Environmental activity-based protein profiling for function-driven enzyme discovery from natural communities.

BACKGROUND: Microbial communities are important drivers of global biogeochemical cycles, xenobiotic detoxification, as well as organic matter decomposition. Their major metabolic role in ecosystem functioning is ensured by a unique set of enzymes, providing a tremendous yet mostly hidden enzymatic potential. Exploring this enzymatic repertoire is therefore not only relevant for a better understanding of how microorganisms function in their natural environment, and thus for ecological research, but further turns microbial communities, in particular from extreme habitats, into a valuable resource for the discovery of novel enzymes with potential applications in biotechnology. Different strategies for their uncovering such as bioprospecting, which relies mainly on metagenomic approaches in combination with sequence-based bioinformatic analyses, have emerged; yet accurate function prediction of their proteomes and deciphering the in vivo activity of an enzyme remains challenging. RESULTS: Here, we present environmental activity-based protein profiling (eABPP), a multi-omics approach that extends genome-resolved metagenomics with mass spectrometry-based ABPP. This combination allows direct profiling of environmental community samples in their native habitat and the identification of active enzymes based on their function, even without sequence or structural homologies to annotated enzyme families. eABPP thus bridges the gap between environmental genomics, correct function annotation, and in vivo enzyme activity. As a showcase, we report the successful identification of active thermostable serine hydrolases from eABPP of natural microbial communities from two independent hot springs in Kamchatka, Russia. CONCLUSIONS: By reporting enzyme activities within an ecosystem in their native state, we anticipate that eABPP will not only advance current methodological approaches to sequence homology-guided enzyme discovery from environmental ecosystems for subsequent biocatalyst development but also contributes to the ecological investigation of microbial community interactions by dissecting their underlying molecular mechanisms.

Natronosalvus hydrolyticus sp. nov., a beta-1,3-glucan utilizing natronoarchaeon from hypersaline soda lakes.

Use of curldlan, an insoluble ß-1,3-glucan, as an enrichment substrate under aerobic conditions resulted in the selection from hypersaline soda lakes of a single natronarchaeon, strain AArc-curdl1. This organism is an obligately aerobic saccharolytic, possessing a poorly explored (in Archaea) potential to utilize beta-1-3 glucans, being only a second example of a haloarchaeon with this ability known in pure culture. The main phenotypic property of the isolate is the ability to grow with insoluble ß-1,3-backboned glucans, i.e. curdlan and pachyman. Furthermore, the strain utilized starch family α-glucans, beta-fructan inulin and a limited spectrum of sugars. The major ether-bound membrane polar phospholipids included PGP-Me and PG. The glyco- and sulfolipids were absent. The major respiratory menaquinone is MK-8:8. According to phylogenomic analysis, AArc-curdl1 represents a separate species in the recently described genus Natronosalvus within the family Natrialbaceae. The closest related species is Natronosalvus amylolyticus (ANI, AAI and DDH values of 90.2, 91.6 and 44 %, respectively). On the basis of its unique physiological properties and phylogenomic distance, strain AArc-curdl1T is classified as a novel species Natronosalvus hydrolyticus sp. nov. (=JCM 34865 = UQM 41566).

Natronoglomus mannanivorans gen. nov., sp. nov., beta-1,4-mannan utilizing natronoarchaea from hypersaline soda lakes.

Beta-mannans are insoluble plant polysaccharides with beta-1,4-linked mannose as the backbone. We used three forms of this polysaccharide, namely, pure mannan, glucomannan, and galactomannan, to enrich haloarchaea, which have the ability to utilize mannans for growth. Four mannan-utilizing strains obtained in pure cultures were closely related to each other on the level of the same species. Furthermore, another strain selected from the same habitats with a soluble beta-1,4-glucan (xyloglucan) was also able to grow with mannan. The phylogenomic analysis placed the isolates into a separate lineage of the new genus level within the family Natrialbaceae of the class Halobacteria. The strains are moderate alkaliphiles, extremely halophilic, and aerobic saccharolytics. In addition to the three beta-mannan forms, they can also grow with cellulose, xylan, and xyloglucan. Functional genome analysis of two representative strains demonstrated the presence of several genes coding for extracellular endo-beta-1,4-mannanase from the GH5_7 and 5_8 subfamilies and the GH26 family of glycosyl hydrolases. Furthermore, a large spectrum of genes encoding other glycoside hydrolases that were potentially involved in the hydrolysis of cellulose and xylan were also identified in the genomes. A comparative genomics analysis also showed the presence of similar endo-beta-1,4-mannanase homologs in the cellulotrophic genera Natronobiforma and Halococcoides. Based on the unique physiological properties and the results of phylogenomic analysis, the novel mannan-utilizing halolarchaea are proposed to be classified into a new genus and species Natronoglomus mannanivorans gen. nov., sp. nov. with the type strain AArc-m2/3/4 (=JCM 34861=UQM 41565).

Halapricum hydrolyticum sp. nov., a beta-1,3-glucan utilizing haloarchaeon from hypersaline lakes.

Two strains of neutrophilic haloaloarchaea were selectively enriched from hypersaline lakes in southwestern Siberia using ß-1,3-glucans as a substrate. The strains were nearly identical in their phenotypes and according to phylogenomic analysis, and represent a distant novel species group in the genus Halapricum of the family Haloarculaceae. The main phenotypic property of the novel isolates is the ability to hydrolyze and grow with the polysaccharides curdlan and pachyman. Such potential has, to date, not been seen in any other haloarchaea in pure cultures. The strains are obligately aerobic saccharolytics. Apart from the insoluble ß-1,3-glucans, they utilized soluble α-glucans (starch, pullulan and glycogen) and a limited number of sugars. The major ether-bound polar phospholipids include PGP-Me and PG. The glyco- and sulfolipids were absent. The major respiratory menaquinone is MK-8:8. On the basis of their unique physiological properties and the results of phylogenomic analysis, the isolates are suggested to be classified into a novel species Halapricum hydrolyticum sp. nov. (type strain HArc-curdl5-1T = DSM 114193T = UQM 41587T).

Cellulose metabolism in halo(natrono)archaea: a comparative genomics study.

Extremely halophilic archaea are one of the principal microbial community components in hypersaline environments. The majority of cultivated haloarchaea are aerobic heterotrophs using peptides or simple sugars as carbon and energy sources. At the same time, a number of novel metabolic capacities of these extremophiles were discovered recently among which is a capability of growing on insoluble polysaccharides such as cellulose and chitin. Still, polysaccharidolytic strains are in minority among cultivated haloarchaea and their capacities of hydrolyzing recalcitrant polysaccharides are hardly investigated. This includes the mechanisms and enzymes involved in cellulose degradation, which are well studied for bacterial species, while almost unexplored in archaea and haloarchaea in particular. To fill this gap, a comparative genomic analysis of 155 cultivated representatives of halo(natrono)archaea, including seven cellulotrophic strains belonging to the genera Natronobiforma, Natronolimnobius, Natrarchaeobius, Halosimplex, Halomicrobium and Halococcoides was performed. The analysis revealed a number of cellulases, encoded in the genomes of cellulotrophic strains but also in several haloarchaea, for which the capacity to grow on cellulose was not shown. Surprisingly, the cellulases genes, especially of GH5, GH9 and GH12 families, were significantly overrepresented in the cellulotrophic haloarchaea genomes in comparison with other cellulotrophic archaea and even cellulotrophic bacteria. Besides cellulases, the genes for GH10 and GH51 families were also abundant in the genomes of cellulotrophic haloarchaea. These results allowed to propose the genomic patterns, determining the capability of haloarchaea to grow on cellulose. The patterns helped to predict cellulotrophic capacity for several halo(natrono)archaea, and for three of them it was experimentally confirmed. Further genomic search revealed that glucose and cellooligosaccharides import occurred by means of porters and ABC (ATP-binding cassette) transporters. Intracellular glucose oxidation occurred through glycolysis or the semi-phosphorylative Entner-Dudoroff pathway which occurrence was strain-specific. Comparative analysis of CAZymes toolbox and available cultivation-based information allowed proposing two possible strategies used by haloarchaea capable of growing on cellulose: so-called specialists are more effective in degradation of cellulose while generalists are more flexible in nutrient spectra. Besides CAZymes profiles the groups differed in genome sizes, as well as in variability of mechanisms of import and central metabolism of sugars.

Fontisphaera persica gen. nov., sp. nov., a thermophilic hydrolytic bacterium from a hot spring of Baikal lake region, and proposal of Fontisphaeraceae fam. nov., and Limisphaeraceae fam. nov. within the Limisphaerales ord. nov. (Verrucomicrobiota).

A novel facultatively anaerobic moderately thermophilic bacterium, strain B-154 T, was isolated from a terrestrial hot spring in the Baikal lake region (Russian Federation). Gram-negative, motile, spherical cells were present singly, in pairs, or aggregates, and reproduced by binary fission. The strain grew at 30-57 °C and within a pH range of 5.1-8.4 with the optimum at 50 °C and pH 6.8-7.1. Strain B-154 T was a chemoorganoheterotroph, growing on mono-, di- and polysaccharides (xylan, starch, galactan, galactomannan, glucomannan, xyloglucan, pullulan, arabinan, lichenan, beta-glucan, pachyman, locust bean gum, xanthan gum). It did not require sodium chloride or yeast extract for growth. Major cellular fatty acids were anteiso-C15:0, iso-C16:0 and iso-C14:0. The respiratory quinone was MK-7. The complete genome of strain B-154 T was 4.73 Mbp in size; its G + C content was 61%. According to the phylogenomic analysis strain B-154 T forms a separate family-level phylogenetic lineage. Moreover, together with Limisphaera ngatamarikiensis and "Pedosphaera parvula" this strain forms a separate order-level phylogenetic lineage within Verrucomicrobiae class. Hence, we propose a novel order, Limisphaerales ord. nov., with two families Limisphaeraceae fam. nov. and Fontisphaeraceae fam. nov., and a novel genus and species Fontisphaera persica gen. nov., sp. nov. with type strain B-154 T. Ecogenomic analysis showed that representatives of the Limisphaerales are widespread in various environments. Although some of them were detected in hot springs the majority of Limisphaerales (54% of the studied metagenome-assembled genomes) were found in marine habitats. This study allowed a better understanding of physiology and ecology of Verrucomicrobiota - a rather understudied bacterial phylum.

Obligate autotrophy at the thermodynamic limit of life in a new acetogenic bacterium.

One of the important current issues of bioenergetics is the establishment of the thermodynamic limits of life. There is still no final understanding of what is the minimum value of the energy yield of a reaction that is sufficient to be used by an organism (the so-called "biological quantum of energy"). A reasonable model for determination of the minimal energy yield would be microorganisms capable of living on low-energy substrates, such as acetogenic prokaryotes. The most prominent metabolic feature of acetogens is autotrophic growth with molecular hydrogen and carbon dioxide as the substrates, which is hardly competitive in environments. Most probably, that is why only facultative autotrophic acetogens have been known so far. Here, we describe the first obligately autotrophic acetogenic bacterium Aceticella autotrophica gen. nov., sp. nov., strain 3443-3AcT. Phylogenetically, the new genus falls into a monophyletic group of heterotrophic bacteria of the genera Thermoanaerobacterium, Thermoanaerobacter, and Caldanaerobacter (hereinafter referred to as TTC group), where the sole acetogenic representative has so far been the facultatively autotrophic Thermoanaerobacter kivui. A. autotrophica and T. kivui both are acetogens employing energy-converting hydrogenase (Ech-acetogens) that are likely to have inherited the acetogenesis capacity vertically from common ancestor. However, their acetogenic machineries have undergone different adjustments by gene replacements due to horizontal gene transfers from different donors. Obligate autotrophy of A. autotrophica is associated with the lack of many sugar transport systems and carbohydrate catabolism enzymes that are present in other TTC group representatives, including T. kivui.

Draft Genome Sequence of Lactococcus lactis Strain 12-16-PSH, Isolated from Prostokvasha from the Arkhangelsk Region of Russia.

A draft genome sequence of Lactococcus lactis strain 12-16-PSH, which was isolated from prostokvasha, is reported. The genome assembly of strain 12-16-PSH contained 63 contigs, with a total length of 2,468,647 bp. A total of 2,421 protein-coding genes were predicted, among which 6 encoded bacteriocins while 15 encoded glycosyl transferases, presumably involved in exopolysaccharide biosynthesis.

Draft Genome Sequence of Thermomicrobium sp. Strain 4228-Ro, a Thermophilic Bacterium Isolated from a Kamchatka Hot Spring.

The genome of Thermomicrobium sp. strain 4228-Ro, an aerobic thermophilic bacterium isolated from a Kamchatka hot spring, was sequenced and analyzed. The genome assembly comprises 13 contigs with a total length of 3,068,448 bp. Genome analysis revealed the pathway of aerobic utilization of sugars, which was corroborated by growth experiments.

Natronogracilivirga saccharolytica gen. nov., sp. nov. and Cyclonatronum proteinivorum gen. nov., sp. nov., haloalkaliphilic organotrophic bacteroidetes from hypersaline soda lakes forming a new family Cyclonatronaceae fam. nov. in the order Balneolales.

Two heterotrophic bacteroidetes strains were isolated as satellites from autotrophic enrichments inoculated with samples from hypersaline soda lakes in southwestern Siberia. Strain Z-1702T is an obligate anaerobic fermentative saccharolytic bacterium from an iron-reducing enrichment culture, while Ca. Cyclonatronum proteinivorum OmegaT is an obligate aerobic proteolytic microorganism from a cyanobacterial enrichment. Cells of isolated bacteria are characterized by highly variable morphology. Both strains are chloride-independent moderate salt-tolerant obligate alkaliphiles and mesophiles. Strain Z-1702T ferments glucose, maltose, fructose, mannose, sorbose, galactose, cellobiose, N-acetyl-glucosamine and alpha-glucans, including starch, glycogen, dextrin, and pullulan. Strain OmegaT is strictly proteolytic utilizing a range of proteins and peptones. The main polar lipid fatty acid in both strains is iso-C15:0, while other major components are various C16 and C17 isomers. According to pairwise sequence alignments using BLAST Gracilimonas was the nearest cultured relative to both strains (<90% of 16S rRNA gene sequence identity). Phylogenetic analysis placed strain Z-1702T and strain OmegaT as two different genera in a deep-branching clade of the new family level within the order Balneolales with genus. Based on physiological characteristics and phylogenetic position of strain Z-1702T it was proposed to represent a novel genus and species Natronogracilivirga saccharolityca gen. nov., sp. nov. (= DSMZ 109061T =JCM 32930T =VKM B 3262T). Furthermore, phylogenetic and phenotypic parameters of N. saccharolityca and C. proteinivorum gen. nov., sp. nov., strain OmegaT (=JCM 31662T, =UNIQEM U979T), make it possible to include them into a new family with a proposed designation Cyclonatronaceae fam. nov..

Draft Genome Sequence of Pyrobaculum sp. Strain 3827-6, a Hyperthermophilic Crenarchaeon, Isolated from a Kamchatka Hot Spring.

The genome of Pyrobaculum sp. strain 3827-6, a facultative autotrophic hyperthermophilic archaeon isolated from a Kamchatka hot spring, was sequenced and analyzed. Genome analysis predicted the dicarboxylate/4-hydroxybutyrate cycle and a [NiFe]-hydrogenase, as well as the tricarboxylic acid cycle, altogether determining the possibility of both autotrophic and heterotrophic growth of this strain.

Taxonomic proposal for a deep branching bacterial phylogenetic lineage: transfer of the family Thermodesulfobiaceae to Thermodesulfobiales ord. nov., Thermodesulfobiia classis nov. and Thermodesulfobiota phyl. nov.

The family Thermodesulfobiaceae, comprising one genus Thermodesulfobium with two validly published species, is currently assigned to order Thermoanaerobacterales within the class Clostridia of the phylum Bacillota. At the same time, the very first 16S rRNA gene sequence-based phylogenetic studies of representatives of the genus pointed out great differences between Thermodesulfobium and other members of the phylum Bacillota. Subsequent studies of new Thermodesulfobium representatives supported deep phylogenetic branching of this lineage within bacterial tree, implying that it represents a novel phylum. The results of the phylogenomic analysis performed in the frames of the present work confirm previous findings and suggest that Thermodesulfobium represents a distinct phylum-level lineage. Thus, we propose the transfer of the family Thermodesulfobiaceae to the new order Thermodesulfobiales within the new class Thermodesulfobiia and the new phylum Thermodesulfobiota.

Metagenomic Insights into the Taxonomic and Functional Features of Traditional Fermented Milk Products from Russia.

Fermented milk products (FMPs) contain probiotics that are live bacteria considered to be beneficial to human health due to the production of various bioactive molecules. In this study, nine artisanal FMPs (kefir, ayran, khurunga, shubat, two cottage cheeses, bryndza, khuruud and suluguni-like cheese) from different regions of Russia were characterized using metagenomics. A metagenomic sequencing of ayran, khurunga, shubat, khuruud and suluguni-like cheese was performed for the first time. The taxonomic profiling of metagenomic reads revealed that Lactococcus species, such as Lc. lactis and Lc. cremoris prevailed in khuruud, bryndza, one sample of cottage cheese and khurunga. The latter one together with suluguni-like cheese microbiome was dominated by bacteria, affiliated to Lactobacillus helveticus (32-35%). In addition, a high proportion of sequences belonging to the genera Lactobacillus, Lactococcus and Streptococcus but not classified at the species level were found in the suluguni-like cheese. Lactobacillus delbrueckii, as well as Streptococcus thermophilus constituted the majority in another cottage cheese, kefir and ayran metagenomes. The microbiome of shubat, produced from camel's milk, was significantly distinctive, and Lentilactobacillus kefiri, Lactobacillus kefiranofaciens and Bifidobacterium mongoliense represented the dominant components (42, 7.4 and 5.6%, respectively). In total, 78 metagenome-assembled genomes with a completeness ≥ 50.2% and a contamination ≤ 8.5% were recovered: 61 genomes were assigned to the Enterococcaceae, Lactobacillaceae and Streptococcaceae families (the Lactobacillales order within Firmicutes), 4 to Bifidobacteriaceae (the Actinobacteriota phylum) and 2 to Acetobacteraceae (the Proteobacteria phylum). A metagenomic analysis revealed numerous genes, from 161 to 1301 in different products, encoding glycoside hydrolases and glycosyltransferases predicted to participate in lactose, alpha-glucans and peptidoglycan hydrolysis as well as exopolysaccharides synthesis. A large number of secondary metabolite biosynthetic gene clusters, such as lanthipeptides, unclassified bacteriocins, nonribosomal peptides and polyketide synthases were also detected. Finally, the genes involved in the synthesis of bioactive compounds like ß-lactones, terpenes and furans, nontypical for fermented milk products, were also found. The metagenomes of kefir, ayran and shubat was shown to contain either no or a very low count of antibiotic resistance genes. Altogether, our results show that traditional indigenous fermented products are a promising source of novel probiotic bacteria with beneficial properties for medical and food industries.

Selective enrichment on a wide polysaccharide spectrum allowed isolation of novel metabolic and taxonomic groups of haloarchaea from hypersaline lakes.

Extremely halophilic archaea (haloarchaea) of the class Halobacteria is a dominant group of aerobic heterotrophic prokaryotic communities in salt-saturated habitats, such as salt lakes and solar salterns. Most of the pure cultures of haloarchaea were enriched, isolated, and cultivated on rich soluble substrates such as amino acids, peptides or simple sugars. So far, the evidences on the capability of haloarchaea to use different polysaccharides as growth substrates remained scarce. However, it is becoming increasingly obvious that these archaea can also actively participate in mineralization of complex biopolymers, in particular cellulose and chitin-two dominant biomass polysaccharides on the planet. Here we used an array of commercially available homo- and heteropolysaccharides to enrich hydrolytic haloarchaea from hypersaline salt lakes with neutral pH and from alkaline soda lakes. This resulted in isolation of a range of halo- and natrono-archaea, respectively, belonging to already described taxa as well as several new genus-level lineages. In some cases, the isolates enriched with different polysaccharides happened to be closely related, thus representing generalistic ecotype, while the others were narrow specialists. In general, soda lakes yielded a broader range of polysaccharide-utilizing specialists in comparison to neutral salt lakes. The results demonstrated a significant diversity of halo(natrono)archaea with a previously unrecognized potential for utilization of a broad range of natural polysaccharides in hypersaline habitats.

Fontivita pretiosa gen. nov., sp. nov., a thermophilic planctomycete of the order Tepidisphaerales from a hot spring of Baikal lake region.

A novel facultatively anaerobic moderately thermophilic bacterium, strain B-254T, was isolated from a terrestrial hot spring near the town of Goryachinsk in the Baikal lake region (Russian Federation). Motile spherical cells of the strain were present as single cocci, in pairs, or aggregates. The cells had a Gram negative cell wall and reproduced by binary fission. The isolate grew at 30-57 °C (opt. 50-54 °C) and at pH 5.1-8.4 (opt. 6.6-7.1). Strain B-254T was a chemoorganoheterotroph, growing on mono-, di- and polysaccharides (xylan, starch, galactan, galactomannan, xyloglucan, arabinan, curdlan, beta-glucan, locust bean gum, xanthan gum). Sodium chloride or yeast extract were not required for growth. Major cellular fatty acids were iso-C16:0, anteiso-C17:0, and C20:0; major polar lipid was phosphatidylethanolamine. The complete genome of strain B-254T was 5.54 Mb; its GC content was 64 %. According to the results of 16S rRNA gene sequence-based phylogenetic analysis and the conserved proteins sequences-based phylogenomic analysis strain B-254T was on a separate lineage within the order Tepidisphaerales (Phycisphaerae, Planctomycetes). Based on phylogenetic and phylogenomic analyses of Phycisphaerae, whole genome comparisons of Tepidisphaerales as well as distinctive phenotypic features of the strain, it was assigned to a novel genus and species for which the name Fontivita pretiosa gen. nov. sp. nov. is proposed. Strain B-254T = KCTC 82380T = VKM B-3507T.

Microbial Communities of Artisanal Fermented Milk Products from Russia.

Fermented milk products (FMPs) have numerous health properties, making them an important part of our nutrient budget. Based on traditions, history and geography, there are different preferences and recipes for FMP preparation in distinct regions of the world and Russia in particular. A number of dairy products, both widely occurring and region-specific, were sampled in the households and local markets of the Caucasus republics, Buryatia, Altai, and the Far East and European regions of Russia. The examined FMPs were produced from cow, camel, mare's or mixed milk, in the traditional way, without adding commercial starter cultures. Lactate and acetate were the major volatile fatty acids (VFA) of the studied FMPs, while succinate, formate, propionate and n-butyrate were present in lower concentrations. Bacterial communities analyzed by 16S rRNA gene V4 fragment amplicon sequencing showed that Firmicutes (Lactococcus, Lactobacillus, Streptococcus, Lentilactobacillus and Leuconostoc) was the predominant phylum in all analyzed FMPs, followed by Proteobacteria (Acetobacter, Klebsiella, Pseudomonas and Citrobacter). Lactobacillus (mainly in beverages) or Lactococcus (mainly in creamy and solid products) were the most abundant community-forming genera in FMPs where raw milk was used and fermentation took place at (or below) room temperature. In turn, representatives of Streptococcus genus dominated the FMPs made from melted or pasteurized milk and fermented at elevated temperatures (such as ryazhenka, cottage cheese and matsoni-like products). It was revealed that the microbial diversity of koumiss, shubat, ryazhenka, matsoni-like products, chegen, sour cream and bryndza varied slightly within each type and correlated well with the same products from other regions and countries. On the other hand, the microbiomes of kefir, prostokvasha, ayran, cottage cheese and suluguni-like cheese were more variable and were shaped by the influence of particular factors linked with regional differences and traditions expressed in specificities in the production process. The microbial diversity of aarts, khurunga, khuruud, tan, ayran and suluguni-like cheese was studied here, to our knowledge, for the first time. The results of this study emphasize the overall similarity of the microbial communities of various FMPs on the one hand, and specificities of regional products on the other. The latter are of particular value in the age of globalization when people have begun searching for new and unusual products and properties. Speaking more specifically, these novel products, with their characteristic communities, might be used for the development of novel microbial associations (i.e., starters) to produce novel products with improved or unique properties.

Natronocalculus amylovorans gen. nov., sp. nov., and Natranaeroarchaeum aerophilus sp. nov., dominant culturable amylolytic natronoarchaea from hypersaline soda lakes in southwestern Siberia.

Several pure cultures of alkaliphilic haloaloarchaea were enriched and isolated from hypersaline soda lakes in southwestern Siberia using amylopectin and fructans as substrates. Phylogenomic analysis placed the isolates into two distinct groups within the class Halobacteria. Four isolates forming group 1 were closely related to a recently described Natranaeroarchaeum sulfidigenes and the other three strains forming group 2 represent a novel genus-level phylogenetic lineage. All isolates are saccharolytic archaea growing with various starch-like alpha-glucans including soluble starch, amylopectin, dextrin, glycogen, pullulane and cyclodextrin. In addition, group 1 can use levan while group 2 - inulin (plant storage beta-fructans). Group 1 strains can also grow anaerobically with either glucose or maltose using elemental sulfur as the electron acceptor. Both groups are moderately alkaliphilic with a pH range for growth from 7.2 to 9.3 (optimum between 8.0-8.8) and low Mg-demanding extreme halophiles growing optimally at 4 M total Na+. The major respiratory menaquinone is MK-8:8 and the core biphytanyl lipids are dominated by archaeol (C20-C20) and a less abundant extended archaeol (C20-C25) with PG and PGP-Me as polar groups. The four isolates of group 1 are suggested to be classified into a new species as Natranaeroarchaeum aerophilus sp. nov. (type strain AArc-St1-1T = JCM 32519T). The three isolates of group 2 are proposed to form a new genus and species for which the name Natronocalculus amylovorans gen. nov., sp. nov. is suggested (type strain AArc-St2T = JCM 32475T).

Natronosporangium hydrolyticum gen. nov., sp. nov., a haloalkaliphilic polyhydrolytic actinobacterium from a soda solonchak soil in Central Asia.

During a cultural diversity survey on hydrolytic bacteria in saline alkaline soils, a hydrolytic actinobacterium strain ACPA39T was enriched and isolated in pure culture from a soda solonchak soil in southwestern Siberia. It forms a substrate mycelium with rod-shaped sporangia containing 1-3 exospores. The isolate is obligately alkaliphilic, growing at pH 7.5-10.3 (optimum at 8.5-9.0) and moderately halophilic, tolerating up to 3 M total Na+ in the form of sodium carbonates. It is an obligately aerobic, organoheteroterophic, saccharolytic bacterium, utilizing various sugars and alpha/beta-glucans as growth substrates. According to the 16S rRNA gene-based phylogenetic analysis, strain ACPA39T forms a distinct branch within the family Micromonosporaceae, with the sequence identities below 94.5% with type strains of other genera. This is confirmed by phylogenomic analysis based on the 120 conserved single copy protein-based markers and genomic indexes (ANI, AAI). The cell-wall of ACPA39T contained meso-DAP, glycine, glutamic acid and alanine in a equimolar ratio, characteristic of the peptidoglycan type A1γ'. The whole-cell sugars include galactose and xylose. The major menaquinone is MK-10(H4). The identified polar lipids consist of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The polar lipid fatty acids were dominated by anteiso-C17:0, iso-C16:0, iso-C17:0, 10 Me-C18:0 and C18:1ω9. Based on the distinct phylogeny, the chemotaxonomy features and unique phenotypic properties, strain ACPA39T (DSM 106523T = VKM 2772T) is classified into a new genus and species in the family Micromonosporaceae for which the name Natronosporangium hydrolitycum gen. nov., sp. nov. is proposed.

Culture-Independent Survey of Thermophilic Microbial Communities of the North Caucasus.

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.