Vadim M. Gumerov

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.

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.

Molecular analysis of microbial diversity in the Zavarzin Spring, Uzon Caldera, Kamchatka

The Zavarzin spring is situated in the caldera of the Uzon volcano, Kamchatka, and is characterized by a temperature of about 60°C, neutral pH, and high concentration of sulfur. The bottom of the spring is covered with a cyanobacterial mat. The structure of the microbial community of the water from the Zavarzin spring was qualitatively and quantitatively characterized by pyrosequencing of the V3 variable region of the 16S rRNA gene, which yielded 37 654 independent sequences. The microbial community includes about 900 bacterial and 90 archaeal genera. Bacteria comprised 95% of the microorganisms and archaea less than 5%. The largest part (32.3%) of the community was constituted by the chemolithoautotrophic bacteria Aquificae from the genera Sulfurihydrogenibium and Thermosulfidibacter. Among autotrophic microorganisms, members of Thermodesulfobacteria (7.3%), the gammaproteobacteria Thiofaba (7.6%), the deltaproteobacteria Desulfurella (2.6%), and the betaproteobacteria Thiomonas (0.6%) were also identified. Heterotrophic bacteria were represented by Calditerrivibrio (12.1%), Thermotogae (6.3%), the betaproteobacteria Tepidimonas (6.0%), Deinococcus-Thermus (4.4%), Caldiserica (1.7%), and Dictyoglomi (1.6%). About 1.9% of microorganisms belonged to the BRC1 phylum, which does not include cultured members, and 0.2% of bacteria formed a new phylogenetic branch of the phylum level, representatives of which have been found only in the Zavarzin spring. Members of all four archaeal phyla were identified: Euryarchaeota (42% of archaeal sequences), Crenarchaeota (50%), Korarchaeota (7.5%), and Nanoarchaeota (0.5%). Thus, in the Zavarzin spring, apart from photosynthesis carried out by the cyanobacterial mat, which covers the bottom, chemolithoautotrophic production of organic matter can occur. In aerobic conditions, it proceeds at the expense of the oxidation of sulfur and its reduced compounds, and in anaerobic conditions, at the expense of the oxidation of hydrogen with sulfur and sulfates as electron acceptors. The organic matter formed by autotrophic bacteria may be utilized by various organotrophic microorganisms, including both fermentative bacteria and organisms that carry out anaerobic respiration with sulfur and nitrate as electron acceptors.

Genome analysis of Chitinivibrio alkaliphilus gen. nov., sp. nov., a novel extremely haloalkaliphilic anaerobic chitinolytic bacterium from the candidate phylum Termite Group 3

Anaerobic enrichments from hypersaline soda lakes with chitin as substrate yielded five closely related anaerobic haloalkaliphilic isolates growing on insoluble chitin by fermentation at pH 10 and salinities up to 3.5 M. The chitinolytic activity was exclusively cell associated. To better understand the biology and evolutionary history of this novel bacterial lineage, the genome of the type strain ACht1 was sequenced. Analysis of the 2.6 Mb draft genome revealed enzymes of chitin-degradation pathways, including secreted cell-bound chitinases. The reconstructed central metabolism revealed pathways enabling the fermentation of polysaccharides, while it lacks the genes needed for aerobic or anaerobic respiration. The Rnf-type complex, oxaloacetate decarboxylase and sodium-transporting V-type adenosine triphosphatase were identified among putative membrane-bound ion pumps. According to 16S ribosomal RNA analysis, the isolates belong to the candidate phylum Termite Group 3, representing its first culturable members. Phylogenetic analysis using ribosomal proteins and taxonomic distribution analysis of the whole proteome supported a class-level classification of ACht1 most probably affiliated to the phylum Fibribacteres. Based on phylogenetic, phenotypic and genomic analyses, the novel bacteria are proposed to be classified as Chitinivibrio alkaliphilus gen. nov., sp. nov., within a novel class Chitinivibrione.

Complete Genome Sequence of the Thermoacidophilic Crenarchaeon Thermoproteus uzoniensis 768-20

Thermoproteus uzoniensis 768-20 is a thermoacidophilic anaerobic crenarchaeon isolated from a solfataric field in Kamchatka, Russia. The complete genome sequence reveals genes for protein and carbohydrate-active enzymes, beta-oxidation of fatty acids, the Embden-Meyerhof and Entner-Doudoroff pathways for glucose metabolism, the tricarboxylic acid cycle, the dicarboxylate/4-hydroxybutyrate cycle, hydrogenase, and sulfur reductase.

Characterization of a thermostable short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus.

ABSTRACT Short-chain alcohol dehydrogenase, encoded by the gene Tsib_0319 from the hyperthermophilic archaeon Thermococcus sibiricus, was expressed in Escherichia coli, purified and characterized as an NADPH-dependent enantioselective oxidoreductase with broad substrate specificity. The enzyme exhibits extremely high thermophilicity, thermostability, and tolerance to organic solvents and salts.

Complete Genome Sequence of Strain 1860, a Crenarchaeon of the Genus Pyrobaculum Able To Grow with Various Electron Acceptors

Strain 1860, a novel member of the genus Pyrobaculum, is a hyperthermophilic organotrophic crenarchaeon growing anaerobically with various electron acceptors. The complete genome sequence reveals genes for several membrane-bound oxidoreductases, the Embden-Meyerhof and Entner-Doudoroff pathways for glucose metabolism, the tricarboxylic acid cycle, the glyoxylate cycle, and the dicarboxylate/4-hydroxybutyrate cycle.

Characteristic of Biodiversity of Thermophilic Microbial Community by Parallel Pyrosequencing Method

Identification of microorganisms is a part of funda� mental and applied studies in microbiology, biotech� nology, and medicine. Microorganisms form various microbial communities associated with certain natural environmental niches, they perform biotechnological processes, and form human microbiome. The struc� tures of microbial communities were conventionally analyzed by microbiological methods, which make it possible to obtain and characterize pure cultures of microorganisms. Later, molecular methods, which do not require cultivation of microorganisms and are based on PCR amplification of 16S rRNA gene frag� ments, their separation, and sequencing, began to be used for this purpose. The use of these methods showed, in particular, that, in some communities, at most 0.1–5% of microorganisms can be cultivated under laboratory conditions and made it possible to reveal new phylogenetic groups of prokaryotes that are evolutionarily distant from the known groups. The methods that have been used until recently usually implied the cloning of 16S rRNA gene fragments and independent sequencing of separate clones using cap� illary electrophoresis. In practice this approach allowed analyzing not more than several dozens or hundreds of 16S rRNA sequences. However, many microbial communities have a more complex compo� sition. For example, human intestinal microbiome may include several hundreds of species of microor�

Influence of intermolecular contacts on the structure of recombinant prolidase from Thermococcus sibiricus

Prolidases are peptidases that are specific for dipeptides with proline as the second residue. The structure of recombinant prolidase from the hyperthermophilic archaeon Thermococcus sibiricus (Tsprol) was determined at 2.6 Å resolution. The homodimer of Tsprol is characterized by a complete lack of interactions between the N- and C-terminal domains of the two subunits and hence can be considered to be the most open structure when compared with previously structurally studied prolidases. This structure exists owing to intermolecular coordination bonds between cadmium ions derived from the crystallization solution and histidine residues of a His tag and aspartate and glutamate residues, which link the dimers to each other. This linking leads to the formation of a crystal with a loose packing of protein molecules and low resistance to mechanical influence and temperature increase.

Phenotypic and Genomic Properties of Chitinispirillum alkaliphilum gen. nov., sp. nov., A Haloalkaliphilic Anaerobic Chitinolytic Bacterium Representing a Novel Class in the Phylum Fibrobacteres

Anaerobic enrichment from sediments of hypersaline alkaline lakes in Wadi el Natrun (Egypt) with chitin resulted in the isolation of a fermentative haloalkaliphilic bacterium, strain ACht6-1, growing exclusively with insoluble chitin as the substrate in a sodium carbonate-based medium at pH 8.5–10.5 and total Na+ concentrations from 0.4 to 1.75 M. The isolate had a Gram-negative cell wall and formed lipid cysts in old cultures. The chitinolytic activity was associated with cells. Analysis of the 4.4 Mb draft genome identified pathways for chitin utilization, particularly, secreted chitinases linked to the cell surface, as well as genes for the hydrolysis of other polysaccharides and fermentation of sugars, while the genes needed for aerobic and anaerobic respiration were absent. Adaptation to a haloalkaliphilic lifestyle was reflected by the gene repertoire encoding sodium rather than proton-dependent membrane-bound ion pumps, including the Rnf-type complex, oxaloacetate decarboxylase, V-type ATPase, and pyrophosphatase. The phylogenetic analysis using 16S rRNA gene and ribosomal proteins indicated that ACht6-1 forms a novel deep lineage at the class level within the bacterial candidate division TG3. Based on phylogenetic, phenotypic and genomic analyses, the novel chitinolytic bacterium is described as Chitinispirillum alkaliphilum gen. nov., sp. nov., within a novel class Chitinispirillia that could be included into the phylum Fibrobacteres.