Endemic to the deep subsurface biosphere sulphate-reducing 'Desulforudis audaxviator' has been called a living microbial fossil due to the high nucleotide sequence identity of its genomes across continents. Evolutionary stasis of this bacterium was established based on the analysis of metagenome assembled genomes, single cell genomes and a single axenic culture. The lack of high-quality reference genomes necessitates efforts to cultivate and isolate pure cultures that could shed light on the hypothetical slow evolution of Desulforudis-clade bacteria deep underground. Molecular signatures demonstrated the presence of Desulforudis-like phylotypes in subsurface environments worldwide. Here we report the isolation of four novel strains of the Desulforudis-clade, all belonging to Desulfosceptrum tomskiensis gen. nov. sp. nov. Four strains of the new species were isolated from deep boreholes in Western Siberia, separated by hundreds of kilometres. Genome comparisons revealed minimal differences between these strains, with average nucleotide sequence identity (ANI) values above 99.9%, low number of SNPs, and near-identical CRISPRs. The bacterium, together with Desulforudis audaxviator BYFT gen. nov. sp. nov., deposited in international culture collections, provides a bases for understanding the slow evolution of Bacillota endemic to the deep biosphere.

The emergence of multidrug-resistant bacteria is a major concern from both epidemiological and ecological perspectives. Studying these microorganisms is crucial for developing prevention strategies. We report the complete genome sequence of the multidrug-resistant Citrobacter portucalensis strain KOS1-1, isolated from wastewater in the city of Moscow.

Terrestrial mud volcanoes are surface geological features where fluidized sediments and gasses from the subsurface are discharged along a fracture network providing a window into the deep biosphere. Although mud volcanoes constitute an important source of methane emission from natural environments, their microbial communities responsible for methane cycling remain poorly characterized. Using a metagenomics approach, we investigated the taxonomic composition and metabolic potential of microbial communities in three active mud volcanoes in the Kerch-Taman mud volcanic province. Despite the volcanoes' close proximity their microbial communities strongly differ. In the Kmv1 and Kmv2 volcanoes surface horizons mostly harbored organotrophic microbial communities, while the relative abundance of anaerobic methanotrophic archaea (ANME) increased with depth. The deep horizons (1.5 m) of Kmv1 were dominated by Ca. Methanoperedenaceae that lacked nitrate reductase and could couple methane oxidation to the reduction of metal oxides, while the abundance of sulfate-reducing bacteria was low. Consistently, with higher sulfate content, the deep horizon in Kmv2 was dominated by Ca. Methanoperedenaceae, ANME-2a/2b clade, sulfate-reducing Desulfobacterota and sulfur-oxidizing Gammaproteobacteria. No clear depth distribution of taxa was observed in the Kmv3 volcano where microorganisms of the methane and sulfur cycles, namely, methanogens, ANME-3 clade, methanotrophic bacteria, and sulfate reducers were simultaneously detected. A high-quality genome of a member of the archaeal candidate phylum EX4484-52 within the DPANN lineage was assembled from metagenomes. This archaeon, named Candidatus Lutivulcanarchaeum fermentans, has complete glycolytic pathway and ATP generation mechanisms, but lacked the biosynthetic pathways for many key cellular compounds, indicating a parasitic or symbiotic lifestyle.

Virus-like particles (VLPs) based on the capsid protein (CP) of the ssRNA bacteriophage Beihai32 represent a promising nanoscale platform for the presentation of heterologous peptides. Previous studies have shown that the C-terminus of the CP tolerates long insertions without compromising VLP assembly. Here, we demonstrate that the N-terminus is similarly permissive to extended insertions. Hybrid CPs with one to four copies of the influenza A virus M2e peptide fused to the N-terminus were expressed in Escherichia coli. Fusion proteins containing four copies of M2e self-assembled into spherical VLPs, displaying the inserted peptides on the surface. Subcutaneous immunization of mice with chimeric VLPs induced high titers of M2e-specific antibodies. Unlike C-terminal fusions, the N-terminal insertion prevented the induction of anti-carrier antibody response indicting masking of the carrier protein in the chimeric VLP. To evaluate the capacity of the N-terminus for larger inserts, green fluorescent protein (GFP, 238 a.a.) was attached to the N-terminus of CP. The hybrid protein was expressed in Escherichia coli and formed VLPs. GFP was displayed on the particle surface and retained fluorescent activity. Overall, the phage Beihai32 CP is a versatile platform for the presentation of peptide antigens, supporting its potential application in VLP-based vaccine design.

Wastewater treatment plants represent a primary source of environmental dissemination of multidrug-resistant (MDR) bacteria, underscoring the urgent need for in-depth investigation of these organisms. While the resistome of MDR bacteria has been extensively studied, there remains a critical gap in understanding the role of plasmid-borne genes encoding adaptive metabolic functions. We isolated two MDR strains from municipal wastewater, Klebsiella sp. KOS9 and Pseudomonas veronii Yu15, both exhibiting resistance to antibiotics, including ampicillin, cefazolin, kanamycin, streptomycin, erythromycin, chloramphenicol, tetracycline, and ciprofloxacin. The plasmids of these strains harbored genes encoding aliphatic amidases, as well as antibiotic resistance genes (ARGs) and enzymes involved in glycogen and dTDP-L-rhamnose biosynthesis, which may contribute to virulence. In Klebsiella sp. KOS9 a single acetamidase operon, was found on the megaplasmid, along with copper and silver resistance genes. P. veronii Yu15 harbored an operon containing the acetamidase and formamidase genes on the chromosome, as well as a phylogenetically distant acetamidase operon on the conjugative megaplasmid. Both strains exhibit acetamidase activity and P. veronii Yu15 was able to utilize acetamide and formamide as sole nitrogen sources. The occurrence of ARGs and adaptive accessory genes on plasmids likely enhances the competitiveness and environmental flexibility of these MDR bacteria.

The genus Singulisphaera accommodates stalk-free planctomycetes with spherical cells, which multiply by budding, possess complex cell organization, large genomes, and colonize a wide spectrum of terrestrial environments. Two described species of this genus, S. acidiphila and S. rosea, were isolated from acidic peatlands and characterized as mildly acidophilic bacteria. Here, we characterize two neutrophilic Singulisphaera isolates from a boreal fen, strains Ch08T and PoTT. We also obtained and analyzed the previously unavailable genome sequence of S. rosea S26T. As confirmed by genome analyses and substrate utilization tests, all strains of Singulisphaera species were capable of growth on amorphous chitin due to the possession of chitinases affiliated with the glycoside hydrolase family GH18. The gene encoding chitinase from strain PoTT was expressed in Escherichia coli, and the endochitinase activity of the recombinant enzyme was confirmed. The genomes also contained gene clusters encoding Planctomycetal-type bacterial microcompartment (BMC) organelles, suggesting participation of Singulisphaera species in degradation of plant-derived polysaccharides. Transcriptome analysis performed with glucose- and L-fucose-grown cells of strain PoTT showed up-regulation of these BMC-encoding genes during growth on L-fucose. Tyrosinases involved in the oxidative removal of phenolic compounds were present only in S. acidiphila MOB10T. All Singulisphaera genomes contained the genes of the recently identified pathway of ornithine lipid biosynthesis. Based on the reported results, we describe two novel species, S. photorubra and S. chitinilytica with the type strains Ch08T (=KCTC 102485T = LMG 34153T) and PoTT (=KCTC 102290T = VKM B-3855T), respectively. The emended description of the genus Singulisphaera is also proposed.

The growing interest in multidrug-resistant (MDR) Citrobacter species stems from their epidemiological significance and their potential to harbor antibiotic resistance genes (ARGs), with mobile genetic elements playing a central role in their dissemination. Wastewater treatment plants play an important role in the formation of such MDR strains due to the high rate of horizontal gene transfer in these environments. In this study, we isolated the MDR strain KOS1-1 of Citrobacter portucalensis from wastewater, sequenced its genome, and characterized its ARG content and plasmid profile. This strain was resistant to ampicillin, cefazolin, cefaclor, cefatrizine, ciprofloxacin, kanamycin, streptomycin, spectinomycin, erythromycin, chloramphenicol, tetracycline, sulfamethoxazole, and trimethoprim. The KOS1-1 strain harbored five low copy number plasmids ranging in size from 77,569 to 289,033 bp. Genome analysis revealed the presence of multiple ARGs both on the chromosome and on plasmids, conferring resistance to β-lactams, quinolones, aminoglycosides, macrolides, sulfonamides, trimethoprim, phenicols, and tetracyclines. Many of these genes were associated with pseudo-composite transposon-like structures, emphasizing the role of mobile elements in ARGs dissemination. Plasmids harbored a bacterial cellulose biosynthesis operon and genes involved in mannose/fucose metabolism that could facilitate biofilm formation and a glycerol dissimilation gene cluster. Bacterial cellulose production was confirmed using electron and atomic force microscopy. Homologus gene clusters were identified on various plasmids of Enterobacteriales, suggesting their distribution via horizontal gene transfer. The presence of plasmids carrying ARGs and adaptive accessory genes increases the competitive fitness of C. portucalensis KOS1-1.IMPORTANCEAntimicrobial resistance represents a silent epidemic that has emerged as a critical global concern in recent years, underscoring the need for further research in this field. This study aimed to isolate and characterize multidrug-resistant bacteria from municipal wastewater, a huge reservoir of antibiotic resistance genes and resistant strains, from which they became disseminated into the environment. The isolated Citrobacter portucalensis strain KOS1-1 exhibits resistance to multiple antibiotics, arsenate, and mercury. It harbors five megaplasmids containing most of the resistance genes, along with laterally acquired bacterial cellulose biosynthesis operon and genes associated with mannose/fucose metabolism, which may facilitate biofilm formation. These plasmids may not only confer a selective advantage to host strains but also promote transfer of resistance determinants in high-density microbial communities of activated sludge at wastewater treatment plants. This work contributes to the understanding of the mechanisms of dissemination of bacterial resistance and virulence factors in municipal wastewater environments.

Virus-like particles (VLPs) formed as a result of self-assembly of viral capsid proteins are widely used as a platform for antigen presentation in vaccine development. However, since the inclusion of a foreign peptide into the capsid protein can alter its spatial structure and interfere with VLP assembly, such insertions are usually limited to short peptides. In this study, we have demonstrated the potential of capsid protein (CP) of single-stranded RNA phage PQ465 to present long peptides using green fluorescent protein (GFP) as a model. GFP was genetically linked to either the N- or C-terminus of PQ465 CP. Hybrid proteins were expressed in Escherichia coli and Nicotiana benthamiana plants. Spherical virus-like particles (~35 nm according to transmission electron microscopy) were successfully formed by both N- and C-terminal fusions expressed in E. coli, and by plant-produced CP with GFP fused to the C-terminus. ELISA revealed that GFP in VLPs was accessible for specific antibodies suggesting that it is exposed on the surface of PQ465-GFP particles. VLPs carrying GFP were recognized by anti-CP antibodies with less efficiency than VLPs formed by empty CP, which indicates shielding of the CP core in PQ465-GFP particles. Therefore, PQ465 CP can be used as a chimeric VLP platform for the display of relatively large protein antigens, which can operate in bacterial and plant expression systems.

Dissimilatory sulfate reduction is the main microbial process that detoxifies metals and increases pH in acid mine drainage. Acidophilic, copper-resistant Desulfosporosinus sp. BG and Desulfosporosinus sp. OT were previously isolated from acidic metalliferous tailings of the Bom-Gorkhon mine in Transbaikalia and Norilsk, respectively. To understand the role of sulfate-reducing bacteria (SRB) in mine tailings, we returned to the Bom-Gorkhon site to measure sulfate-reduction rate (SRR) with radioactive tracer and to estimate the proportion of SRB in the microbial community using 16 SrRNA gene profiling and metagenomic analysis. The SRR measured under ambient temperature conditions was high, reaching 9.86 ± 0.89 µmol SO4 cm− 3 day− 1. Unexpectedly for a temperate biotope, SRR values of the same order of magnitude were recorded at 60 °C. Thermophilic spore-forming Desulfotomaculum and Desulfofundulus are likely involved in the thermophilic process. The spores of thermophilic Desulfofundulus germinating at 20 °C may input into sulfate reduction at in-situ temperature conditions. Metagenomic analysis by dsr gene mapping and 16 S rRNA gene profiling revealed low abundance of Desulfosporosinus and other SRBs, indicating that geochemically important active sulfate reduction in acidic wetland sediments is carried out by a “rare biosphere” consortium. The cultivated BGT and OT strains are described as Desulfosporosinus cupriresistens sp. nov.

Schizosaccharomyces pombe is a non-Saccharomyces yeast that is widely used in winemaking due to its ability to ferment malic acid, thus improving organoleptic properties of wine. We report the draft genome sequence of S. pombe strain I-540, isolated from grape must in Russia.

Background: The extracellular domain of the M2 protein (M2e) and the conserved region of the second subunit of the hemagglutinin (HA2, 76–130 а.а.) of the influenza A virus, could be used to develop broad-spectrum influenza vaccines. However, these antigens have low immunogenicity and require the use of special carriers to enhance it. Virus-like particles (VLPs) formed from viral capsid proteins are among the most effective carriers. Methods: In this work, we obtained and characterized VLPs based on capsid proteins (CPs) of single-stranded RNA bacteriophages Beihai32 and PQ465, simultaneously displaying M2e and HA2 peptides. Results: Fusion proteins expressed in Escherichia coli formed spherical VLPs of about 30 nm in size. Subcutaneous immunization of mice with chimeric VLPs elicited a robust humoral immune response against M2e and the whole influenza A virus, and promoted the formation of cytokine-secreting antigen-specific CD4+ and CD8+ effector memory T cells. Conclusions: VLPs based on CPs of phages Beihai32 and PQ465 carrying conserved peptides M2e and HA2 of the influenza A virus can be used for the development of universal influenza vaccines.

The Sphaerotilus-Leptothrix group comprises two genera studied for 200 years. Attempts to unite these genera were made repeatedly, but have caused controversy due to differences in phenotypic characters identified in a small number of pure cultures. According to the latest release of Genome Taxonomy Database, Sphaerotilus and Leptothrix are combined into a single genus, Sphaerotilus. In this study, we analyzed 20 high-quality genomes forming 7 clusters on a phylogenetic tree constructed from concatenated sequences of 120 conserved genes, and investigated the distribution of metabolic genes. The Sphaerotilus-Leptothrix group formed a distinct monophyletic lineage within the family Sphaerotilaceae, where Leptothrix species were distributed among Sphaerotilus species. The average amino acid identities between the Sphaerotilus-Leptothrix genomes vary from 65 to 94.21%. All analyzed genomes contained genes of glycolysis, tricarboxylic acid cycle, and glyoxylate cycle. Genes for enzymes of dissimilatory sulfur metabolism were found, including oxidation of sulfide (sqr, fccAB), thiosulfate (soxAXBYZCD), elemental sulfur (rDSR) and sulfite (soeABC). Genes for manganese oxidation (mnxG, mofABC), previously considered unique to Leptothrix, were found in a number of other representatives of the combined genus. Also, some representatives contained genes for dissimilatory nitrate reduction and nitrogen fixation; autotrophic CO₂ fixation via the Calvin cycle; potential iron oxidation (cyc2, mtoAB). The identified metabolic pathways indicate a wide range of ecological strategies and occupied niches, determined by chemoorganoheterotrophic, chemolithoautotrophic and chemolithoheterotrophic nutritional types. Thus, the data obtained from phylogenetic analysis, large-scale genome analysis and assessment of metabolic potential support the unification of the genera Sphaerotilus and Leptothrix as a single genus Sphaerotilus.

The yeast Lachancea thermotolerans of the family Saccharomycetaceae is used in winemaking to increase the acidity of wine and improve its aromatic profile. We report the complete genome sequence of L. thermotolerans strain L-84, isolated from Sauvignon grapes from a vineyard in Crimea.

We report the complete genome sequence of the wine yeast strain Saccharomyces cerevisiae I-118. It was isolated from yeast sediment formed after spontaneous fermentation of Rkatsiteli grapes and is used to produce white wines. Genomic data provide the basis for linking the genotype of this strain with its oenological properties.

Microbial communities in wastewater treatment plants (WWTPs) play a crucial role in the decontamination of polluted water. An uncultured order-level lineage AKYH767 of the phylum Bacteroidota has been consistently detected in microbial consortia of activated sludge at WWTPs worldwide, but its functional role remains elusive. Representatives of AKYH767 were also detected in soils and freshwater bodies, which may be their natural reservoirs. Here, we obtained ten high-quality metagenome-assembled genomes, including one closed circular genome, of AKYH767 bacteria from metagenomes of the wastewater and activated sludge and used genomic data to uncover the metabolic potential of these bacteria and to predict their functional role. The cells of the AKYH767 bacteria were inferred to be rod-shaped and non-motile. Genome-based metabolic reconstruction predicted the Embden–Meyerhof pathway, the non-oxidative stage of the pentose phosphate pathway, and the complete tricarboxylic acid cycle. A facultatively anaerobic chemoheterotrophic lifestyle with the capacity to oxidize low organic substrates through aerobic respiration was suggested. Under anaerobic conditions AKYH767 bacteria can perform different steps of denitrification. They have limited capacities to hydrolyze carbohydrates and proteinaceous substrates but can utilize fatty acids. A peculiar property of AKYH767 bacteria is the presence of the phenylacetyl-CoA pathway for the utilization of phenylacetate, and about half of the genomes encoded the benzoate degradation pathway. Apparently, in bioreactors at WWTPs, the AKYH767 bacteria could be involved in the denitrification and biodegradation of aromatic compounds. Based on phylogenetic and genomic analyses, the novel AKYH767 bacterium is proposed to be classified as Candidatus Pollutiaquabacter aromativorans, within the candidate order Pollutiaquabacterales.

Flagellin is the main structural protein of the bacterial flagellum, responsible for the movement of flagellated bacteria. Flagellin activates Toll-like receptor 5, inducing both innate and adaptive immune reactions, which highlights its potential as a vaccine adjuvant, particularly efficient in case of administration via mucosal routes. Genetic fusion of an antigen to flagellin has been shown to enhance the immune responses against the antigen. The molecular architecture of flagellin provides versatile and robust adjuvant functionality, facilitating the development of diverse vaccination strategies against multiple diseases as recombinant protein-based vaccines demonstrate substantial advantages over conventional live-attenuated and inactivated vaccines in both developmental efficiency and safety profiles. We present a comprehensive overview of vaccine design strategies employing genetic fusion of antigens to flagellin for protection against various infectious diseases. The proven effectiveness of flagellin-based delivery has enabled several vaccine candidates to enter clinical trials.

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, the development of efficient serological tests for monitoring the dynamics of the disease as well as the immune response after illness or vaccination was critical. In this regard, low-cost and fast production of immunogenic antigens is essential for the rapid development of diagnostic serological kits. This study assessed the plant-based production of nucleoprotein (N) of SARS-CoV-2 and chimeric receptor-binding domain (RBD) of SARS-CoV-2 presented by hepatitis E virus capsid (HEV/RBD) and validation of the plant-derived proteins as diagnostic antigens for serological tests. The target proteins were expressed in and purified from Nicotiana benthamiana plants. The resulting yield of chimeric HEV/RBD protein reached 100 mg/kg fresh weight and 30 mg/kg fresh weight for N protein. The purified N protein and HEV/RBD protein were used to develop an indirect enzyme-linked immunosorbent assay (iELISA) for the detection of antibodies to SARS-CoV-2 in human sera. To validate the iELISA tests, a panel of 84 sera from patients diagnosed with COVID-19 was used, and the results were compared to those obtained by another commercially available ELISA kit (Dia.Pro D. B., Sesto San Giovanni, Italy). The performance of an HEV/RBD in-house ELISA showed a sensitivity of 89.58% (95% Cl: 75.23–95.37) and a specificity of 94.44% (95% Cl: 76.94–98.2). Double Recognition iELISA based on HEV/RBD and N protein is characterized by a lower sensitivity of 85.42% (95% Cl: 72.24–93.93) and specificity of 94.44% (95% Cl: 81.34–99.32) at cut-off = 0.154, compared with iELISA based on HEV/RBD. Our study confirms that N and fusion HEV/RBD proteins, which are transiently expressed in plants, can be used to detect responses to SARS-CoV-2 in human sera reliably. Our research validates the commercial potential of using plants as an expression system for recombinant protein production and their application as diagnostic reagents for serological detection of infectious diseases, hence lowering the cost of diagnostic kits.

Wine yeast Saccharomyces cerevisiae strain I-25, isolated from yeast sediment formed after spontaneous fermentation of Cabernet Sauvignon grapes in the Krasnodar region, is used to produce regional red wines in Russia. To characterize its genetic properties and biotechnological potential, we obtained a complete genome sequence.

Genome reduction and associated metabolic deficiencies have been described in various lineages of parasitic and symbiotic microorganisms that obtain essential nutrients from their partners, and in some free-living microorganisms that inhabit stable environments. The animal gut is a relatively stable ecosystem, characterized by an abundance of organic substances and a high concentration of microorganisms, which provides favorable conditions for the survival of microorganisms with reduced genomes. Metagenomic analysis of 49 samples of feces of farm animals (cows, sheep, yaks, and horses) revealed uncultured lineages of bacteria with reduced genomes (<1 Mbp): family UBA1242 (Christensenellales, Firmicutes), order Rs-D84 (Alphaproteobacteria), and family UBA9783 (Opitutales, Verrucomicrobiota), defined in genome-taxonomy database. Analysis of the genomes showed that these bacteria lacked pathways for the biosynthesis of amino acids, nucleotides, lipids, and many other essential metabolites. The UBA9783 genomes encoded a near-complete Embden-Meyerhof glycolytic pathway and the non-oxidative phase of the pentose phosphate pathway, while in UBA1242 and Rs-D84, these pathways are incomplete. All bacteria are limited to fermentative metabolism and lack aerobic and anaerobic respiratory pathways. All UBA9783 and some Rs-D84 genomes encoded F0F1-type ATP synthase and pyrophosphate-energized proton pump; they also can import and utilize peptides and some amino acids. While UBA9783 bacteria could thrive as specialized free-living organisms in the organic-rich gut environment, the UBA1242 and Rs-D84 lineages appear to have adopted the lifestyle of an obligate symbiont/parasite, obtaining metabolites from other cells.IMPORTANCEThe microbiota of the animal gastrointestinal tracts is a complex community of microorganisms which interact in a synergistic or antagonistic relationship and play key nutritional and metabolic roles. However, despite its importance, the gut microbiota of farm animals, especially its uncultured majority, remains largely unexplored. We performed a metagenomic analysis of the gut microbiome of farm animals and characterized three uncultured lineages of bacteria with reduced genomes (<1 Mbp) from the phyla Firmicutes, Proteobacteria, and Verrucomicrobiota. These bacteria were predicted to possess key metabolic deficiencies such as the inability to synthesize essential cell metabolites, suggesting their adaptation to the lifestyle of a symbiont/parasite, or a scavenger obtaining nutrients from the organic-rich gut environment. This study shows that genome reduction with metabolic specialization and adaptation to a partner-dependent lifestyle occurred through convergent evolution in several phylogenetically distant lineages of gut microbiota.