohols can also be the fermentation products. The strain possessed superoxide dismutase and peroxidase activities and the ability to consume O2, which is in full accordance with the presence of corresponding genes of antioxidant defense in the genome. The phylogenetic analysis suggested that the strain WBS is the closest relative of Fusibacter bizertensis LTF Kr01T (16S rRNA gene sequence similarity 98.78%). Based on biochemical and genomic characteristics, the strain WBS is proposed to represent a novel aero-, halo- and psychrotolerant strain from the genus Fusibacter, isolated for the first time among its members from cold oxygenated marine bottom sediments.

The natural combustion of underground coal seams leads to the formation of gas, which contains molecular hydrogen and carbon monoxide. In places where hot coal gases are released to the surface, specific thermal ecosystems are formed. Here, 16S rRNA gene profiling and shotgun metagenome sequencing were employed to characterize the taxonomic diversity and genetic potential of prokaryotic communities of the near-surface ground layer near hot gas vents in an open quarry heated by a subsurface coal fire. The communities were dominated by only a few groups of spore-forming Firmicutes, namely the aerobic heterotroph Candidatus Carbobacillus altaicus, the aerobic chemolitoautotrophs Kyrpidia tusciae and Hydrogenibacillus schlegelii, and the anaerobic chemolithoautotroph Brockia lithotrophica. Genome analysis predicted that these species can obtain energy from the oxidation of hydrogen and/or carbon monoxide in coal gases. We assembled the first complete closed genome of a member of uncultured class-level division DTU015 in the phylum Firmicutes. This bacterium, ‘Candidatus Fermentithermobacillus carboniphilus’ Bu02, was predicted to be rod-shaped and capable of flagellar motility and sporulation. Genome analysis showed the absence of aerobic and anaerobic respiration and suggested chemoheterotrophic lifestyle with the ability to ferment peptides, amino acids, N-acetylglucosamine, and tricarboxylic acid cycle intermediates. Bu02 bacterium probably plays the role of a scavenger, performing the fermentation of organics formed by autotrophic Firmicutes supported by coal gases. A comparative genome analysis of the DTU015 division revealed that most of its members have a similar lifestyle.

Flor yeast strains represent a specialized group of Saccharomyces cerevisiae yeasts used for the production of sherry-like wines by biological wine aging. We sequenced the genome of the industrial flor yeast strain I-329 from a collection of microorganisms for winemaking “Magarach” and the metagenomes of two flor velums based on this strain and continuously maintained for several decades. The winery uses two processes for the production of sherry-like wine: batch aging and a continuous process similar to the criaderas–solera system. The 18S rRNA gene profiling and sequencing of metagenomes of flor velums revealed the presence of the yeasts Pichia membranifaciens and Malassezia restricta in minor amounts along with the dominant S. cerevisiae I-329 flor yeast. Bacteria Oenococcus oeni and Lentilactobacillus hilgardii together accounted for approximately 20% of the velum microbiota in the case of a batch process, but less than 1% in the velum used in the continuous process. Collection strain I-329 was triploid for all chromosomes except diploid chromosomes I and III, while the copy numbers of all chromosomes were equal in industrial velums. A comparative analysis of the genome of strain I-329 maintained in the collection and metagenomes of industrial velums revealed only several dozens of single nucleotide polymorphisms, which indicates a long-term genetic stability of this flor yeast strain under the harsh conditions of biological wine aging.

Sulphate-reducing bacteria, primarily Desulfovibrio, are responsible for the active generation of H2S in swine production waste. The model species for sulphate reduction studies, Desulfovibrio vulgaris strain L2, was previously isolated from swine manure characterized by high rates of dissimilatory sulphate reduction. The source of electron acceptors in low-sulphate swine waste for the high rate of H2S formation remains uncertain. Here, we demonstrate the ability of the L2 strain to use common animal farming supplements including L-lysine-sulphate, gypsum and gypsum plasterboards as electron acceptors for H2S production. Genome sequencing of strain L2 revealed the presence of two megaplasmids and predicted resistance to various antimicrobials and mercury, which was confirmed in physiological experiments. Most of antibiotic resistance genes (ARG) are carried by two class 1 integrons located on the chromosome and on the plasmid pDsulf-L2-2. These ARGs, predicted to confer resistance to beta-lactams, aminoglycosides, lincosamides, sulphonamides, chloramphenicol and tetracycline, were probably laterally acquired from various Gammaproteobacteria and Firmicutes. Resistance to mercury is likely enabled by two mer operons also located on the chromosome and on pDsulf-L2-2 and acquired via horizontal gene transfer. The second megaplasmid, pDsulf-L2-1, encoded nitrogenase, catalase and type III secretion system suggesting close contact of the strain with intestinal cells in the swine gut. The location of ARGs on mobile elements allows us to consider D. vulgaris strain L2 as a possible vector transferring antimicrobials resistance determinants between the gut microbiote and microbial communities in environmental biotopes.

The diversity and activity of sulfate-reducing bacteria (SRB) in the camel gut remains largely unexplored. An abundant SRB community has been previously revealed in the feces of Bactrian camels (Camelus bactrianus). This study aims to combine the 16S rRNA gene profiling, sulfate reduction rate (SRR) measurement with a radioactive tracer, and targeted cultivation to shed light on SRB activity in the camel gut. Fresh feces of 55 domestic Bactrian camels grazing freely on semi-arid mountain pastures in the Kosh-Agach district of the Russian Altai area were analyzed. Feces were sampled in early winter at an ambient temperature of −15 °C, which prevented possible contamination. SRR values measured with a radioactive tracer in feces were relatively high and ranged from 0.018 to 0.168 nmol S cm−3 day−1. The 16S rRNA gene profiles revealed the presence of Gram-negative Desulfovibrionaceae and spore-forming Desulfotomaculaceae. Targeted isolation allowed us to obtain four pure culture isolates belonging to Desulfovibrio and Desulforamulus. An active SRB community may affect the iron and copper availability in the camel intestine due to metal ions precipitation in the form of sparingly soluble sulfides. The copper-iron sulfide, chalcopyrite (CuFeS2), was detected by X-ray diffraction in 36 out of 55 analyzed camel feces. In semi-arid areas, gypsum, like other evaporite sulfates, can be used as a solid-phase electron acceptor for sulfate reduction in the camel gastrointestinal tract.

Bottom sediments at methane discharge sites of the Laptev Sea shelf were investigated. The rates of microbial methanogenesis and methane oxidation were measured, and the communities responsible for these processes were analyzed. Methane content in the sediments varied from 0.9 to 37 µmol CH4 dm−3. Methane carbon isotopic composition (δ13C-CH4) varied from −98.9 to −77.6‰, indicating its biogenic origin. The rates of hydrogenotrophic methanogenesis were low (0.4–5.0 nmol dm−3 day−1). Methane oxidation rates varied from 0.4 to 1.2 µmol dm−3 day−1 at the seep stations. Four lineages of anaerobic methanotrophic archaea (ANME) (1, 2a–2b, 2c, and 3) were found in the deeper sediments at the seep stations along with sulfate-reducing Desulfobacteriota. The ANME-2a-2b clade was predominant among ANME. Aerobic ammonium-oxidizing Crenarchaeota (family Nitrosopumilaceae) predominated in the upper sediments along with heterotrophic Actinobacteriota and Bacteroidota, and mehtanotrophs of the classes Alphaproteobacteria (Methyloceanibacter) and Gammaproteobacteria (families Methylophilaceae and Methylomonadaceae). Members of the genera Sulfurovum and Sulfurimonas occurred in the sediments of the seep stations. Mehtanotrophs of the classes Alphaproteobacteria (Methyloceanibacter) and Gammaproteobacteria (families Methylophilaceae and Methylomonadaceae) occurred in the sediments of all stations. The microbial community composition was similar to that of methane seep sediments from geographically remote areas of the global ocean.

Two strains of filamentous, colorless sulfur bacteria were isolated from bacterial fouling in the outflow of hydrogen sulfide-containing waters from a coal mine (Thiothrix sp. Ku-5) and on the seashore of the White Sea (Thiothrix sp. AS). Metagenome-assembled genome (MAG) A52 was obtained from a sulfidic spring in the Volgograd region, Russia. Phylogenetic analysis based on the 16S rRNA gene sequences showed that all genomes represented the genus Thiothrix. Based on their average nucleotide identity and digital DNA-DNA hybridization data these new isolates and the MAG represent three species within the genus Thiothrix with the proposed names Thiothrix subterranea sp. nov. Ku-5T, Thiothrix litoralis sp. nov. AST, and “Candidatus Thiothrix anitrata” sp. nov. A52. The complete genome sequences of Thiothrix fructosivorans QT and Thiothrix unzii A1T were determined. Complete genomes of seven Thiothrix isolates, as well as two MAGs, were used for pangenome analysis. The Thiothrix core genome consisted of 1,355 genes, including ones for the glycolysis, the tricarboxylic acid cycle, the aerobic respiratory chain, and the Calvin cycle of carbon fixation. Genes for dissimilatory oxidation of reduced sulfur compounds, namely the branched SOX system (SoxAXBYZ), direct (soeABC) and indirect (aprAB, sat) pathways of sulfite oxidation, sulfur oxidation complex Dsr (dsrABEFHCEMKLJONR), sulfide oxidation systems SQR (sqrA, sqrF), and FCSD (fccAB) were found in the core genome. Genomes differ in the set of genes for dissimilatory reduction of nitrogen compounds, nitrogen fixation, and the presence of various types of RuBisCO.

The spread of antibiotic resistance genes (ARGs) that are present in livestock manures, which are discharged into the environment, is a severe threat to human and animal health. Here, we used 16S rRNA gene profiling and metagenomic analysis to characterize microbial community composition and antibiotic resistance in a manure storage lagoon from a large-scale swine finishing facility. Manure samples were collected at intervals of two years. Both the prokaryotic community and the resistome were dominated by the Firmicutes, Proteobacteria and Bacteroidota. Metagenomic analysis of two samples revealed 726 and 641 ARGs classified into 59 and 46 AMR gene families. Besides multidrug efflux pumps, the predominating ARGs potentially encoded resistance to tetracyclines, macrolide–lincosamide–streptogramin, aminoglycosides, peptide antibiotics, rifamycin, chloramphenicol, and beta-lactams. Genes from all predominant AMR gene families were found in both samples indicating overall long-term stability of the resistome. Antibiotic efflux pumps were the primary type of ARGs in the Proteobacteria, while antibiotic target alteration or protection was the main mechanism of resistance in the Firmicutes, Actinobacteriota and Bacteroidota. Metagenome-assembled genomes (MAG) of four multidrug-resistant strains were assembled. The first MAG, assigned to Escherichia flexneri, contained 46 ARGs, including multidrug efflux pumps, modified porins, beta-lactamases, and genes conferring resistance to peptide antibiotics. The second MAG, assigned to the family Alcaligenaceae, contained 18 ARGs encoding resistance to macrolide–lincosamide–streptogramin, tetracyclines, aminoglycosides and diaminopyrimidins. Two other MAGs representing the genera Atopostipes and Prevotella, contained four and seven ARGs, respectively. All these MAGs represented minor community members and accounted for less than 0.3% of the whole metagenome. Overall, a few lineages originated from the gut but relatively rare in the manure storage lagoon, are the main source of ARGs and some of them carry multiple resistance determinants.

Viral particles have been detected in the underground biosphere where they could be one of the main factors impacting microbial diversity, biogeochemistry and evolution. To characterize the viral component in the deep subsurface biosphere, we sequenced the metagenome of subsurface aquifer located in the Tomsk region of Russia, sampled via 2.8-km-deep borehole 5P. The de novo assembly of metagenomics sequences yielded three circular genomes assigned to bacteriophages of the order Caudovirales. The annotated genome sequences of these bacteriophages have been deposited in the GenBank database under the accession numbers MK113949, MK113950 and MK113951.

The authors wish to make the following corrections to this paper [...]

The emergence of the carnivory syndrome and traps in plants is one of the most intriguing questions in evolutionary biology. In the present study, we addressed it by comparative transcriptomics analysis of leaves and leaf-derived pitcher traps from a predatory plant Nepenthes ventricosa × Nepenthes alata. Pitchers were collected at three stages of development and a total of 12 transcriptomes were sequenced and assembled de novo. In comparison with leaves, pitchers at all developmental stages were found to be highly enriched with upregulated genes involved in stress response, specification of shoot apical meristem, biosynthesis of sucrose, wax/cutin, anthocyanins, and alkaloids, genes encoding digestive enzymes (proteases and oligosaccharide hydrolases), and flowering-related MADS-box genes. At the same time, photosynthesis-related genes in pitchers were transcriptionally downregulated. As the MADS-box genes are thought to be associated with the origin of flower organs from leaves, we suggest that Nepenthes species could have employed a similar pathway involving highly conserved MADS-domain transcription factors to develop a novel structure, pitcher-like trap, for capture and digestion of animal prey during the evolutionary transition to carnivory. The data obtained should clarify the molecular mechanisms of trap initiation and development and may contribute to solving the problem of its emergence in plants.

The metagenome of foulings from sulfidic spring “Serovodorodny” (Tatarstan, Russia), where members of the genus Thiothrix was observed, was sequenced. Representatives of the phyla Gammaproteobacteria, Cyanobacteria and Campilobacteriota dominated in the microbial community. The complete genome of Thiothrix sp. KT was assembled from the metagenome. It displayed 93.93–99.72% 16S rRNA gene sequence identity to other Thiothrix species. The average nucleotide identity (ANI) и digital DNA-DNA hybridization (dDDH) showed that the genome designated KT represents a new species within the genus Thiothrix, ‘Candidatus Thiothrix sulfatifontis’ sp. nov. KT. The taxonomic status has been determined of the strain Thiothrix sp. CT3, isolated about 30 years ago and not assigned to any of Thiothrix species due to high 16S rRNA gene sequence identity with related species (i.e., 98.8–99.4%). The complete genome sequence of strain CT3 was determined. The ANI between CT3 and other Thiothrix species was below 82%, and the dDDH values were less than 40%, indicating that strain CT3 belongs to a novel species, Thiothrix winogradskyi sp. nov. A genome analysis showed that both strains are chemo-organoheterotrophs, chemolithotrophs (in the presence of hydrogen sulfide and thiosulfate) and chemoautotrophs. For the first time, representatives of Thiothrix showed anaerobic growth in the presence of thiosulfate.

To get insights into microbial diversity and biogeochemical processes in the terrestrial deep subsurface aquifer, we sequenced the metagenome of artesian water collected at a 2.8 km deep oil exploration borehole 5P in Western Siberia, Russia. We obtained 71 metagenome-assembled genomes (MAGs), altogether comprising 93% of the metagenome. Methanogenic archaea accounted for about 20% of the community and mostly belonged to hydrogenotrophic Methanobacteriaceae; acetoclastic and methylotrophic lineages were less abundant. ANME archaea were not found. The most numerous bacteria were the Firmicutes, Ignavibacteriae, Deltaproteobacteria, Chloroflexi, and Armatimonadetes. Most of the community was composed of anaerobic heterotrophs. Only six MAGs belonged to sulfate reducers. These MAGs accounted for 5% of the metagenome and were assigned to the Firmicutes, Deltaproteobacteria, Candidatus Kapabacteria, and Nitrospirae. Organotrophic bacteria carrying cytochrome c oxidase genes and presumably capable of aerobic respiration mostly belonged to the Chloroflexi, Ignavibacteriae, and Armatimonadetes. They accounted for 13% of the community. The first complete closed genomes were obtained for members of the Ignavibacteriae SJA-28 lineage and the candidate phylum Kapabacteria. Metabolic reconstruction of the SJA-28 bacterium, designated Candidatus Tepidiaquacella proteinivora, predicted that it is an anaerobe growing on proteinaceous substrates by fermentation or anaerobic respiration. The Ca. Kapabacteria genome contained both the sulfate reduction pathway and cytochrome c oxidase. Presumably, the availability of buried organic matter of Mesozoic marine sediments, long-term recharge of the aquifer with meteoric waters and its spatial heterogeneity provided the conditions for the development of microbial communities, taxonomically and functionally more diverse than those found in oligotrophic underground ecosystems.

Microbiological, molecular ecological, biogeochemical, and isotope geochemical research was carried out at the polar Lake Bol’shie Khruslomeny at the coast of the Kandalaksha Bay, White Sea in March and September 2017. The uppermost mixolimnion was oxic, with low salinity (3–5%). The lower chemocline layer was brown-green colored, with very high content of particulate organic matter (up to 11.8 mg C L–1). The lowermost monimolimnion had marine salinity (22–24%) and very high concentrations of sulfide (up to 18 mmol L–1) and CH4 (up to 1.8 mmol L–1). In the chemocline, total microbial abundance and the rate of anoxygenic photosynthesis were 8.8 × 106 cells mL–1 and 34.4 μmol C L–1 day–1, respectively. Both in March and September, sulfate reduction rate increased with depth, peaking (up to 0.6–1.1 μmol S L–1 day–1) in the lower chemocline. Methane oxidation rates in the chemocline were up to 85 and 180 nmol CH4 L–1 day–1 in March and September, respectively; stimulation of this process by light was observed in September. The percentages of cyanobacteria and methanotrophs in the layer where light-induced methane oxidation occurred were similar, ∼2.5% of the microbial community. Light did not stimulate methane oxidation in deeper layers. The carbon isotope composition of particulate organic matter (δ13C-Corg), dissolved carbonates (δ13C-DIC), and methane (δ13C- CH4) indicated high microbial activity in the chemocline. Analysis of the 16S rRNA gene sequences revealed predominance of Cyanobium cyanobacteria (order Synechococcales) in the mixolimnion. Green sulfur bacteria Chlorobium phaeovibrioides capable of anoxygenic photosynthesis constituted ∼20% of the chemocline community both in March and in September. Methyloprofundus gammaptoteobacteria (family Methylomonaceae) were present in the upper chemocline, where active methane oxidation occurred. During winter, cyanobacteria were less abundant in the chemocline, while methanotrophs occurred in higher horizons, including the under-ice layer. Chemolithotrophic gammaproteobacteria of the genus Thiomicrorhabdus, oxidizing reduced sulfur compounds at low oxygen concentrations, were revealed in the chemocline in March. Both in March and September archaea constituted up to 50% of all microorganisms in the hypolimnion. The percentage of putative methanogens in the archaeal community was low, and they occurred mainly in near-bottom horizons.

Flor strains of Saccharomyces cerevisiae are principal microbial agents responsible for biological wine aging used for production of sherry-like wines. The flor yeast velum formed on the surface of fortified fermented must is a major adaptive and technological characteristic of flor yeasts that helps them to withstanding stressful winemaking conditions and ensures specific biochemical and sensory oxidative alterations typical for sherry wines. We have applied RNAseq technology for transcriptome analysis of an industrial flor yeast strain at different steps of velum development over 71 days under experimental winemaking conditions. Velum growth and maturation was accompanied by accumulation of aldehydes and acetales. We have identified 1490 differentially expressed genes including 816 genes upregulated and 674 downregulated more than 2-fold at mature biofilm stage as compared to the early biofilm. Distinct expression patterns of genes involved in carbon and nitrogen metabolism, respiration, cell cycle, DNA repair, cell adhesion, response to various stresses were observed. Many genes involved in response to different stresses, oxidative carbon metabolism, high affinity transport of sugars, glycerol utilization, sulfur metabolism, protein quality control and recycling, cell wall biogenesis, apoptosis were induced at the mature biofilm stage. Strong upregulation was observed for FLO11 flocculin while expression of other flocculins remained unaltered or moderately downregulated. Downregulated genes included those for proteins involved in glycolysis, transportation of ions, metals, aminoacids, sugars, indicating repression of some major transport and metabolic process at the mature biofilm stage. Presented results are important for in-depth understanding of cell response elicited by velum formation and sherry wine manufacturing conditions, and for the comprehension of relevant regulatory mechanisms. Such knowledge may help to better understand the molecular mechanisms that flor yeasts use to adapt to winemaking environments, establish the functions of previously uncharacterized genes, improve the technology of sherry- wine production, and find target genes for strain improvement.

Large areas in the northern hemisphere are covered by extensive wetlands, which represent a complex mosaic of raised bogs, eutrophic fens, and aapa mires all in proximity to each other. Aapa mires differ from other types of wetlands by their concave surface, heavily watered by the central part, as well as by the presence of large-patterned string-flark complexes. In this paper, we characterized microbial diversity patterns in the surface peat layers of the neighboring string and flark structures located within the mire site in the Vologda region of European North Russia, using 16S rRNA gene sequencing. The microbial communities in raised strings were clearly distinct from those in submerged flarks. Strings were dominated by the Alpha- and Gammaproteobacteria. Other abundant groups were the Acidobacteriota, Bacteroidota, Verrucomicrobiota, Actinobacteriota, and Planctomycetota. Archaea accounted for only 0.4% of 16S rRNA gene sequences retrieved from strings. By contrast, they comprised about 22% of all sequences in submerged flarks and mostly belonged to methanogenic lineages. Methanotrophs were nearly absent. Other flark-specific microorganisms included the phyla Chloroflexi, Spirochaetota, Desulfobacterota, Beijerinckiaceae- and Rhodomicrobiaceae-affiliated Alphaproteobacteria, and uncultivated groups env.OPS_17 and vadinHA17 of the Bacteroidota. Such pattern probably reflects local anaerobic conditions in the submerged peat layers in flarks.

Abstract Thermal ecosystems associated with underground coal combustion sites are rare and less studied than geothermal features. Here we analysed microbial communities of near-surface ground layer and bituminous substance in an open quarry heated by subsurface coal fire by metagenomic DNA sequencing. Taxonomic classification revealed dominance of only a few groups of Firmicutes. Near-complete genomes of three most abundant species, ‘Candidatus Carbobacillus altaicus’ AL32, Brockia lithotrophica AL31, and Hydrogenibacillus schlegelii AL33, were assembled. According to the genomic data, Ca. Carbobacillus altaicus AL32 is an aerobic heterotroph, while B. lithotrophica AL31 is a chemolithotrophic anaerobe assimilating CO2 via the Calvin cycle. H. schlegelii AL33 is an aerobe capable of both growth on organic compounds and carrying out CO2 fixation via the Calvin cycle. Phylogenetic analysis of the large subunit of RuBisCO of B. lithotrophica AL31 and H. schlegelii AL33 showed that it belongs to the type 1-E. All three Firmicutes species can gain energy from aerobic or anaerobic oxidation of molecular hydrogen, produced as a result of underground coal combustion along with other coal gases. We propose that thermophilic Firmicutes, whose spores can spread from their original geothermal habitats over long distances, are the first colonizers of this recently formed thermal ecosystem.

Methanotrophic verrucomicrobia of the order Methylacidiphilales are known as extremely acidophilic, thermophilic or mesophilic bacteria that inhabit acidic geothermal ecosystems. The occurrence of verrucomicrobial methanotrophs in other types of acidic environments remains an open question. Notably, Methylacidiphilales-affiliated 16S rRNA gene sequences are commonly retrieved from acidic (pH 3.5–5.5) peatlands. In this study, we compared the patterns of verrucomicrobial diversity in four acidic raised bogs and six neutral fens located in European North Russia. Methylacidiphilales-like 16S rRNA gene reads displaying 83–86% similarity to 16S rRNA gene sequences of currently described verrucomicrobial methanotrophs were recovered exclusively from raised bogs. Laboratory incubation of peat samples with 10% methane for 3 weeks resulted in the pronounced increase of a relative abundance of alphaproteobacterial methanotrophs, while no response was detected for Methylacidiphilales-affiliated bacteria. Three metagenome-assembled genomes (MAGs) of peat-inhabiting Methylacidiphilales bacteria were reconstructed and examined for the presence of genes encoding methane monooxygenase enzymes and autotrophic carbon fixation pathways. None of these genomic determinants were detected in assembled MAGs. Metabolic reconstructions predicted a heterotrophic metabolism, with a potential to hydrolyze several plant-derived polysaccharides. As suggested by our analysis, peat-inhabiting representatives of the Methylacidiphilales are acidophilic aerobic heterotrophs, which comprise a sister family of the methanotrophic Methylacidiphilaceae.

A combination of physicochemical and radiotracer analysis, high-throughput sequencing of the 16S rRNA, and particulate methane monooxygenase subunit A (pmoA) genes was used to link a microbial community profile with methane, sulfur, and nitrogen cycling processes. The objects of study were surface sediments sampled at five stations in the northern part of the Barents Sea. The methane content in the upper layers (0–5 cm) ranged from 0.2 to 2.4 µM and increased with depth (16–19 cm) to 9.5 µM. The rate of methane oxidation in the oxic upper layers varied from 2 to 23 nmol CH4 L−1 day−1 and decreased to 0.3 nmol L−1 day−1 in the anoxic zone at a depth of 16–19 cm. Sulfate reduction rates were much higher, from 0.3 to 2.8 µmol L−1 day−1. In the surface sediments, ammonia-oxidizing Nitrosopumilaceae were abundant; the subsequent oxidation of nitrite to nitrate can be carried out by Nitrospira sp. Aerobic methane oxidation could be performed by uncultured deep-sea cluster 3 of gamma-proteobacterial methanotrophs. Undetectable low levels of methanogenesis were consistent with a near complete absence of methanogens. Anaerobic methane oxidation in the deeper sediments was likely performed by ANME-2a-2b and ANME-2c archaea in consortium with sulfate-reducing Desulfobacterota. Sulfide can be oxidized by nitrate-reducing Sulfurovum sp. Thus, the sulfur cycle was linked with the anaerobic oxidation of methane and the nitrogen cycle, which included the oxidation of ammonium to nitrate in the oxic zone and denitrification coupled to the oxidation of sulfide in the deeper sediments. Methane concentrations and rates of microbial biogeochemical processes in sediments in the northern part of the Barents Sea were noticeably higher than in oligotrophic areas of the Arctic Ocean, indicating that an increase in methane concentration significantly activates microbial processes.

Microbial life can be supported at subzero temperatures in permafrost up to several million years old. Genome analysis of strains isolated from permafrost provides a unique opportunity to study microorganisms that have not previously come into contact with the human population. Acinetobacter lwoffii is a typical soil bacterium that has been increasingly reported as hospital pathogens associated with bacteremia. In order to identify the specific genetic characteristics of ancient permafrost-conserved strains of A. lwoffii and their differences from present-day clinical isolates, we carried out a genome-wide analysis of five strains of A. lwoffii isolated from permafrost aged from 15 thousand to 1.8 million years. Surprisingly, we did not identify chromosomal genetic determinants that distinguish permafrost strains from clinical A. lwoffii isolates and strains from other natural habitats. Phylogenetic analysis based on whole genome sequences showed that permafrost strains do not form a separate cluster and some of them are most closely related to clinical isolates. The genomes of clinical and permafrost strains contain similar mobile elements and prophages, which indicates an intense horizontal transfer of genetic material. Comparison of plasmids of modern and permafrost strains showed that plasmids from the modern strains are enriched with antibiotic resistance genes, while the content of genes for resistance to heavy metals and arsenic is nearly the same. The thawing of permafrost caused by global warming could release new potentially pathogenic strains of Acinetobacter.

Application of seawater for secondary oil recovery stimulates the development of sulfidogenic bacteria in the oil field leading to microbially influenced corrosion of steel equipment, oil souring, and environmental issues. The aim of this work was to investigate potential sulfide producers in the high-temperature Uzen oil field (Republic of Kazakhstan) exploited with seawater flooding and the possibility of suppressing growth of sulfidogens in both planktonic and biofilm forms. Approaches used in the study included 16S rRNA and dsrAB gene sequencing, scanning electron microscopy, and culture-based techniques. Thermophilic hydrogenotrophic methanogens of the genus Methanothermococcus (phylum Euryarchaeota) predominated in water from the zone not affected by seawater flooding. Methanogens were accompanied by fermentative bacteria of the genera Thermovirga, Defliviitoga, Geotoga, and Thermosipho (phylum Thermotogae), which are potential thiosulfate- or/and sulfur-reducers. In the sulfate- and sulfide-rich formation water, the share of Desulfonauticus sulfate-reducing bacteria (SRB) increased. Thermodesulforhabdus, Thermodesulfobacterium, Desulfotomaculum, Desulfovibrio, and Desulfoglaeba were also detected. Mesophilic denitrifying bacteria of the genera Marinobacter, Halomonas, and Pelobacter inhabited the near-bottom zone of injection wells. Nitrate did not suppress sulfidogenesis in mesophilic enrichments because denitrifiers reduced nitrate to dinitrogen; however, thermophilic denitrifiers produced nitrite, an inhibitor of SRB. Enrichments and a pure culture Desulfovibrio alaskensis Kaz19 formed biofilms highly resistant to biocides. Our results suggest that seawater injection and temperature of the environment determine the composition and functional activity of prokaryotes in the Uzen oil field.

Abstract Background The multigene family encoding the 5S rRNA, one of the most important structurally-functional part of the large ribosomal subunit, is an obligate component of all eukaryotic genomes. 5S rDNA has long been a favored target for cytological and phylogenetic studies due to the inherent peculiarities of its structural organization, such as the tandem arrays of repetitive units and their high interspecific divergence. The complex polyploid nature of the genome of bread wheat, Triticum aestivum, and the technically difficult task of sequencing clusters of tandem repeats mean that the detailed organization of extended genomic regions containing 5S rRNA genes remains unclear. This is despite the recent progress made in wheat genomic sequencing. Using pyrosequencing of BAC clones, in this work we studied the organization of two distinct 5S rDNA-tagged regions of the 5BS chromosome of bread wheat. Results Three BAC-clones containing 5S rDNA were identified in the 5BS chromosome-specific BAC-library of Triticum aestivum. Using the results of pyrosequencing and assembling, we obtained six 5S rDNA- containing contigs with a total length of 140,417 bp, and two sets (pools) of individual 5S rDNA sequences belonging to separate, but closely located genomic regions on the 5BS chromosome. Both regions are characterized by the presence of approximately 70–80 copies of 5S rDNA, however, they are completely different in their structural organization. The first region contained highly diverged short-type 5S rDNA units that were disrupted by multiple insertions of transposable elements. The second region contained the more conserved long-type 5S rDNA, organized as a single tandem array. FISH using probes specific to both 5S rDNA unit types showed differences in the distribution and intensity of signals on the chromosomes of polyploid wheat species and their diploid progenitors. Conclusion A detailed structural organization of two closely located 5S rDNA-tagged genomic regions on the 5BS chromosome of bread wheat has been established. These two regions differ in the organization of both 5S rDNA and the neighboring sequences comprised of transposable elements, implying different modes of evolution for these regions.

Abstract Background In the course of replication of eukaryotic chromosomes, the telomere length is maintained due to activity of telomerase, the ribonucleoprotein reverse transcriptase. Abolishing telomerase function causes progressive shortening of telomeres and, ultimately, cell cycle arrest and replicative senescence. To better understand the cellular response to telomerase deficiency, we performed a transcriptomic study for the thermotolerant methylotrophic yeast Hansenula polymorpha DL-1 lacking telomerase activity. Results Mutant strain of H. polymorpha carrying a disrupted telomerase RNA gene was produced, grown to senescence and analyzed by RNA-seq along with wild type strain. Telomere shortening induced a transcriptional response involving genes relevant to telomere structure and maintenance, DNA damage response, information processing, and some metabolic pathways. Genes involved in DNA replication and repair, response to environmental stresses and intracellular traffic were up-regulated in senescent H. polymorpha cells, while strong down-regulation was observed for genes involved in transcription and translation, as well as core histones. Conclusions Comparison of the telomerase deletion transcription responses by Saccharomyces cerevisiae and H. polymorpha demonstrates that senescence makes different impact on the main metabolic pathways of these yeast species but induces similar changes in processes related to nucleic acids metabolism and protein synthesis. Up-regulation of a subunit of the TORC1 complex is clearly relevant for both types of yeast.

The nitritation-anammox process, which involves partial aerobic oxidation of the ammonium to nitrite and following oxidation of ammonium by nitrite to molecular nitrogen, is an efficient and cost-effective approach for biological nitrogen removal from wastewater. To characterize the microbial communities involved in the nitrogen and carbon cycles in wastewater treatment bioreactors employing this process, we sequenced the metagenome of a sludge sample collected from the lab-scale nitritation-anammox sequencing-batch reactor. At the phylum level, Proteobacteria and Chloroflexi were the most numerous groups. Anammox bacteria belonged to the genus Candidatus Brocadia. The obtained data will help to investigate the taxonomical and functional diversity the microbial communities involved in nitritation-anammox process, and will be used for genome-based analysis of uncultured bacterial lineages. The raw sequencing data is available from the NCBI Sequence Read Archive (SRR9831403) database under the BioProject PRJN0A55627. Keywords: Anammox, Metagenome, Candidatus brocadia

The microbial community of a laboratory-scale bioreactor based on the anammox process was investigated by using metagenomic approaches and fluorescent in situ hybridization (FISH). The bioreactor was initially inoculated with activated sludge from the denitrifying bioreactor of a municipal wastewater treatment station. By constantly increasing the ammonium and nitrite load, a microbial community containing the novel species of anammox bacteria “Candidatus Jettenia ecosi” developed in the bioreactor after 5 years when the maximal daily nitrogen removal rate reached 8.5 g/L. Sequencing of the metagenome of anammox granules and the binning of the contigs obtained, allowed a high quality draft genome of the dominant anammox bacterium, “Candidatus Jettenia ecosi” to be assembled. Annotation of the 3.9 Mbp long genome revealed 3970 putative protein-coding genes, 45 tRNA genes, and genes for 16S/23S rRNAs. Analysis of the genome of “Candidatus Jettenia ecosi” revealed genes involved in anammox metabolism, including nitrite and ammonium transporters, copper-containing nitrite reductase, a nitrate reductase complex, hydrazine synthase, and hydrazine dehydrogenase. Autotrophic carbon fixation could be accomplished through the Wood Ljungdahl pathway. The composition of the community was investigated through a search of 16S rRNA sequences in the metagenome and FISH analysis of the anammox granules. The presence of the members of Ignavibacteriae, Betaproteobacteria, Chloroflexi and other microbial lineages reflected the complexity of the microbial processes in the studied bioreactor performed by anammox Planctomycetes, fermentative bacteria, and denitrifiers.

Background Parasitic plants have the ability to obtain nutrients from their hosts and are less dependent on their own photosynthesis or completely lose this capacity. The reduction in plastid genome size and gene content in parasitic plants predominantly results from loss of photosynthetic genes. Plants from the family Orobanchaceae are used as models for studying plastid genome evolution in the transition from an autotrophic to parasitic lifestyle. Diphelypaea is a poorly studied genus of the Orobanchaceae, comprising two species of non-photosynthetic root holoparasites. In this study, we sequenced the plastid genome of Diphelypaea coccinea and compared it with other Orobanchaceae, to elucidate patterns of plastid genome evolution. In addition, we used plastid genome data to define the phylogenetic position of Diphelypaea spp. Methods The complete nucleotide sequence of the plastid genome of D. coccinea was obtained from total plant DNA, using pyrosequencing technology. Results The D. coccinea plastome is only 66,616 bp in length, and is highly rearranged; however, it retains a quadripartite structure. It contains only four rRNA genes, 25 tRNA genes and 25 protein-coding genes, being one of the most highly reduced plastomes among the parasitic Orobanchaceae. All genes related to photosynthesis, including the ATP synthase genes, had been lost, whereas most housekeeping genes remain intact. The plastome contains two divergent, but probably intact clpP genes. Intron loss had occurred in some protein-coding and tRNA genes. Phylogenetic analysis yielded a fully resolved tree for the Orobanchaceae, with Diphelypaea being a sister group to Orobanche sect. Orobanche.

The Lake Bolshie Hruslomeny is located on the shores of the Kandalaksha Bay of the White Sea, North European Russia. This lake, formed from the sea bay and still retaining the subsurface connection with the sea, is meromictic, with a fresh oxygenated upper layer and an anoxic brackish hypolimnion with high concentrations of methane and hydrogen sulphide. To characterize the microbial communities involved in the carbon and sulfur cycles in the lake, we sequenced the metagenome of a water sample collected at the chemocline level. At the phylum level, Chlorobi, Proteobacteria, Bacteroidetes and Firmicutes were the most numerous groups. The obtained data will help investigate the diversity and ecological role of the microbial community in the Lake Bolshie Hruslomeny and provide insight into the biogeochemical processes in subarctic lakes. The raw sequencing data is available from the NCBI Sequence Read Archive (SRA) database under the BioProject PRJNA503531.

Thermal ecosystems associated with areas of underground burning coal seams are rare and poorly understood in comparison with geothermal objects. We studied the microbial communities associated with gas vents from the coal-fire in the mining wastes in the Kemerovo region of the Russian Federation. The temperature of the ground heated by the hot coal gases and steam coming out to the surface was 58 °C. Analysis of the composition of microbial communities revealed the dominance of Ktedonobacteria (the phylum Chloroflexi), known to be capable of oxidizing hydrogen and carbon monoxide. Thermophilic hydrogenotrophic Firmicutes constituted a minor part of the community. Among the well-known thermophiles, members of the phyla Aquificae, Deinococcus-Thermus and Bacteroidetes were also found. In the upper ground layer, Acidobacteria, Verrucomicrobia, Actinobacteria, Planctomycetes, as well as Proteobacteria of the alpha and gamma classes, typical of soils, were detected; their relative abundancies decreased with depth. The phylum Verrucomicrobia was dominated by Candidatus Udaeobacter, aerobic heterotrophs capable of generating energy through the oxidation of hydrogen present in the atmosphere in trace amounts. Archaea made up a small part of the communities and were represented by thermophilic ammonium-oxidizers. Overall, the community was dominated by bacteria, whose cultivated relatives are able to obtain energy through the oxidation of the main components of coal gases, hydrogen and carbon monoxide, under aerobic conditions.

Abstract Background Chloroplasts of most plants are responsible for photosynthesis and contain a conserved set of about 110 genes that encode components of housekeeping gene expression machinery and photosynthesis-related functions. Heterotrophic plants obtaining nutrients from other organisms and their plastid genomes represent model systems in which to study the effects of relaxed selective pressure on photosynthetic function. The most evident is a reduction in the size and gene content of the plastome, which correlates with the loss of genes encoding photosynthetic machinery which become unnecessary. Transition to a non-photosynthetic lifestyle is expected also to relax the selective pressure on photosynthetic machinery in the nuclear genome, however, the corresponding changes are less known. Results Here we report the complete sequence of the plastid genome of Monotropa hypopitys, an achlorophyllous obligately mycoheterotrophic plant belonging to the family Ericaceae. The plastome of M. hypopitys is greatly reduced in size (35,336 bp) and lacks the typical quadripartite structure with two single-copy regions and an inverted repeat. Only 45 genes remained presumably intact– those encoding ribosomal proteins, ribosomal and transfer RNA and housekeeping genes infA, matK, accD and clpP. The clpP and accD genes probably remain functional, although their sequences are highly diverged. The sets of genes for ribosomal protein and transfer RNA are incomplete relative to chloroplasts of a photosynthetic plant. Comparison of the plastid genomes of two subspecies-level isolates of M. hypopitys revealed major structural rearrangements associated with repeat-driven recombination and the presence of isolate-specific tRNA genes. Analysis of the M. hypopitys transcriptome by RNA-Seq showed the absence of expression of nuclear-encoded components of photosystem I and II reaction center proteins, components of cytochrome b6f complex, ATP synthase, ribulose bisphosphate carboxylase components, as well as chlorophyll from protoporphyrin IX biosynthesis pathway. Conclusions With the complete loss of genes related to photosynthesis, NADH dehydrogenase, plastid-encoded RNA polymerase and ATP synthase, the M. hypopitys plastid genome is among the most functionally reduced ones characteristic of obligate non-photosynthetic parasitic species. Analysis of the M. hypopitys transcriptome revealed coordinated evolution of the nuclear and plastome genomes and the loss of photosynthesis-related functions in both genomes.

Here we report the draft genome sequence of the acid-tolerant Desulfovibrio sp. DV isolated from the sediments of a Pb-Zn mine tailings dam in the Chita region, Russia. The draft genome has a size of 4.9 Mb and encodes multiple K+-transporters and proton-consuming decarboxylases. The phylogenetic analysis based on concatenated ribosomal proteins revealed that strain DV clusters together with the acid-tolerant Desulfovibrio sp. TomC and Desulfovibrio magneticus. The draft genome sequence and annotation have been deposited at GenBank under the accession number MLBG00000000.

Here, we report on the draft genome of a copper-resistant and acidophilic Desulfosporosinus sp. BG, isolated from the tailings of a molybdenum-tungsten mine in Transbaikal area. The draft genome has a size of 4.52 Mb and encodes transporters of heavy metals. The phylogenetic analysis based on concatenated ribosomal proteins revealed that strain BG clusters together with the other acidophilic copper-resistant strains Desulfosporosinus sp. OT and Desulfosporosinus sp. I2. The K+-ATPase, Na+/H+ antiporter and amino acid decarboxylases may participate in enabling growth at low pH. The draft genome sequence and annotation have been deposited at GenBank under the accession number NZ_MASS00000000.

Two metagenome-assembled genomes (MAGs), obtained from laboratory-scale enhanced biological phosphorus removal bioreactors, were analyzed. The values of 16S rRNA gene sequence identity, average nucleotide identity, and average amino acid identity indicated that these genomes, designated as RT and SSD2, represented two novel species within the genus Thiothrix, ‘Candidatus Thiothrix moscowensis’ and ‘Candidatus Thiothrix singaporensis’. A complete set of genes for the tricarboxylic acid cycle and electron transport chain indicates a respiratory type of metabolism. A notable feature of RT and SSD2, as well as other Thiothrix species, is the presence of a flavin adenine dinucleotide (FAD)-dependent malate:quinone oxidoreductase instead of nicotinamide adenine dinucleotide (NAD)-dependent malate dehydrogenase. Both MAGs contained genes for CO2 assimilation through the Calvin–Benson–Bassam cycle; sulfide oxidation (sqr, fccAB), sulfur oxidation (rDsr complex), direct (soeABC) and indirect (aprBA, sat) sulfite oxidation, and the branched Sox pathway (SoxAXBYZ) of thiosulfate oxidation to sulfur and sulfate. All these features indicate a chemoorganoheterotrophic, chemolithoautotrophic, and chemolithoheterotrophic lifestyle. Both MAGs comprise genes for nitrate reductase and NO-reductase, while SSD2 also contains genes for nitrite reductase. The presence of polyphosphate kinase and exopolyphosphatase suggests that RT and SSD2 could accumulate and degrade polyhosphates during the oxic-anoxic growth cycle in the bioreactors, such as typical phosphate-accumulating microorganisms.

Mud volcanoes are prominent geological structures where fluids and gases from the deep subsurface are discharged along a fracture network in tectonically active regions. Microbial communities responsible for sulfur and methane cycling and organic transformation in terrestrial mud volcanoes remain poorly characterized. Using a metagenomics approach, we analyzed the microbial community of bubbling fluids retrieved from an active mud volcano in eastern Crimea. The microbial community was dominated by chemolithoautotrophic Campylobacterota and Gammaproteobacteria, which are capable of sulfur oxidation coupled to aerobic and anaerobic respiration. Methane oxidation could be enabled by aerobic Methylococcales bacteria and anaerobic methanotrophic archaea (ANME), while methanogens were nearly absent. The ANME community was dominated by a novel species of Ca. Methanoperedenaceae that lacked nitrate reductase and probably couple methane oxidation to the reduction of metal oxides. Analysis of two Ca. Bathyarchaeota genomes revealed the lack of mcr genes and predicted that they could grow on fatty acids, sugars, and proteinaceous substrates performing fermentation. Thermophilic sulfate reducers indigenous to the deep subsurface, Thermodesulfovibrionales (Nitrospirae) and Ca. Desulforudis (Firmicutes), were found in minor amounts. Overall, the results obtained suggest that reduced compounds delivered from the deep subsurface support the development of autotrophic microorganisms using various electron acceptors for respiration.

Large areas in Northern Russia are covered by extensive mires, which represent a complex mosaic of ombrotrophic raised bogs, minerotrophic and eutrophic fens, all in a close proximity to each other. In this paper, we compared microbial diversity patterns in the surface peat layers of the neighbouring raised bogs and eutrophic fens that are located within two geographically remote mire sites in Vologda region using 16S rRNA gene sequencing. Regardless of location, the microbial communities in raised bogs were highly similar to each other but were clearly distinct from those in eutrophic fens. Bogs were dominated by the Acidobacteria (30%–40% of total 16S rRNA gene reads), which belong to the orders Acidobacteriales and Bryobacterales. Other bog-specific bacteria included the Phycisphaera-like group WD2101 and the families Isosphaeraceae and Gemmataceae of the Planctomycetes, orders Opitutales and Pedosphaerales of the Verrucomicrobia and a particular group of alphaproteobacteria within the Rhizobiales. In contrast, fens hosted Anaerolineae-affiliated Chloroflexi, Vicinamibacteria- and Blastocatellia-affiliated Acidobacteria, Rokubacteria, uncultivated group OM190 of the Planctomycetes and several groups of betaproteobacteria. The Patescibacteria were detected in both types of wetlands but their relative abundance was higher in fens. A number of key parameters that define the distribution of particular bacterial groups in mires were identified.

The candidate phyla radiation is a large monophyletic lineage comprising unculturable bacterial taxa with small cell and genome sizes, mostly known from genomes obtained from environmental sources without cultivation. Here, we present the closed complete genome of a member of the superphylum Microgenomates obtained from the metagenome of a deep subsurface thermal aquifer. Phylogenetic analysis indicates that the new bacterium, designated Ch65, represents a novel phylum-level lineage within the Microgenomates group, sibling to the candidate phylum Collierbacteria. The Ch65 genome has a highly unusual nucleotide composition with one strand of highly enriched in cytosine versus guanine throughout the whole length. Such nucleotide composition asymmetry, also detected in the members of Ca. Collierbacteria and Ca. Beckwithbacteria, suggests that most of the Ch65 chromosome is replicated in one direction. A genome analysis predicted that the Ch65 bacterium has fermentative metabolism and could produce acetate and lactate. It lacks respiratory capacity, as well as complete pathways for the biosynthesis of lipids, amino acids, and nucleotides. The Embden−Meyerhof glycolytic pathway and nonoxidative pentose phosphate pathway are mostly complete, although glucokinase, 6-phosphofructokinase, and transaldolase were not found. The Ch65 bacterium lacks secreted glycoside hydrolases and conventional transporters for importing sugars and amino acids. Overall, the metabolic predictions imply that Ch65 adopts the lifestyle of a symbiont/parasite, or a scavenger, obtaining resources from the lysed microbial biomass. We propose the provisional taxonomic assignment ‘Candidatus Chazhemtobacterium aquaticus’, genus ‘Chazhemtobacterium‘, family ‘Chazhemtobacteraceae‘ in the Microgenomates group.

Members of the Acidobacteria are among the most efficient colonizers of acidic terrestrial habitats but the key traits underlying their environmental fitness remain to be understood. We analyzed indigenous assemblages of Acidobacteria in a lichen-covered acidic (pH 4.1) soil of forested tundra dominated by uncultivated members of subdivision 1. An isolate of these bacteria with cells occurring within saccular chambers, strain SBC82T, was obtained. The genome of strain SBC82T consists of a 7.11-Mb chromosome and four megaplasmids, and encodes a wide repertoire of enzymes involved in degradation of chitin, cellulose, and xylan. Among those, four secreted chitinases affiliated with the glycoside hydrolase family GH18 were identified. Strain SBC82T utilized amorphous chitin as a source of carbon and nitrogen; the respective enzyme activities were detected in tests with synthetic substrates. Chitinolytic capability was also confirmed for another phylogenetically related acidobacterium isolated from a Sphagnum peat bog, strain CCO287. As revealed by metatranscriptomic analysis of chitin-amended peat, 16S rRNA reads from these acidobacteria increased in response to chitin availability. Strains SBC82T and CCO287 were assigned to a novel genus and species, Acidisarcina polymorpha gen. nov., sp. nov. Members of this genus colonize acidic soils and peatlands and specialize in degrading complex polysaccharides.

Flor yeast strains represent a specialized group of Saccharomyces cerevisiae yeasts used for biological wine aging. We have sequenced the genomes of three flor strains originated from different geographic regions and used for production of sherry-like wines in Russia. According to the obtained phylogeny of 118 yeast strains, flor strains form very tight cluster adjacent to the main wine clade. SNP analysis versus available genomes of wine and flor strains revealed 2,270 genetic variants in 1,337 loci specific to flor strains. Gene ontology analysis in combination with gene content evaluation revealed a complex landscape of possibly adaptive genetic changes in flor yeast, related to genes associated with cell morphology, mitotic cell cycle, ion homeostasis, DNA repair, carbohydrate metabolism, lipid metabolism, and cell wall biogenesis. Pangenomic analysis discovered the presence of several well-known “non-reference” loci of potential industrial importance. Events of gene loss included deletions of asparaginase genes, maltose utilization locus, and FRE-FIT locus involved in iron transport. The latter in combination with a flor-yeast-specific mutation in the Aft1 transcription factor gene is likely to be responsible for the discovered phenotype of increased iron sensitivity and improved iron uptake of analyzed strains. Expansion of the coding region of the FLO11 flocullin gene and alteration of the balance between members of the FLO gene family are likely to positively affect the well-known propensity of flor strains for velum formation. Our study provides new insights in the nature of genetic variation in flor yeast strains and demonstrates that different adaptive properties of flor yeast strains could have evolved through different mechanisms of genetic variation.

Monotropa hypopitys is a mycoheterotrophic, nonphotosynthetic plant acquiring nutrients from the roots of autotrophic trees through mycorrhizal symbiosis, and, similar to other extant plants, forming asymmetrical lateral organs during development. The members of the YABBY family of transcription factors are important players in the establishment of leaf and leaf-like organ polarity in plants. This is the first report on the identification of YABBY genes in a mycoheterotrophic plant devoid of aboveground vegetative organs. Seven M. hypopitys YABBY members were identified and classified into four clades. By structural analysis of putative encoded proteins, we confirmed the presence of YABBY-defining conserved domains and identified novel clade-specific motifs. Transcriptomic and qRT-PCR analyses of different tissues revealed MhyYABBY transcriptional patterns, which were similar to those of orthologous YABBY genes from other angiosperms. These data should contribute to the understanding of the role of the YABBY genes in the regulation of developmental and physiological processes in achlorophyllous leafless plants.