The Greater Caucasus is a part of seismically active Alpine–Himalayan orogenic belt and has been a center of significant volcanic activity during the Quaternary period. That led to the formation of the number of hydrothermal habitats, including subterranean thermal aquifers and surface hot springs. However, there are only a limited number of scientific works reporting on the microbial communities of these habitats. Moreover, all these reports concern only studies of specific microbial taxa, carried out using classical cultivation approaches. In this work, we present first culture-independent study of hydrotherms in the Republic of North Ossetia-Alania, located in the southern part of the North Caucasus. Using 16S metabarcoding, we analyzed the composition of the microbial communities of two subterranean thermal aquifers and terrestrial hot springs of the Karmadon valley. Analysis of correlations between the chemical composition of water and the representation of key taxa allowed us to identify the key factors determining the formation of microbial communities. In addition, we were able to identify a significant number of highly abundant deep phylogenetic lineages. Our study represents a first glance on the thermophilic microbial communities of the North Caucasus and may serve as a basis for further microbiological studies of the extreme habitats of this region.

In this study, we used culture-independent analysis based on 16S rRNA gene amplicons and metagenomics to explore in depth the microbial communities and their metabolic capabilities of artisanal brine cheeses made in the North Caucasus. Additionally, analysis of organic acid profiles was carried out for cheese characterization. Twelve cheese samples (designated as 05SR–16SR) from various artisanal producers were taken from five different villages located in Northern Ossetia–Alania (Russia). These cheeses were made using methods based on cultural traditions inherited from previous generations and prepared using a relatively uncontrolled fermentation process. The microbial diversity of Caucasus artisanal cheeses was studied for the first time. The results showed a diverse composition in all cheeses, with Bacillota (synonym Firmicutes) (9.1–99.3%) or Pseudomonadota (synonym Proteobacteria) (0.2–89.2%) prevalence. The microbial communities of the majority of the studied cheeses were dominated by lactic acid bacteria (LAB) genera, like Lactococcus (10.3–77.1% in 07SR, 09SR, 10SR, 11SR, 13SR, 15SR, 16SR), Lactobacillus (54.6% in 09SR), Streptococcus (13.9–93.9% in 11SR, 13SR, 14SR, 15SR), Lactiplantibacillus (13.4–30.6% in 16SR and 07SR) and Lentilactobacillus (5.9–14.2% in 09SR, 10SR and 13SR). Halophilic lactic acid bacteria belonging to the Tetragenococcus genus accounted for 7.9–18.6% in 05SR and 06SR microbiomes. A distinctive feature of Ossetia cheese microbiomes was the large variety of halophilic proteobacteria, and in some cheeses they prevailed, e.g., Chromohalobacter (63–76.5% in 05SR and 06SR), Psychrobacter (10–47.1% in 08SR, 11SR, 12SR), Halomonas (2.9–33.5% in 06SR, 08SR, 11SR and 12SR), Marinobacter (41.9% in 12SR) or Idiomarina (2.9–14.4% in 06SR, 08SR and 11SR samples). Analysis of the genomes assembled from metagenomes of three cheeses with different bacterial composition revealed the presence of genes encoding a variety of enzymes, involved in milk sugar, proteins and lipid metabolism in genomes affiliated with LAB, as well as genes responsible for beneficial bioamine and bacteriocin synthesis. Also, most of the LAB did not contain antibiotic resistance genes, which makes them potential probiotics, so highly demanded nowadays. Analysis of the genomes related to halophilic proteobacteria revealed that they are not involved in milk fermentation; however, the search for “useful” genes responsible for the synthesis of beneficial products/metabolites was partially positive. In addition, it has been shown that some halophiles may be involved in the synthesis of inappropriate bioactive components. The results obtained by culture-independent analyses confirm the importance of using such techniques both to clarify the quality and health-promoting properties of the product, and to look for probiotic strains with specified unique properties. This study has shown that traditional dairy foods may be a source of such beneficial strains.

A novel planctomycete, strain T20PH1^T, was isolated from alpine meadow soil collected at an altitude of 1800 m in the North Caucasus Mountains, Russia. Phylogenomic analysis placed it within the family Tepidisphaeraceae of the class Phycisphaerae. The closest relatives based on 16S rRNA gene sequence similarity are Fontivita pretiosa B-254^T (88.9%), ‘Humisphaera borealis’ M1803^T (88.6%), and Tepidisphaera mucosa 2842^T (88.4%). Strain T20PH1^T is the first soil-derived representative of the class Phycisphaerae. Cells of strain T20PH1^T are motile, pink-pigmented cocci reproducing by binary fission. This planctomycete is an obligately aerobic chemoorganotroph with growth optima at 25–30 °C and pH 6.0–7.0. A notable functional trait of strain T20PH1^T is its ability to grow on cellulose, including microcrystalline, fibrous and carboxymethyl cellulose, as well as on xylan, starch, lichenan and xanthan. The genome comprised a 5.15 Mb chromosome and a 125 kb plasmid with G + C contents of 65.97 and 66.75%, respectively. Genome analysis identified a GH5_5 subfamily cellulase as the most likely enzyme responsible for cellulose degradation by this planctomycete. Based on phenotypic and phylogenomic evidence, strain T20PH1^T (=LMG 34157^T = KCTC 102499^T) was classified as representing a novel genus and species, Terrisphaera alpina gen. nov., sp. nov.

The phylum Armatimonadota represents one of the least characterized bacterial lineages, with only three formally described species despite its widespread distribution in various environments. Deep subsurface thermal environments harbor significant microbial diversity and represent promising sources for novel enzyme discovery through metagenomic approaches. This study reports the identification, cloning, and biochemical characterization of Cel7465, a novel endoglucanase from an uncultured GBS-DC family within the order Fimbriimonadales. The enzyme was identified through metagenomic analysis of microbial mats from the Biragzang deep thermal well (North Ossetia, Russia, 48 °C) and successfully expressed in cells of Escherichia coli strain ArcticExpress (DE3). Phylogenetic analysis assigned Cel7465 to glycoside hydrolase family 5 subfamily 36. The purified recombinant enzyme exhibited optimal activity at 55 °C and pH 8.0, with high specific activity of 4347 U/mg. The enzyme demonstrated broad pH tolerance (50% activity retained from pH 4.0 to 10.0) and notable thermal stability, retaining 60% activity after one hour at 80 °C and 20% after four hours. Remarkably, the presence of Mn²⁺ ions enhanced enzyme activity more than 7-fold, while Mg²⁺ and Ca²⁺ ions provided moderate activation. Cel7465 represents the first biochemically characterized glycoside hydrolase from the order Fimbriimonadales, expanding our understanding of enzymatic capabilities within the understudied phylum Armatimonadota and demonstrating the continued potential of extreme environments as sources of novel industrial biocatalysts.

Two novel verrucomicrobial strains, RX08ᵀ and T20Ac3, were isolated from a boreal fen in northern European Russia and alpine meadow soil in the North Caucasus, respectively. Both strains were Gram-stain-negative, aerobic, non-motile rods growing at 15–30°C (optimum 25°C) and pH 6.5–8.5. A reversible light-dependent transition from yellow to pink pigmentation was observed in both strains during growth on solid and in liquid media. Phylogenetic analysis placed the isolates within the recently established genus Neoluteolibacter, with Neoluteolibacter rhizosphaerae GHJ8T representing the closest described relative (98.4% 16S rRNA gene sequence similarity). The complete genome of strain RX08ᵀ comprised a single 6.34 Mb chromosome with a G+C content of 62.0 mol% and 5,106 predicted protein-coding genes. Genome analysis revealed a complete C30 carotenoid biosynthesis pathway, while HPLC demonstrated that pigmentation change was accompanied by the accumulation of glycosylated diapocarotenoid derivatives, consistent with the observed pigmentation shift. Both strains utilized a wide range of sugars and polysaccharides, including chitin and inulin, as growth substrates. Consistent with these phenotypic characteristics, the genome encoded multiple glycoside hydrolases, including a distinct GH18 chitinase and four GH32 fructanases, suggesting adaptation to the degradation of structurally distinct environmental polysaccharides. Comparative phylogenomic analyses and overall genome relatedness indices supported the classification of strains RX08ᵀ and T20Ac3 as representing a novel species, for which the name Neoluteolibacter xanthoroseus sp. nov. is proposed. The type strain is RX08ᵀ (= LMG 34154ᵀ = KCTC 102489ᵀ).

Mineral springs of the Northern Caucasus remain largely uncharacterized by culture-independent approaches. At the high-altitude Dzhily-Su spring complex, where isotope data indicate deep-seated magmatic fluid inputs, planktonic and sediment-associated communities were analyzed using 16S rRNA gene profiling to determine whether these inputs shape the local microbiome. Rather than a distinct magmatically influenced community, the results revealed a pronounced water–sediment partitioning of taxa with known iron-cycling representatives, microaerophilic Gallionellaceae dominating the water fractions and facultatively anaerobic Aeromonas and Shewanella dominating the sediments. Furthermore, analysis of the fine-size (0.22 µm) water fraction highlighted a notable enrichment of candidate lineages and archaea, including a prominent Nanoarchaeota population (58.6% of archaeal reads) lacking identified mesophilic hosts. The predominance of a typical neutrophilic iron-cycling assemblage, together with the observed water–sediment partitioning, is consistent with an important role of local physicochemical gradients in structuring the microbial community despite the unusual geochemical setting of the parent aquifer.