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Microbial communities are significant drivers of global biogeochemical cycles, yet accurate function prediction of their proteome and discerning their activityin situfor bioprospecting remains challenging. Here, we present environmental activity-based protein profiling (eABPP) as a novel proteomics-based approach bridging the gap between environmental genomics, correct function annotation andin situenzyme activity. As a showcase, we report the successful identification of active thermostable serine hydrolases by combining genome-resolved metagenomics and mass spectrometry-based eABPP of natural microbial communities from two independent hot springs in Kamchatka, Russia. eABPP does not only advance current methodological approaches by providing evidence for enzyme and microbial activityin situbut also represents an alternative approach to sequence homology-guided biocatalyst discovery from environmental ecosystems.

A moderately thermophilic, neutrophilic, aerobic, Gram-negative bacterium, strain 3729kT, was isolated from a thermal spring of the Chukotka Peninsula, Arctic region, Russia. It grew chemoorganoheterotrophically, utilizing proteinaceous substrates, including highly rigid keratins as well as various polysaccharides (glucomannan, locust bean gum, gum guar and xanthan gum). The major fatty acids of strain 3729kTwere iso-C15 : 0(60.9%), iso-C17 : 0(12.0%), C16 : 0(9.9%) and iso-C16 : 0(7.4%). Isoprenoid quinones were Q-8 (95%) and Q-9 (5%). The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine and three unidentified polar lipids. Strain 3729kTwas inhibited by chloramphenicol, neomycin, novobiocin, kanamycin, tetracycline, ampicillin and polymyxin B, but resistant to rifampicin, vancomycin and streptomycin. At the same time, strain 3729kTinhibited growth ofMicrococcus luteusand its genome possessed genes for antimicrobial activity against Gram-positive bacteria (a single putative bacteriocin and several secreted lysozymes and peptidoglycan lytic transglycosylases). The DNA G+C content was 69.8 mol%. 16S rRNA gene sequence-based phylogenetic analysis placed strain 3729kTinto a distinct species/genus-level branch within the familyXanthomonadaceae(Proteobacteria). Phylogenetic analysis of 120 conservative protein sequences of allXanthomonadaceaewith validly published names and publicly available genomic sequences supported a species-level position of strain 3729kTwithin the genusArenimonas. Pairwise ANI values between strain 3729kTand otherArenimonasspecies were of 75–80 %, supporting the proposal of a novel species. Accordingly,Arenimonas fontissp. nov., with the type strain 3729kT(=VMK В−3232Т=DSM 105847T), was proposed.

A novel hyperthermophilic crenarchaeon, strain 3507LTT, was isolated from a terrestrial hot spring near Tinguiririca volcano, Chile. Cells were non-motile thin, slightly curved filamentous rods. It grew at 73–93 °C and pH range of 5 to 7.5 with an optimum at 85 °C and pH 6.0–6.7. The presence of culture broth filtrate of another hyperthemophilic archaeon as well as yeast extract was obligatory for growth of the novel isolate. Strain 3507LTT is an anaerobic chemoorganoheterotroph, fermenting monosaccharides, disaccharides and polysaccharides (lichenan, starch, xanthan gum, xyloglucan, alpha-cellulose and amorphous cellulose). No growth stimulation was detected when nitrate, thiosulfate, selenate or elemental sulfur were added as the electron acceptors. The complete genome of strain 3507LTT consisted of a single circular chromosome with size of 1.63 Mbp. The DNA G+C content was 53.9%. According to the 16S rRNA gene sequence as well as conserved protein sequences phylogenetic analyses, strain 3507LTT together with Thermofilum uzonense formed a separate cluster within a Thermofilaceae family (Thermoproteales/Thermoprotei/Crenarchaeota). Based on phenotypic characteristics, phylogeny as well as AAI comparisons, a novel genus and species Infirmifilum lucidum strain 3507LTT (=VKM B-3376T = KCTC 15797T) gen. nov. sp. nov. was proposed. Its closest relative, Thermofilum uzonense strain 1807–2T should be reclassified as Infirmifilum uzonense strain 1807–2T comb. nov. Finally, based on phylogenomic and comparative genome analyses of representatives of Thermofilaceae family and other representatives of Thermoproteales order, a proposal of transfer of the family Thermofilaceae into a separate order Thermofilales ord. nov. was made.

Chukotka is an arctic region located in the continuous permafrost zone, but thermal springs are abundant there. In this study, for the first time, the microbial communities of the Chukotka hot springs (CHS) biofilms and sediments with temperatures 54&ndash, 94 &deg, C were investigated and analyzed by NGS sequencing of 16S rRNA gene amplicons. In microbial mats (54&ndash, 75 &deg, C), phototrophic bacteria of genus Chloroflexus dominated (up to 89% of all prokaryotes), while Aquificae were the most numerous at higher temperatures in Fe-rich sediments and filamentous &ldquo, streamers&rdquo, (up to 92%). The electron donors typical for Aquificae, such as H2S and H2, are absent or present only in trace amounts, and the prevalence of Aquificae might be connected with their ability to oxidize the ferrous iron present in CHS sediments. Armatimonadetes, Proteobacteria, Deinococcus-Thermus, Dictyoglomi, and Thermotogae, as well as uncultured bacteria (candidate divisions Oct-Spa1-106, GAL15, and OPB56), were numerous, and Cyanobacteria were present in low numbers. Archaea (less than 8% of the total community of each tested spring) belonged to Bathyarchaeota, Aigarchaeota, and Thaumarchaeota. The geographical location and the predominantly autotrophic microbial community, built on mechanisms other than the sulfur cycle-based ones, make CHS a special and unique terrestrial geothermal ecosystem.

Using a sample from a terrestrial hot spring (pH 6.8, 60 °C), we enriched a thermophilic microbial consortium performing anaerobic autotrophic oxidation of hydrothermal siderite (FeCO3), with CO2/bicarbonate as the electron acceptor and the only carbon source, producing green rust and acetate. In order to reproduce Proterozoic environmental conditions during the deposition of banded iron formation (BIF), we incubated the microbial consortium in a bioreactor that contained an unmixed anoxic layer of siderite, perfectly mixed N2/CO2-saturated liquid medium and microoxic (2% O2) headspace. Long-term incubation (56 days) led to the formation of magnetite (Fe3O4) instead of green rust as the main product of Fe(II) oxidation, the precipitation of newly formed metabolically induced siderite in the anoxic zone, and the deposition of hematite (Fe2O3) on bioreactor walls over the oxycline boundary. Acetate was the only metabolic product of CO2/bicarbonate reduction. Thus, we have demonstrated the ability of autotrophic thermophilic microbial consortium to perform a short cycle of iron minerals transformation: siderite–magnetite–siderite, accompanied by magnetite and hematite accumulation. This cycle is believed to have driven the evolution of the early biosphere, leading to primary biomass production and deposition of the main iron mineral association of BIF.

A novel hyperthermophilic, anaerobic filamentous archaeon, Thermofilum adornatum strain 1910bT, is capable of growing with cellulose as its sole carbon and energy source. This strain was isolated from a terrestrial hot spring in Kamchatka, Russia. The isolate 1910bT grew optimally at a temperature of 80°C and a pH of 5.5-6.0, producing cell-bound inducible cellulases. During genome analysis, genes, encoding various glycosidases (GHs) involved in oligo- and polysaccharide hydrolysis and genes for the fermentation of sugars were identified. No homologs of currently known cellulase families were found among the GHs encoded by the 1910bT genome, suggesting that novel proteins are involved. To figure this out, a proteomic analysis of cells grown on cellulose or pyruvate (as a control) was performed. Both in-depth genomic and proteomic analyses revealed four proteins (Cel25, Cel30, Cel40, and Cel45) that were the most likely to be involved in the cellulose hydrolysis in this archaeon. Two of these proteins (Cel30 and Cel45) were hypothetical according to genome analysis, while the other two (Cel25 and Cel40) have GH3 and GH1 domains, respectively. The respective genes were heterologously expressed in Escherichia coli BL21 (DE3), and enzymatic activities of recombinant proteins were measured with carboxymethyl cellulose (CMC), Avicel and cellobiose as substrates. It was revealed that the Cel30 and Cel25 proteins were likely exoglucanases with side beta-glucosidase and endoglucanase activities, that Cel40 was a multifunctional glucanase capable of hydrolyzing beta-1,4-glucosides of various lengths, and that Cel45 was an endoglucanase with side exoglucanase activity. Taking into account that the cellulolytic activity of T. adornatum 1910bT surface protein fractions was inducible, that recombinant Cel25 and Cel30 were much less active than Cel40 and Cel45, and that their gene expressions were (almost) non-induced by CMC, we suggest that Cel40 and Cel45 play a major role in the degradation of cellulose, while Cel25 and Cel30 act only as accessory enzymes.

A novel hyperthermophilic, anaerobic filamentous archaeon, Thermofilum adornatum strain 1910bT, is capable of growing with cellulose as its sole carbon and energy source. This strain was isolated from a terrestrial hot spring in Kamchatka, Russia. The isolate 1910bT grew optimally at a temperature of 80°C and a pH of 5.5–6.0, producing cell-bound inducible cellulases. During genome analysis, genes, encoding various glycosidases (GHs) involved in oligo- and polysaccharide hydrolysis and genes for the fermentation of sugars were identified. No homologs of currently known cellulase families were found among the GHs encoded by the 1910bT genome, suggesting that novel proteins are involved. To figure this out, a proteomic analysis of cells grown on cellulose or pyruvate (as a control) was performed. Both in-depth genomic and proteomic analyses revealed four proteins (Cel25, Cel30, Cel40, and Cel45) that were the most likely to be involved in the cellulose hydrolysis in this archaeon. Two of these proteins (Cel30 and Cel45) were hypothetical according to genome analysis, while the other two (Cel25 and Cel40) have GH3 and GH1 domains, respectively. The respective genes were heterologously expressed in Escherichia coli BL21 (DE3), and enzymatic activities of recombinant proteins were measured with carboxymethyl cellulose (CMC), Avicel and cellobiose as substrates. It was revealed that the Cel30 and Cel25 proteins were likely exoglucanases with side beta-glucosidase and endoglucanase activities, that Cel40 was a multifunctional glucanase capable of hydrolyzing beta-1,4-glucosides of various lengths, and that Cel45 was an endoglucanase with side exoglucanase activity. Taking into account that the cellulolytic activity of T. adornatum 1910bT surface protein fractions was inducible, that recombinant Cel25 and Cel30 were much less active than Cel40 and Cel45, and that their gene expressions were (almost) non-induced by CMC, we suggest that Cel40 and Cel45 play a major role in the degradation of cellulose, while Cel25 and Cel30 act only as accessory enzymes.

The ability to grow by anaerobic CO oxidation with production of H

We present the results of a study of mesophilic anoxygenic phototrophic Chloroflexota bacteria from Mechigmen hot spring (the Chukotka Peninsula) and Siberia. According to 16S rRNA phylogenetic analysis, these bacteria belong to Oscillochloris trichoides. However, sequencing the draft genome of the bacterium from the Chukotka and analysis of the average nucleotide identity, as well as in silico DNA-DNA hybridization, reveal that this bacterium belongs to a novel species within the Oscillochloris genus. We, therefore, propose ‘Candidatus Oscillochloris fontis’ as a novel taxon to represent this mesophilic alkaliphilic anaerobic anoxygenic phototrophic bacterium. Spectrophotometry and high-performance liquid chromatography analysis show that the bacterium possesses bacteriochlorophylls c and a, as well as lycopene, β-carotene and γ-carotene. In addition, transmission electron microscopy shows the presence of chlorosomes, polyhydroxyalkanoate- and polyphosphate-like granules. The genome of ‘Ca. Oscillochloris fontis’ and the Siberian strains of Oscillochloris sp. possess the key genes for nitrogenase complex (nifH) and ribulose-1,5-bisphosphate carboxylase/oxygenase (cbbL), as previously described for O. trichoides DG-6. The results presented here, and previously published data, show that Oscillochloris bacteria from different aquatic environments have the potential for CO2 and N2 fixation. Additionally, we describe a new primer system for the detection of RuBisCo form I.

An obligately anaerobic, hyperthermophilic, organoheterotrophic archaeon, strain 1633T, was isolated from a terrestrial hot spring of the Uzon Caldera (Kamchatka Peninsula, Russia). Cells were regular cocci, 0.5–0.9 μm in diameter, with one flagellum. The temperature range for growth was 80–95 °C, with an optimum at 84 °C. Strain 1633T grew on yeast extract, beef extract, peptone, cellulose and cellobiose. No growth was detected on other sugars or carbohydrates, organic acids, or under autotrophic conditions. The only detected growth products were CO2, acetate, and H2. The growth rate was stimulated by elemental sulfur, which was reduced to hydrogen sulfide. The in silico-calculated G+C content of the genomic DNA of strain 1633T was 55.64 mol%. 16S rRNA gene sequence analysis placed strain 1633T together with the non-validly published ‘Thermogladius shockii’ strain WB1 in a separate genus-level cluster within the family Desulfurococcaceae. Average nucleotide identity (ANI) results revealed 75.72 % identity between strain 1633T and ‘Thermogladius shockii’ WB1. Based on these results we propose a novel genus and species with the name Thermogladius calderae gen. nov., sp. nov. The type strain of the type species is 1633T ( = DSM 22663T = VKM B-2946T).

A novel anaerobic moderately thermophilic bacterium, designated strain 38H-strT, was isolated from a 12m deep hot spring of the Kunashir Island shore. Gram-negative cells were non-spore-forming, motile, straight or curved filamentous rods, occasionally forming loops and knots. The strain grew at 20-65°C and pH range of 4.0-9.0 with an optimum at 50°C and pH 6.5-7.0. Strain 38H-strT required 0.5-2.5% NaCl (1.5% is an optimum) for growth. It was a chemoorganoheterotroph, growing on carbohydrates (starch, pullulan, alginate, laminarin, beta-glucan) or peptide mixtures and proteins (peptone, tryptone, gelatin, and α- or β- keratins). Major products of glucose fermentation were acetate, hydrogen, and carbon dioxide. Major cellular fatty acids were iso- and anteiso-C15:0. Phosphatidylethanolamine, an unidentified phospholipid, and three unidentified polar lipids were detected in cellular lipids fractions. The quinone was MK-7. The size of complete genome of strain 38H-strT was 3.2 Mb; DNA G+C content was 38.3mol%. According to 16S rRNA gene sequence and conserved protein sequences phylogenies, strain 38H-strT represented a deeply branched lineage near the root of the class Bacteroidia. Based on phylogenetic analysis and phenotypic features the novel isolate was assigned to a novel family within the order Bacteroidales for which the name Tenuifilaceae fam. nov. is proposed. Strain 38H-strT (=DSM 100343T =VKM B-2964T) represents the first genus and species Tenuifilum thalassicum gen. nov., sp. nov.

The ability to grow by anaerobic CO oxidation with production of H2 from water is known for some thermophilic bacteria, most of which belong to Firmicutes, as well as for a few hyperthermophilic Euryarchaeota isolated from deep-sea hydrothermal habitats. A hyperthermophilic, neutrophilic, anaerobic filamentous archaeon strain 1505=VKM B-3180=KCTC 15798 was isolated from a terrestrial hot spring in Kamchatka (Russia) in the presence of 30% CO in the gas phase. Strain 1505 could grow lithotrophically using carbon monoxide as the energy source with the production of hydrogen according to the equation CO+H2O→CO2+H2; mixotrophically on CO plus glucose; and organotrophically on peptone, yeast extract, glucose, sucrose, or Avicel. The genome of strain 1505 was sequenced and assembled into a single chromosome. Based on 16S rRNA gene sequence analysis and in silico genome-genome hybridization, this organism was shown to be closely related to the Thermofilum adornatum species. In the genome of Thermofilum sp. strain 1505, a gene cluster (TCARB_0867-TCARB_0879) was found that included genes of anaerobic (Ni,Fe-containing) carbon monoxide dehydrogenase and genes of energy-converting hydrogenase ([Ni,Fe]-CODH-ECH gene cluster). Compared to the [Ni,Fe]-CODH-ECH gene clusters occurring in the sequenced genomes of other H2-producing carboxydotrophs, the [Ni,Fe]-CODH-ECH gene cluster of Thermofilum sp. strain 1505 presented a novel type of gene organization. The results of the study provided the first evidence of anaerobic CO oxidation coupled with H2 production performed by a crenarchaeon, as well as the first documented case of lithotrophic growth of a Thermofilaceae representative.

Strain 1633, a novel member of the genus Thermogladius , isolated from a freshwater hot spring, is an anaerobic hyperthermophilic crenarchaeon capable of fermenting proteinaceous and cellulose substrates. The complete genome sequence reveals genes for protein and carbohydrate-active enzymes, the Embden-Meyerhof pathway for glucose metabolism, cytoplasmic NADP-dependent hydrogenase, and several energy-coupling membrane-bound oxidoreductases.

Carbon monoxide (CO) is one of the common gaseous compounds found in hot volcanic environments. It is known to serve as the growth substrate for a number of thermophilic prokaryotes, both aerobic and anaerobic. The goal of this work was to study the process of anaerobic transformation of CO by microbial communities inhabiting natural thermal environments: hot springs of Uzon Caldera, Kamchatka. The anaerobic microbial community of Treshchinny Spring (80°C, pH 6.5) was found to exhibit two peaks of affinity for CO (K S1 = 54 nM and K S2 = 1 μM). The actual rate of anaerobic CO transformation by the microbial community of this spring, calculated after obtaining the concentration dependence curve and extrapolated to the natural concentration of CO dissolved in the hot spring water (20 nM), was found to be 120 μmol l−1 of sediment day−1. In all the hot springs studied, more than 90% of the carbon of 14CO upon anaerobic incubation was recovered as 14CO2. From 1 to 5% of 14CO was transformed to volatile fatty acids (VFA). The number of microorganisms capable of anaerobic CO oxidation determined by dilution-to-extinction method reached 106 cells ml−1 of sediment. CO-transforming anaerobic thermophilic microorganisms isolated from the springs under study exhibited hydrogenogenic type of CO oxidation and belonged to the bacterial genera Carboxydocella and Dictyoglomus. These data suggest a significant role of hydrogenogenic carboxydotrophic prokaryotes in anaerobic CO transformation in Uzon Caldera hot springs.