Anna A. Perevalova

Factors Controlling the Distribution of Archaeal Tetraethers in Terrestrial Hot Springs

ABSTRACT Glycerol dialkyl glycerol tetraethers (GDGTs) found in hot springs reflect the abundance and community structure of Archaea in these extreme environments. The relationships between GDGTs, archaeal communities, and physical or geochemical variables are underexamined to date and when reported often result in conflicting interpretations. Here, we examined profiles of GDGTs from pure cultures of Crenarchaeota and from terrestrial geothermal springs representing a wide distribution of locations, including Yellowstone National Park (United States), the Great Basin of Nevada and California (United States), Kamchatka (Russia), Tengchong thermal field (China), and Thailand. These samples had temperatures of 36.5 to 87°C and pH values of 3.0 to 9.2. GDGT abundances also were determined for three soil samples adjacent to some of the hot springs. Principal component analysis identified four factors that accounted for most of the variance among nine individual GDGTs, temperature, and pH. Significant correlations were observed between pH and the GDGTs crenarchaeol and GDGT-4 (four cyclopentane rings, m/z 1,294); pH correlated positively with crenarchaeol and inversely with GDGT-4. Weaker correlations were observed between temperature and the four factors. Three of the four GDGTs used in the marine TEX86 paleotemperature index (GDGT-1 to -3, but not crenarchaeol isomer) were associated with a single factor. No correlation was observed for GDGT-0 (acyclic caldarchaeol): it is effectively its own variable. The biosynthetic mechanisms and exact archaeal community structures leading to these relationships remain unknown. However, the data in general show promise for the continued development of GDGT lipid-based physiochemical proxies for archaeal evolution and for paleo-ecology or paleoclimate studies.

Uncultured archaea dominate in the thermal groundwater of Uzon Caldera, Kamchatka

The thermoacidophilic microbial community inhabiting the groundwater with pH 4.0 and temperature 50°C at the East Thermal Field of Uzon Caldera, Kamchatka, was examined using pyrosequencing of the V3 region of the 16S rRNA gene. Bacteria comprise about 30% of microorganisms and are represented primarily by aerobic lithoautotrophs using the energy sources of volcanic origin—thermoacidophilic methanotrophs of the phylum Verrucomicrobia and Acidithiobacillus spp. oxidising metals and reduced sulfur compounds. More than 70% of microbial population in this habitat were represented by archaea, in majority affiliated with “uncultured” lineages. The most numerous group (39% of all archaea) represented a novel division in the phylum Euryarchaeota related to the order Thermoplasmatales. Another abundant group (33% of all archaea) was related to MCG1 lineage of the phylum Crenarchaeota, originally detected in the Yellowstone hot spring as the environmental clone pJP89. The organisms belonging to these two groups are widely spread in hydrothermal environments worldwide. These data indicate an important environmental role of these two archaeal groups and should stimulate the investigation of their metabolism by cultivation or metagenomic approaches.

Fervidicoccus fontis gen. nov., sp. nov., an anaerobic, thermophilic crenarchaeote from terrestrial hot springs, and proposal of Fervidicoccaceae fam. nov. and Fervidicoccales ord. nov.

Two novel thermophilic and slightly acidophilic strains, Kam940(T) and Kam1507b, which shared 99 % 16S rRNA gene sequence identity, were isolated from terrestrial hot springs of the Uzon caldera on the Kamchatka peninsula. Cells of both strains were non-motile, regular cocci. Growth was observed between 55 and 85 degrees C, with an optimum at 65-70 degrees C (doubling time, 6.1 h), and at pH 4.5-7.5, with optimum growth at pH 5.5-6.0. The isolates were strictly anaerobic organotrophs and grew on a narrow spectrum of energy-rich substrates, such as beef extract, gelatin, peptone, pyruvate, sucrose and yeast extract, with yields above 10(7) cells ml(-1). Sulfate, sulfite, thiosulfate and nitrate added as potential electron acceptors did not stimulate growth when tested with peptone. H(2) at 100 % in the gas phase inhibited growth on peptone. Glycerol dibiphytanyl glycerol tetraethers (GDGTs) with zero to four cyclopentyl rings were present in the lipid fraction of isolate Kam940(T). The G+C content of the genomic DNA of strain Kam940(T) was 37 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolates were archaea of the phylum Crenarchaeota, only distantly related to the cultured members of the class Thermoprotei (no more than 89 % identity), and formed an independent lineage adjacent to the orders Desulfurococcales and Acidilobales and clustering only with uncultured clones from hot springs of Yellowstone National Park and Iceland as the closest relatives. On the basis of their phylogenetic position and novel phenotypic features, isolates Kam940(T) and Kam1507b are proposed to be assigned to a new genus and species, Fervidicoccus fontis gen. nov., sp. nov. The type strain of Fervidicoccus fontis is strain Kam940(T) (=DSM 19380(T) =VKM B-2539(T)). The phylogenetic data as well as phenotypic properties suggest that the novel crenarchaeotes form the basis of a new family, Fervidicoccaceae fam. nov., and order, Fervidicoccales ord. nov., within the class Thermoprotei.

Microbial diversity and autotrophic activity in Kamchatka hot springs

Microbial communities of Kamchatka Peninsula terrestrial hot springs were studied using molecular, radioisotopic and cultural approaches. Analysis of 16S rRNA gene fragments performed by means of high-throughput sequencing revealed that aerobic autotrophic sulfur-oxidizing bacteria of the genus Sulfurihydrogenibium (phylum Aquificae) dominated in a majority of streamers. Another widely distributed and abundant group was that of anaerobic bacteria of the genus Caldimicrobium (phylum Thermodesulfobacteria). Archaea of the genus Vulcanisaeta were abundant in a high-temperature, slightly acidic hot spring, where they were accompanied by numerous Nanoarchaeota, while the domination of uncultured Thermoplasmataceae A10 was characteristic for moderately thermophilic acidic habitats. The highest rates of inorganic carbon assimilation determined by the in situ incubation of samples in the presence of 14C-labeled bicarbonate were found in oxygen-dependent streamers; in two sediment samples taken from the hottest springs this process, though much weaker, was found to be not dependent on oxygen. The isolation of anaerobic lithoautotrophic prokaryotes from Kamchatka hot springs revealed a wide distribution of the ability for sulfur disproportionation, a new lithoautotrophic process capable to fuel autonomous anaerobic ecosystems.

Brockia lithotrophica gen. nov., sp. nov., an anaerobic thermophilic bacterium from a terrestrial hot spring

A novel thermophilic bacterium, strain Kam1851(T), was isolated from a terrestrial hot spring of the Uzon Caldera, Kamchatka Peninsula, Russia. Cells of strain Kam1851(T) were spore-forming rods with a gram-positive type of cell wall. Growth was observed between 46 and 78 °C, and pH 5.5-8.5. The optimal growth (doubling time, 6.0 h) was at 60-65 °C and pH 6.5. The isolate was an obligate anaerobe growing in pre-reduced medium only. It grew on mineral medium with molecular hydrogen or formate as electron donors, and elemental sulfur, thiosulfate or polysulfide as electron acceptors. The main cellular fatty acids were C(16 : 0) (34.2 %), iso-C(16 : 0) (18 %), C(18 : 0) (12.8 %) and iso-C(17 : 0) (11.1 %). The G+C content of the genomic DNA of strain Kam1851(T) was 63 mol%. 16S rRNA gene sequence analysis showed that strain Kam1851(T) belonged to the order Thermoanaerobacterales, but it was not closely related to representatives of any genera with validly published names. The most closely related strains, which had no more than 89.2 % sequence similarity, were members of the genera Ammonifex and Caldanaerobacter. On the basis of its phylogenetic position and novel phenotypic features, isolate Kam1851(T) is proposed to represent a novel species in a new genus, Brockia lithotrophica gen. nov., sp. nov.; the type strain of Brockia lithotrophica is Kam1851(T) ( = DSM 22653(T) = VKM B-2685(T)).

Activity and Distribution of Thermophilic Prokaryotes in Hydrothermal Fluid, Sulfidic Structures, and Sheaths of Alvinellids (East Pacific Rise, 13°N)

ABSTRACT Processes of inorganic carbon assimilation, methanogenesis, sulfate reduction, and acetate oxidation to CO2 occurring in samples from the East Pacific Rise at 13°N were traced, using radioisotopically labeled substrates, at temperatures ranging from 65 to 100°C. Molecular hydrogen stimulated lithotrophic methanogenesis and sulfate reduction but inhibited inorganic carbon assimilation. Active mineralization of acetate was observed in an organic-rich Alvinella-associated system at 80°C. Members of the Thermococcales were the most numerous hyperthermophilic archaea in these samples, their density achieving 108 cells per cm3, while the numbers of cultured hydrogen-utilizing thermophilic lithotrophs were several orders of magnitude lower.

Calorithrix insularis gen. nov., sp. nov., a novel representative of the phylum Calditrichaeota.

A moderately thermophilic, anaerobic bacterium designated as strain KRT was isolated from a shallow-water submarine hydrothermal vent (Kunashir Island, Southern Kurils, Russia). Cells of strain KRT were thin (0.2-0.3 µm), flexible, motile, Gram-stain-negative rods of variable length. Optimal growth conditions were pH 6.6, 55 °C and 1-3 % (w/v) NaCl. Strain KRT was able to ferment a wide range of proteinaceous substrates, pyruvate, and mono-, di- and polysaccharides. The best growth occurred with proteinaceous compounds. Nitrate significantly stimulated the growth on proteinaceous substrates decreasing H2 formation, ammonium being the main product of nitrate reduction. Strain KRT did not need the presence of a reducing agent in the medium and tolerated the presence of oxygen in the gas phase up to 3 % (v/v). In the presence of nitrate, aerotolerance of isolate KRT was enhanced up to 6-8 % O2 (v/v). Strain KRT was able to grow chemolithoheterotrophically, oxidizing H2 and reducing nitrate to ammonium. Yeast extract (0.05 g l-1) was required for growth. The G+C content of the genomic DNA of strain KRT was 47.3 mol%. 16S rRNA gene sequence analysis placed isolate KRT in the phylum Calditrichaeota where it represented a novel species of a new genus, for which the name Calorithrix insularis gen. nov., sp. nov. is proposed. The type strain of Calorithrix insularis is KRT (=DSM 101605T=VKM B-3022T).

Phylogenetic Diversity of the Sulfur Cycle Bacteria in the Bottom Sediments of the Chersonesus Bay

The Black Sea is the largest meromictic basin, in the bottom sediments of which a powerful biogenic process of sulfide production occurs. The goal of the present work was to obtain data on phylogenetic diversity of the sulfur cycle microorganisms (sulfate-reducing and sulfur-oxidizing bacteria) in the Black Sea coastal gas-saturated bottom sediments. The samples were collected in the Chersonesus (Blue) Bay near Sevastopol from whitish bacterial mats of sulfurettes, and from the upper layer of the nearby seabed. Using DNA isolated from the native samples and obtained enrichment cultures, PCR analysis was performed with oligonucleotide primers specific to the fragments of the 16S rRNA genes of the main subgroups of sulfatereducing bacteria (SRB) and to the fragments of the dsrB gene (both reductive and oxidative types), encoding the β-subunit of dissimilatory (bi)sulfite reductase, the key enzyme in the sulfur cycle, inherent in both sulfate- reducing and sulfur-oxidizing microorganisms. The presence of 16S rRNA gene fragments specific to the genera Desulfobacterium, Desulfobacter, Desulfococcus–Desulfonema–Desulfosarcina, and Desulfovibrio–Desulfomicrobium was detected in the DNA samples isolated from coastal bottom bacterial mats. Usage of denaturing gradient gel electrophoresis (DGGE) with subsequent sequencing of reamplified dsrB gene fragments revealed that according to deduced amino acid sequences encoded by the dsrB gene (reductive type), SRB from the coastal gas-saturated bottom sediments of the Black Sea had the highest homology (92−99%) with the dsrB gene of cultured SRB belonging to the genera Desulfovibrio, Desulfatitalea, Desulfobacter, and Desulfobacterium, as well as with uncultured SRB strains from various marine habitats, such as bottom sediments of the Northern and Japanese seas. Deduced amino acid sequences encoded by the oxidative dsrB gene had the highest homology (90−99%) with the relevant sequences of the genera Thiocapsa, Thiobaca, Thioflavicoccus, and Thiorhodococcus.