Bioinformatics-Structural Approach to the Search for New D-Amino Acid Oxidases.

D-amino acid oxidase (DAAO, EC 1.2.1.2) plays an important role in the functioning of prokaryotes as well as of lower (yeast and fungi) and higher eukaryotes (mammals). DAAO genes have not yet been found in archaean genomes. D-amino acid oxidase is increasingly used in various fields, which requires the development of new variants of the enzyme with specific properties. However, even within one related group (bacteria, yeasts and fungi, mammals), DAAOs show very low homology between amino acid sequences. In particular, this fact is clearly observed in the case of DAAO from bacteria. The high variability in the primary structures of DAAO severely limits the search for new enzymes in known genomes. As a result, many (if not most) DAAO genes remain either unannotated or incorrectly annotated. We propose an approach that uses bioinformatic methods in combination with general 3D structure and active center structure analysis to confirm that the gene found encodes D-amino acid oxidase and to predict the possible type of its substrate specificity. Using a homology search, we obtained a set of candidate sequences, modelled the tertiary structure of the selected enzymes, and compared them with experimental and model structures of known DAAOs. The effectiveness of the proposed approach for discrimination of DAAOs and glycine oxidases is shown. Using this approach, new DAAO genes were found in the genomes of six strains of extremophilic bacteria, and for the first time in the world, one gene was identified in the genome of halophilic archaea. Preliminary experiments confirmed the predicted specificity of DAAO from Natronosporangium hydrolyticum ACPA39 with D-Leu and D-Phe.

Data on draft genome sequence of Caldanaerobacter sp. strain 1523vc, a thermophilic bacterium, isolated from a hot spring of Uzon Caldera, (Kamchatka, Russia).

The draft genome sequence of Caldanaerobacter sp. strain 1523vc, a thermophilic bacterium, isolated from a hot spring of Uzon Caldera, (Kamchatka, Russia) is presented. The complete genome assembly was of 2 713 207 bp with predicted completeness of 99.38%. Genome structural annotation revealed 2674 protein-coding genes, 127 pseudogenes and 77 RNA genes. Pangenome analysis of 7 currently available high quality Caldanaerobacter spp. genomes including 1523vc revealed 4673 gene clusters. Of them, 1130 clusters formed a core genome of genus Caldanaerobacter. Of the rest 3543 Caldanaerobacter pangenome genes, 385 were exclusively represented in 1523vc genome. 101 of 2801 Caldanaerobacter CDS were found to be encoding carbohydrate-active enzymes (CAZymes). The majority of CAZymes were predicted to be involved in degradation of beta-linked polysaccharides as chitin, cellulose and hemicelluloses, reflecting the metabolism of strain 1523vc, isolated on cellulose. 5 of 101 CAZyme genes were found to be unique for the strain 1523vc and belonged to GH23, GT56, GH15 and two CE9 family proteins. The draft genome of strain 1523vc was deposited at DBJ/EMBL/GenBank under the accessions JABEQB000000000, PRJNA629090 and SAMN14766777 for Genome, Bioproject and Biosample, respectively.

Draft genome sequence data of Bifidobacterium longum strain VKPM Ac-1636, a prospective probiotic isolated from human gut.

Data on the draft genome sequence of Bifidobacterium longumsubsp. longum strain Ac-1636 is presented in this report. This strain, isolated from the digestive tract of one-year old healthy infant, was deposited in the Russian National Collection of Industrial Microorganisms as a prospective candidate for development of probiotics and probiotic foods. The 2,321,741 bp draft genome consists of 73 scaffolds with N50 of 162,253 bp. Genome annotation revealed the presence of multiple determinants of probiotic properties of this strain. The draft genome sequence data of strainAc-1636 is available in DBJ/EMBL/GenBank under the accession nos. RZHL00000000, PRJNA511803 and SAMN10644101 for Genome, Bioproject and Biosample, respectively.

Data on draft genome sequence of Bacillus sp. strain VKPM B-3276 isolated from Culex pipiens larvae.

The draft genome sequence of Bacillus sp. VKPM B-3276, a mesophilic, gram-positive bacterium, isolated from dead Culex pipiens larvae is presented. This strain was deposited in the Russian National Collection of Industrial Microorganisms as a prospective candidate for development of new entomopathogenic agents. The genome of Bacillus sp. VKPM B-3276 was 6,126,346 bp in length with predicted completeness of 99.43%. Genome analysis identified 6518 protein-coding sequences and 111 RNAs genes. 13% (271) of the protein-coding genes were assigned to "Carbohydrates" subsystem category, according to RAST/SEED. Among them about 50 enzymes, responsible for chitin, peptidoglycan and related molecules decomposition, were found. The draft genome of strain VKPM B-3276 was deposited at DBJ/EMBL/GenBank under the accession nos. RZHM00000000, PRJNA511803 and SAMN10644103 for Genome, Bioproject and Biosample, respectively.

Desulfothermobacter acidiphilus gen. nov., sp. nov., a thermoacidophilic sulfate-reducing bacterium isolated from a terrestrial hot spring.

An anaerobic sulfate-reducing micro-organism, strain 3408-1T, was isolated from a terrestrial hot spring in Kamchatka peninsula (Russia). The cells were spore-forming rods with a Gram-positive type of cell wall. The new isolate was a moderately thermoacidophilic anaerobe able to grow either by sulfate or thiosulfate respiration with H2 or formate as substrates, or by fermenting yeast extract, maltose, sucrose, glucose and pyruvate. The fermentation products were acetate, CO2 and H2. The pH range for growth was 2.9-6.5, with an optimum at 4.5. The temperature range for growth was 42-70 °C, with an optimum at 55 °C. The G+C content of DNA was 58 mol%. Phylogenetic analysis of the 16S rRNA gene showed that strain 3408-1T belongs to the family Thermoanaerobacteraceae, order Thermoanaerobacterales and was distantly related to the species of the genus Ammonifex(93-94 % sequence similarity). On the basis of physiological properties and results of phylogenetic analysis, strain 3408-1T is considered to represent a novel species of a new genus, for which the name Desulfothermobacter acidiphilus gen. nov., sp. nov. is proposed. The type strain is 3408-1T (=DSM 105356T=VKM B-3183T).

Mobilitalea sibirica gen. nov., sp. nov., a halotolerant polysaccharide-degrading bacterium.

A novel strictly anaerobic, halotolerant, organotrophic bacterium, strain P3M-3(T), was isolated from a microbial mat formed under the flow of hot water emerging from a 2775 m-deep well in Tomsk region (western Siberia, Russia). Cells of strain P3M-3(T) were straight and curved rods, 0.2-0.4 µm in width and 1.5-20 µm in length. Strain P3M-3(T) grew optimally at 37 °C, pH 7.0-7.5 and in a NaCl concentration of 15 g l(-1). Under optimum growth conditions, the doubling time was 1 h. The isolate was able to ferment a variety of mono-, di- and polysaccharides, including microcrystalline cellulose. Acetate, ethanol, H2 and CO2 were the main products of glucose fermentation. The DNA G+C content was 33.4 mol%. 16S rRNA gene-based phylogenetic analysis showed that strain P3M-3(T) was a member of family Lachnospiraceae, whose representatives are also found in Clostridium cluster XIVa. 16S rRNA gene sequence similarity with Clostridium jejuense HY-35-12(T), the closest relative, was 93.9%. A novel genus and species, Mobilitalea sibirica gen. nov., sp. nov., are proposed based on phylogenetic analysis and physiological properties of the novel isolate. The type strain of the type species is P3M-3(T) ( = DSM 26468(T) = VKM B-2804(T)).

[Capacity of hyperthermophilic Crenarchaeota for decomposition of refractory protiens (α- and ß-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 ß-keratin decomposition was also found in collection strains (type strains of Desulfurococcus amylolyticus subsp. amylolyticus, D. mucosus subsp. mobilis, and D. fermentans).

Complete Genomic Sequence of "Thermofilum adornatus" Strain 1910bT, a Hyperthermophilic Anaerobic Organotrophic Crenarchaeon.

The complete genomic sequence of a novel hyperthermophilic crenarchaeon, strain 1910b(T), was determined. The genome comprises a 1,750,259-bp circular chromosome containing single copies of 3 rRNA genes, 43 tRNA genes, and 1,896 protein-coding sequences. In silico genome-genome hybridization suggests the proposal of a novel species, "Thermofilum adornatus" strain 1910b(T).

Complete Genome Sequence of Salinarchaeum sp. Strain HArcht-Bsk1T, Isolated from Hypersaline Lake Baskunchak, Russia.

The complete genome sequence of a novel halophilic archaeon, Salinarchaeum sp. strain HArcht-Bsk1(T), was determined using next-generation sequencing. The genome comprises a 3,255,260-bp circular chromosome with a G+C content of 66.7%. Automatic annotation of the genome revealed a single rRNA operon, 45 tRNAs, and 3,013 protein-coding gene sequences.

Ornatilinea apprima gen. nov., sp. nov., a cellulolytic representative of the class Anaerolineae.

A novel obligately anaerobic, mesophilic, organotrophic bacterium, strain P3M-1(T), was isolated from a microbial mat formed in a wooden bath filled with hot water emerging from a 2775 m-deep well in the Tomsk region of western Siberia, Russia. Cells of strain P3M-1(T) were rod-shaped, 0.3-0.7 µm in width and formed multicellullar filaments that reached up to 400 µm in length. Strain P3M-1(T) grew optimally at 42-45 °C, pH 7.5-8.0, and with 0.1% (w/v) NaCl. Under optimal conditions, the doubling time was 6 h. The isolate was able to ferment a variety of proteinaceous substrates and sugars, including microcrystalline cellulose. Acetate, ethanol and H(2) were the main products of glucose fermentation. The genomic DNA G+C content was 55 mol%. 16S rRNA gene sequence-based phylogenetic analyses showed that strain P3M-1(T) was a member of the class Anaerolinea, with 92.8 % sequence similarity to Levilinea saccharolytica KIBI-1(T). Based on phylogenetic analysis and physiological properties, strain P3M-1(T) represents a novel species in a new genus, for which the name Ornatilinea apprima gen. nov., sp. nov. is proposed; the type strain of O. apprima is P3M-1(T) (= DSM 23815(T)=VKM B-2669(T)).

Thermosipho affectus sp. nov., a thermophilic, anaerobic, cellulolytic bacterium isolated from a Mid-Atlantic Ridge hydrothermal vent.

A novel obligately anaerobic, extremely thermophilic, organotrophic bacterium, strain ik275mar(T), was isolated from a Mid-Atlantic Ridge deep-sea hydrothermal vent. Cells were rods surrounded by a sheath-like structure (toga), 0.4-0.9 µm in width and 1.2-6.0 µm in length. Strain ik275mar(T) grew at 37-75 °C, pH 5.6-8.2 and at NaCl concentrations of 10-55 g l(-1). Under optimum conditions (70 °C, pH 6.6, NaCl 20 g l(-1)), doubling time was 32 min. The isolate was able to ferment carbohydrates including starch, cellulose and cellulose derivatives. Acetate, H(2) and CO(2) were the main products of glucose fermentation. G+C content of DNA was 27 mol%. Phylogenetic analysis of 16S rRNA gene sequences showed that strain ik275mar(T) is a member of the genus Thermosipho. 16S rRNA gene sequence identity with the other species of the genus Thermosipho ranged from 93.7 to 94.5 %. Based on the phylogenetic analysis and physiological properties of the novel isolate, we propose a novel species, Thermosipho affectus sp. nov., with type strain ik275mar(T) ( = DSM 23112(T)  = VKM B-2574(T)).

Desulfurococcus kamchatkensis sp. nov., a novel hyperthermophilic protein-degrading archaeon isolated from a Kamchatka hot spring.

A novel obligately anaerobic, hyperthermophilic, organotrophic archaeon, designated strain 1221n(T), was isolated from a hot spring of Uzon Caldera (Kamchatka Peninsula, Russia). Cells of strain 1221n(T) were non-motile regular cocci, 0.6-1 mum in diameter. The temperature range for growth at pH 6.5 was 65-87 degrees C, with an optimum at 85 degrees C. The pH range for growth at 85 degrees C was 5.5-7.5, with an optimum at pH 6.5. Growth was not observed at or below 6 degrees C or at or above 90 degrees C, as well as at or below pH 5.0 and at or above pH 8.0. The isolate fermented a wide range of substrates including proteins: alpha-keratin, albumin and gelatin. Elemental sulfur was not essential for growth, but stimulated growth. Strain 1221n(T) synthesized 40 and 120 kDa proteinases localized on the cell envelope. The G+C content of the DNA was 44.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparison indicated that strain 1221n(T) was affiliated with the genus Desulfurococcus. The level of 16S rRNA gene sequence similarity with other Desulfurococcus species was 96.7-98.1 %, and Desulfurococcus amylolyticus was found to be the most closely related organism. Based on the data from the phylogenetic analysis and the physiological properties of the novel isolate, strain 1221n(T) should be classified as representing a novel species, for which the name Desulfurococcus kamchatkensis sp. nov. is proposed. The type strain is 1221n(T) (=DSM 18924(T)=VKM B-2413(T)).

[Keratinase of an anaerobic thermophilic bacterium Thermoanaerobacter sp. strain 1004-09 isolated from a hot spring in the Baikal Rift zone].

A thermophilic anaerobic bacterial strain 1004-09 belonging to the genus Thermoanaerobacter and capable of growth on protein substrates such as albumin, gelatin, casein, and alpha and beta-keratins was isolated from the Urinskii hot spring (Barguzin river valley, Republic of Buryatia, Russia). A 150-kDa serine proteinase was revealed in the strain supernatant; it exhibited optimal activity at 60 degrees C and pH 9.3 and was capable of keratin hydrolysis. A number of characteristics for the strain 1004-09 keratinase were established including activation by SDS and NaCl and residual activity (15% to the activity of the intact protein) in the presence of 10% ethanol and acetone.

Thermoanaerobacterium aciditolerans sp. nov., a moderate thermoacidophile from a Kamchatka hot spring.

An anaerobic, moderately thermoacidophilic bacterium, strain 761-119T, was isolated from an acidic hot spring in the Orange Field of the Uzon Caldera (Kamchatka, far-eastern Russia). Cells were spore-forming, Gram-positive rods, possessing one polar flagellum. Growth of strain 761-119T was observed between 37 and 68 degrees C and in the pH(20 degrees C) range 3.2-7.1. No growth was observed within 5 days of incubation at or below 35 degrees C and at or above 70 degrees C, as well as at or below pH(20 degrees C) 2.8 and at or above pH(20 degrees C) 7.5. The optimal temperature and pH(20 degrees C) for growth were 55 degrees C and pH(20 degrees C) 5.7, respectively. A wide range of carbohydrates and polysaccharides were fermented, as well as peptides and proteinaceous substrates. The main products of glucose fermentation were acetate, ethanol, lactate, H2 and CO2. The DNA G+C content was 34 (+/-0.5) mol%. 16S rRNA gene sequence analysis indicated that strain 761-119T belonged to the genus Thermoanaerobacterium. The level of 16S rRNA gene sequence similarity with other Thermoanaerobacterium species was 86.5-97.8 %, with the only moderately acidophilic member of this genus, Thermoanaerobacterium aotearoense, being one of its closest relatives. DNA-DNA hybridization with T. aotearoense showed 33 % relatedness. Thus, morphological (one polar flagellum) and physiological characteristics (lower pH limit of growth at pH(20 degrees C) 3.2 compared with T. aotearoense) and 16S rRNA gene sequence analyses revealed that strain 761-119T represents a novel species in the genus Thermoanaerobacterium, for which the name Thermoanaerobacterium aciditolerans sp. nov. is proposed, with the type strain 761-119T (=DSM 16487T=VKM B-2363T).

Desulfurococcus fermentans sp. nov., a novel hyperthermophilic archaeon from a Kamchatka hot spring, and emended description of the genus Desulfurococcus.

An obligately anaerobic, hyperthermophilic, organoheterotrophic archaeon, strain Z-1312(T), was isolated from a freshwater hot spring of the Uzon caldera (Kamchatka Peninsula, Russia). The cells were regular cocci, 1-4 microm in diameter, with one long flagellum. The cell envelope was composed of a globular layer attached to the cytoplasmic membrane. The temperature range for growth was 63-89 degrees C, with an optimum between 80 and 82 degrees C. The pH range for growth at 80 degrees C was 4.8-6.8, with an optimum at pH 6.0. Strain Z-1312(T) grew by hydrolysis and/or fermentation of a wide range of polymeric and monomeric substrates, including agarose, amygdalin, arabinose, arbutin, casein hydrolysate, cellulose (filter paper, microcrystalline cellulose, carboxymethyl cellulose), dextran, dulcitol, fructose, lactose, laminarin, lichenan, maltose, pectin, peptone, ribose, starch and sucrose. No growth was detected on glucose, xylose, mannitol or sorbitol. Growth products when sucrose or starch were used as the substrate were acetate, H(2) and CO(2). Elemental sulfur, thiosulfate and nitrate added as potential electron acceptors for anaerobic respiration did not stimulate growth when tested with starch as the substrate. H(2) at 100 % in the gas phase did not inhibit growth on starch or peptone. The G+C content of the DNA was 42.5 mol%. 16S rRNA gene sequence analysis placed the isolated strain Z-1312(T) as a member of the genus Desulfurococcus, where it represented a novel species, for which the name Desulfurococcus fermentans sp. nov. (type strain Z-1312(T) = DSM 16532 (T) = VKM V-2316(T)) is proposed.

[Oligonucleotide probes for the detection of Thermoanaerobacter]. / Oligonukleotidnye zondy dlia detektsii predstavitelei roda Thermoanaerobacter.

Based on the analysis of 16S rRNA nucleotide sequences, oligonucleotide probes were designed for the detection and identification of representatives of the genus Thermoanaerobacter. To increase the specificity level of detection, the genus Thermoanaerobacter was divided into three groups. The probe Tab 827 (5'-GCTTCCGCDYCCCACACCTA-3') detected all known representatives of the genus Thermoanaerobacter; the probe Tab_1 844 (5'-TTAACTACGGCACGRAATGCTTC-3') was specific for the first group of the species of the genus (T. wiegelii, T. siderophilus, T. sulfurophilus, T. brockii, T. kivui, T. ethanolicus, T. acetoethylicus, and T. thermohydrosulfuricus); the probe Tab_2 424 (5'-CACTAMYGGGGTTTACAACC-3') targeted the second group (T. thermocopriae, T. mathranii, and T. italicus); and the probe Tab_3 184 (5'-TC-CTCCATCAGGATGCCCTA-3') was specific for the third group (T. tengcongensis, T. yonseiensis, T. subterraneus, and Carboxydibrachium pacificum, an organism related to the genus Thermoanaerobacter according to its 16S rRNA sequence). The oligonucleotide probes were labeled with Dig-11-dUTP. Hybridization with the probes showed the affiliation with Thermoanaerobacter of several pure cultures that were morphologically similar to representatives of this genus but possessed metabolic features unusual for it (capacity for agarose hydrolysis, anaerobic oxidation of CO, growth at low pH values) or were isolated from habitats previously unknown for Thermoanaerobacter (deep-sea hydrothermal vents).