Hot Spring In Kamchatka Research Articles

Water and Sediments of an Acidic Hot Spring—Distinct Differentiation with Regard to the Microbial Community Composition and Functions

Over the last half-century, microbial communities of the Kamchatka hot springs have been largely studied using molecular, radioisotopic, and cultural approaches. Generally, these results were obtained for mixed samples of water with sediments, for only hydrothermal water, or for only sediment samples. Simultaneous comparative analysis of the microbial communities of water and sediments was performed for only one Kamchatka hot spring with circumneutral pH. Here, the microbial communities of both sediments and water (separately) of hot spring #4229 (the Uzon Caldera, Kamchatka) with a temperature of 50–56 °C and pH of 3.2 were analyzed by 16S rRNA gene V4 fragment amplicon sequencing. It was revealed that the microbial community of sediments was represented by uncultured phylogenetically deep-branching lineages of archaea, such as ARK-15 within Thermoplasmatota and ‘Ca. Marsarchaeales’ from the Thermoproteota phyla. Metagenome analysis showed that these archaea most probably carried out the degradation of organic matter. The microbial community of the hot water is represented by thermoacidophilic, (micro)aerobic, chemolithoautotrophic, hydrogen- and sulfur-oxidizing bacteria of the genera Hydrogenobaculum (phylum Aquificota) and Acidithiobacillus (phylum Pseudomonadota). Radioisotopic tracing and DNA-stable-isotope probing techniques proved their role as primary producers in the hot spring. The experiment revealed significant differences in the composition and functions of the microbial communities of sediments and water through the example of a typical acidic hot spring in Kamchatka.

Major Carotenoids of Meiothermus ruber Are Deinoxanthin Glucoside Esters, Not Meiothermoxanthin Glucoside Esters

Meiothermus ruber DSMZ 1279T was isolated from a hot spring in Kamchatka and was red in color. The major carotenoid present was reported to be 1'-(β-d-glucopyranosyloxy)-3,4,3',4'-tetradehydro-1',2'-dihydro-β,ψ-caroten-2-one after saponification (Burgess et al. J. Nat. Prod. 1999, 62, 859-863). In this study, we purified the major carotenoids in this species without saponification. We then reidentified the major carotenoids present using spectroscopic data, including electronic circular dichroism (ECD), 1H NMR, rotating-frame nuclear Overhauser effect spectroscopy (ROESY), 13C NMR, heteronuclear single-quantum correlation spectroscopy (HSQC), heteronuclear multiple-bond correlation spectroscopy (HMBC), and MS, and enzymatic hydrolysis of fatty acid moieties and found deinoxanthin glucoside iso fatty acid esters. The bound fatty acids present included four iso types, and their composition differed from cellular lipids. Moreover, the previously identified carotenoid glucoside was a saponification artifact of deinoxanthin glucoside esters. Ketomyxocoxanthin glucoside esters and 1'-hydroxytorulene glucoside esters were also present. On the basis of the identification of carotenoids and the whole genome sequence of M.ruber, we propose a carotenoid biosynthetic pathway and note the corresponding genes.

Ecological and biochemical properties of thermophilic bacteria taken from the Dachnie hot springs of Kamchatka (Far East, Russia)

In this work, we studied some ecological and biochemical characteristics (cultural, morphological, physiological and biochemical properties, extracellular enzymatic activity, bacterial growth in various ranges of temperatures, pH, NaCl concentrations, resistance to heavy metals) of 14 strains of thermophilic bacteria taken from thermal waters and microbial mats of Kamchatka Dachnie springs. On selective environment the strains formed mostly transparent, beige, white-gray, brown, yellow and pink colonies. It was shown that the isolates were represented most of all by asporogenic, catalase-positive and oxidase-positive, mobile, gram-negative rods of various sizes. Most of the cultures were capable of forming exozymes, especially amylases and proteinases. The strains grew in a wide range of temperatures (3550C), pH (58), NaCl concentration (15%) and were characterized by increased resistance to a number of heavy metals. 4 strains of thermophilic heterotrophic bacteria exhibited multiple multidrug resistance to metals. They were identified and it was shown that the isolates were represented by bacteria of the genus Bacillus sp. (№ 19), Thermus sp. (№ 40), Pseudomonas sp. (№ 15), Rhizobium sp. (№ 125). The strains with high enzymatic activity and increased resistance to pollutants are promising for use in the field of ecology for cleaning contaminated environmental objects.

The first crenarchaeon capable of growth by anaerobic carbon monoxide oxidation coupled with H2 production

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.

Novel Hyperthermophilic Crenarchaeon Thermofilum adornatum sp. nov. Uses GH1, GH3, and Two Novel Glycosidases for Cellulose Hydrolysis.

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.

Metagenomic mining for thermostable esterolytic enzymes uncovers a new family of bacterial esterases.

Biocatalysts exerting activity against ester bonds have a broad range of applications in modern biotechnology. Here, we have identified a new esterolytic enzyme by screening a metagenomic sample collected from a hot spring in Kamchatka, Russia. Biochemical characterization of the new esterase, termed EstDZ2, revealed that it is highly active against medium chain fatty acid esters at temperatures between 25 and 60 °C and at pH values 7–8. The new enzyme is moderately thermostable with a half-life of more than six hours at 60 °C, but exhibits exquisite stability against high concentrations of organic solvents. Phylogenetic analysis indicated that EstDZ2 is likely an Acetothermia enzyme that belongs to a new family of bacterial esterases, for which we propose the index XV. One distinctive feature of this new family, is the presence of a conserved GHSAG catalytic motif. Multiple sequence alignment, coupled with computational modelling of the three-dimensional structure of EstDZ2, revealed that the enzyme lacks the largest part of the “cap” domain, whose extended structure is characteristic for the closely related Family IV esterases. Thus, EstDZ2 appears to be distinct from known related esterolytic enzymes, both in terms of sequence characteristics, as well as in terms of three-dimensional structure.

Capacity of hyperthermophilic Crenarchaeota for decomposition of refractory proteins (α- and β-keratins)

Anaerobic thermophilic archaea of the genera Thermogladius and Desulfurococcus capable of a- and P3-keratin decomposition were isolated from hot springs of Kamchatka and Kunashir Island. For two of them (strains 2355k and 3008g), the presence of high-molecular mass, cell-bound endopeptidases active against nonhydrolyzed and partially hydrolyzed proteins at high values of temperature and pH was shown. Capacity for β-keratin decomposition was also found in collection strains (type strains of Desulfurococcus amylolyticus subsp. amylolyticus, D. mucosus subsp. mobilis, and D. fermentans).

Patterns of Gene Flow Define Species of Thermophilic Archaea

Author SummaryMicroorganisms from the bacterial and archaeal domains of the tree of life comprise the greatest breadth of biodiversity on earth. Yet the essential evolutionary process of speciation (through which biodiversity is generated) is poorly understood in microbes. At issue is the fundamental question of whether gene flow among individuals of clonally reproducing microorganisms is rapid enough to provide coherence within—and prevent speciation between—coexisting lineages. We use complete sequencing of microbial genomes to observe speciation in action. We focus on Archaea called Sulfolobus islandicus gathered from a geothermal hot spring from the Mutnovsky Volcano in Kamchatka, Russia, whose physical isolation allows us to pinpoint evolutionary processes to one location. Contrary to the theoretical predictions for microbes, we provide evidence that two novel lineages are in the process of becoming ecologically distinct and evolutionarily independent despite the fact that they recombine. The divergence we observe is not happening uniformly across the genome because certain genomic regions are more prone to become differentiated between species than others. This genomic view of the process of speciation occurring within a single natural microbial population contributes to our understanding of the generation of biodiversity in Archaea and furthers our understanding of speciation across the tree of life.

Single Archaeal Species Caught Splitting into Two Distinct Groups

A single archaeal species, Sulfolobus islandicus, from an isolated volcanic hot spring in Kamchatka, Russia, is at the point of diverging into two new species, according to microbiologist Rachel Whitaker from the University of Illinois in Urbana-Champaign and her collaborators at the University of California, Davis, and Oxford University in the United Kingdom. Their findings, apparently the first observed instance of sympatric speciation—one lineage becoming two—in a microorganism, provide an illuminating glimpse of how strains in the same niche can diverge. Details appear in the February 2012 PLoS Biology 10(2):e1001265 (doi:10.1371/journal.pbio. e1001265).

Molecular detection of methanotrophic bacteria in the hot springs of the Uzon caldera, Kamchatka

867 Microbial communities inhabiting such extreme ecosystems as continental volcanogenic hot springs are of considerable interest for both basic and applied biotechnological studies. The currently available data on methanotrophs from hightemperature habitats are scarce (1). The validly described species of thermo� philic and thermotolerant methanotrophic bacteria include members of the genera Methylothermus (2, 3), Methylococcus (4), and Methylocaldum (5, 6). The microbial communities inhabiting hightemperature springs of Kamchatka are of great interest to Russian scientists (7); however, the data on the microorgan� isms utilizing methane and/or other C1 compounds are fragmentary. Acidothermophilic methaneoxidiz� ing representatives of the phylum Verrucomicrobia were isolated from the acidic hot springs of Kam� chatka (pH 3.5) (8). Methaneoxidizing activity was detected by the radioisotope method in the silt sam� ples collected from the springs of the Vostochnoe Field of the Uzon Caldera, with the temperature not exceeding 60°C (Pimenov, unpublished data). There is, however, no data on the presence of methanotrophs in other hot springs of Kamchatka. Thus, the goal of the present work was to detect aerobic methanotrophic bacteria in the sediments of the hot springs of the Uzon caldera using two molecu� lar biological techniques: fluorescent in situ hybridiza� tion (FISH) and analysis of the pmoA gene encoding the β subunit of the particulate methane monooxyge� nase.

Complete Genome Sequence of “Vulcanisaeta moutnovskia” Strain 768-28, a Novel Member of the Hyperthermophilic Crenarchaeal Genus Vulcanisaeta

Strain 768-28 was isolated from a hot spring in Kamchatka, Russia, and represents a novel member of the Vulcanisaeta genus. The complete genome sequence of this thermoacidophilic anaerobic crenarchaeon reveals genes for protein and carbohydrate-active enzymes, the Embden-Meyerhof and Entner-Doudoroff pathways for glucose metabolism, the tricarboxylic acid cycle, beta-oxidation of fatty acids, and sulfate reduction.

Ammonifex thiophilus sp. nov., a hyperthermophilic anaerobic bacterium from a Kamchatka hot spring

A novel anaerobic, extremely thermophilic, facultatively chemolithoautotrophic bacterium designated strain SR(T) was isolated from a terrestrial hot spring in Kamchatka (Russia). The cells of the novel strain were spore-forming rods with a Gram-positive type of cell wall. The novel isolate grew at 60-82 degrees C (optimum 75 degrees C) and pH 6.0-7.5 (optimum 6.8). Molecular hydrogen and formate were used as electron donors. Thiosulfate, sulfate or elemental sulfur served as electron acceptors yielding hydrogen sulfide. No growth was observed on either substrate in the presence of nitrate as the electron acceptor. The G+C content of the DNA was 56.2 mol%. Phylogenetic analyses of the 16S rRNA gene showed that strain SR(T) was most closely related to Ammonifex degensii (96.4 % gene sequence similarity). However, the novel isolate possessed phenotypic characteristics that differed significantly from those of A. degensii, the only other recognized species of the genus Ammonifex. It is concluded that strain SR(T) (=DSM 19636(T)=VKM B-2461(T)) represents the type strain of a novel species of the genus Ammonifex, for which the name Ammonifex thiophilus sp. nov. is proposed. An emendation of the genus Ammonifex is proposed based on the phenotypic characteristics of the novel species.

Mesophilic and thermotolerant actinomycetes in strongly heated soils

Mesophilic and thermotolerant actinomycetes were identified in strongly heated desert-steppe soils of Mongolia, mountainous meadow soils of the Central Caucasus, and cyanobacterial films on volcanic ash near hot springs of Kamchatka. Thermotolerant actinomycetes in these soil objects were more abundant and had a greater taxonomic diversity in comparison with mesophilic actinomycetes. Thermotolerant Streptomyces were present in all the objects, except for sample 117 from the desert-steppe soil and the cyanobacterial film on volcanic ash. Thermotolerant actinomycetes from the Micromonospora and Actinomadura genera predominated in the desert-steppe soil; representatives of the Micromonospora genus predominated in the cyanobacterial film on volcanic ash, and representatives of the Microtetraspora genus predominated in the samples of geyserite near hot springs of Kamchatka.

Viruses in acidic geothermal environments of the Kamchatka Peninsula

Screening for viruses in samples taken from acidic hot springs of Kamchatka (Russia) revealed a collection of morphotypes, including linear, spherical and complex fusiform shapes, which show partial similarity to those found in acidic geothermal environments in other geographical locations. One of the viruses, Acidianus filamentous virus 9, AFV9, was isolated and its structure and genome were studied in detail.

Methane oxidation at 55°C and pH 2 by a thermoacidophilic bacterium belonging to the Verrucomicrobia phylum

Methanotrophic bacteria constitute a ubiquitous group of microorganisms playing an important role in the biogeochemical carbon cycle and in control of global warming through natural reduction of methane emission. These bacteria share the unique ability of using methane as a sole carbon and energy source and have been found in a great variety of habitats. Phylogenetically, known methanotrophs constitute a rather limited group and have so far only been affiliated with the Proteobacteria. Here, we report the isolation and initial characterization of a nonproteobacterial obligately methanotrophic bacterium. The isolate, designated Kam1, was recovered from an acidic hot spring in Kamchatka, Russia, and is more thermoacidophilic than any other known methanotroph, with optimal growth at approximately 55 degrees C and pH 3.5. Kam1 is only distantly related to all previously known methanotrophs and belongs to the Verrucomicrobia lineage of evolution. Genes for methane monooxygenases, essential for initiation of methane oxidation, could not be detected by using standard primers in PCR amplification and Southern blot analysis, suggesting the presence of a different methane oxidation enzyme. Kam1 also lacks the well developed intracellular membrane systems typical for other methanotrophs. The isolate represents a previously unrecognized biological methane sink, and, due to its unusual phylogenetic affiliation, it will shed important light on the origin, evolution, and diversity of biological methane oxidation and on the adaptation of this process to extreme habitats. Furthermore, Kam1 will add to our knowledge of the metabolic traits and biogeochemical roles of the widespread but poorly understood Verrucomicrobia phylum.

Radioisotopic tracing of carbon monoxide conversion by anaerobic thermophilic prokaryotes

The rate of CO conversion by a pure culture of a thermophilic CO-oxidizing, H2-producing bacterium Carboxydocella sp. strain 1503 was determined by the radioisotopic method. The overall daily uptake of 14CO by the bacterium was estimated at 38–56 μmol CO per 1 ml of the culture. A radioisotopic method was developed to separate and quantitatively determine the products of anaerobic CO conversion by microbial communities in hot springs. The new method was first tested on the microbial community from a sample obtained from a hot spring in Kamchatka. The potential rate of CO conversion by the anaerobic microbial community was found to be 40.75 nmol CO/cm3 sediment per day. 85% of the utilized 14CO was oxidized to carbon dioxide; 14.5% was incorporated into dissolved organic matter, including 0.2% that went into volatile fatty acids; 0.5% was used for cell biomass production; and only just over 0.001% was converted to methane.

Anoxybacillus voinovskiensis sp. nov., a moderately thermophilic bacterium from a hot spring in Kamchatka.

A novel moderately thermophilic bacterium, strain TH13T, was isolated from a hot spring in Kamchatka. It was found to be a Gram-positive, facultative aerobe; the straight, non-motile rods grew at 30-64 degrees C (optimum 54 degrees C). The isolate was positive for catalase and oxidase tests and reduced nitrate to nitrite, but was negative for H2S production and growth in more than 3% NaCl (w/v). The isolate grew at pH 7-8, but not at pH values higher than 9. The DNA G+C content was 43.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that strain TH13T was a member of the genus Anoxybacillus. DNA-DNA hybridization revealed a low relatedness (less than 30.2%) between the isolate and its close phylogenetic neighbours Anoxybacillus pushchinoensis and Anoxybacillus flavithermus. On the basis of phenotypic characteristics, phylogenetic data and DNA-DNA hybridization data, it was concluded that the isolate merited classification as a novel species, for which the name Anoxybacillus voinovskiensis sp. nov. is proposed. The type strain of this species is TH13T (=NCIMB 13956T=JCM 12111T).

Phylogenetic position of desulfurococcus amylolyticus

Seven strains of extremely thermophilic, obligately anaerobic cocci were isolated from the terrestrial hot springs of Kamchatka and Kunashir [1]. The temperature optimum for growth of the new isolates was 90‐ 92 ° C. Investigation of the new isolates revealed their similarity in all major diagnostic features. The beststudied representative of this group, strain Z-533, was described as the type strain of a new species of sulfurreducing archaea, Desulfurococcus amylolyticus [2]. In order to determine the phylogenetic position of this organism and confirm its affiliation to the genus Desulfurococcus , the nucleotide sequence of its 16S rRNA gene selectively amplified in vitro with the use of universal primers was analyzed [3]. The amplification was carried out in a reaction buffer composed of 1.5 mM MgCl 2 , 50 mM KCl, 10 mM Tris‐HCl (pH 8.3), and 0.001% gelatin. The reaction mixture (100 μ l) contained standard concentrations of dNTPs and equimolar concentrations of pA and pH' primers. Thirty amplification cycles with the following temperature profile

Thermoanaerobium lactoethylicum spec. nov. a new anaerobic bacterium from a hot spring of Kamchatka

A new anaerobic thermophilic Gram-positive, nonsporeforming bacterium strain ZE-1 was isolated from a hot spring of Kamchatka (USSR). The cells are rod-shaped, (0.5–0.8 · 2.0–20 μm), non-motile. The bacterium can grow between 42 and 75°C; the optimal temperature is 65°C. The growth is possible between pH values 5.0 and 8.5; optimal pH is 7.0. The cultures grow on the media containing peptone, yeast extract, or casein hydrolysate as nitrogen sources in the presence of glucose or some other sugars, mannitol or starch. The main fermentation products of glucose are ethanol, acetate, lactate, H2, CO2; byproducts are propionic, butyric and isovaleric acids. Glucose is metabolized via Embden-Meyerhoff-Parnas pathway. Molecular hydrogen does not inhibit growth. The bacterium does not reduce aceton to isopropanol, but is able to form H2S from elemental sulfur. The bacterium contains a soluble hydrogenase. This enzyme catalyzes both evolution and uptake of H2 and is active in the presence of methyl viologen. The DNA-base composition is 34.6 mol%; the genome size 2.08x109 D. The name proposed for the isolated bacterium strain ZE-1 is Thermoanaerobium lactoethylicum spec. nov.