ArdA genes from pKM101 and from B. bifidum chromosome have a different range of regulated genes.

The ardA genes are present in a wide variety of conjugative plasmids and play an important role in overcoming the restriction barrier. To date, there is no information on the chromosomal ardA genes. It is still unclear whether they keep their antirestriction activity and why bacterial chromosomes contain these genes. In the present study, we confirmed the antirestriction function of the ardA gene from the Bifidobacterium bifidum chromosome. Transcriptome analysis in Escherichia coli showed that the range of regulated genes varies significantly for ardA from conjugative plasmid pKM101 and from the B. bifidum chromosome. Moreover, if the targets for both ardA genes match, they often show an opposite effect on regulated gene expression. The results obtained indicate two seemingly mutually exclusive conclusions. On the one hand, the pleiotropic effect of ardA genes was shown not only on restriction-modification system, but also on expression of a number of other genes. On the other hand, the range of affected genes varies significally for ardA genes from different sources, which indicates the specificity of ardA to inhibited targets. Author Summary. Conjugative plasmids, bacteriophages, as well as transposons, are capable to transfer various genes, including antibiotic resistance genes, among bacterial cells. However, many of those genes pose a threat to the bacterial cells, therefore bacterial cells have special restriction systems that limit such transfer. Antirestriction genes have previously been described as a part of conjugative plasmids, and bacteriophages and transposons. Those plasmids are able to overcome bacterial cell protection in the presence of antirestriction genes, which inhibit bacterial restriction systems. This work unveils the antirestriction mechanisms, which play an important role in the bacterial life cycle. Here, we clearly show that antirestriction genes, which are able to inhibit cell protection, exist not only in plasmids but also in the bacterial chromosomes themselves. Moreover, antirestrictases have not only an inhibitory function but also participate in the regulation of other bacterial genes. The regulatory function of plasmid antirestriction genes also helps them to overcome the bacterial cell protection against gene transfer, whereas the regulatory function of genomic antirestrictases has no such effect.

Mid-holocene Brown Bear (Ursus arctos) from the Bolshoy Lyakhovsky Island (New Siberian Islands).

A morphological description is provided for a unique find of a frozen mummified subfossil brown bear (Ursus arctos L., 1758), found for the first time ever. The find is a well-preserved bear carcass of approximately 3500 years in age. Results of computed tomography and DNA testing are discussed.

Development of Resistance to Clarithromycin and Amoxicillin-Clavulanic Acid in Lactiplantibacillus plantarum In Vitro Is Followed by Genomic Rearrangements and Evolution of Virulence.

Ensuring the safety of the use of probiotics is a top priority. Obviously, in addition to studying the beneficial properties of lactic acid bacteria, considerable attention should be directed to assessing the virulence of microorganisms as well as investigating the possibility of its evolution under conditions of selective pressure. To assess the virulence of probiotics, it is now recommended to analyze the genomes of bacteria in relation to the profiles of the virulome, resistome, and mobilome as well as the analysis of phenotypic resistance and virulence in vitro. However, the corresponding procedure has not yet been standardized, and virulence analysis of strains in vivo using model organisms has not been performed. Our study is devoted to testing the assumption that the development of antibiotic resistance in probiotic bacteria under conditions of selective pressure of antimicrobial drugs may be accompanied by the evolution of virulence. In this regard, special attention is required for the widespread in nature commensals and probiotic bacteria actively used in pharmacology and the food industry. As a result of step-by-step selection from the Lactiplantibacillus plantarum 8p-a3 strain isolated from the "Lactobacterin" probiotic (Biomed, Russia), the L. plantarum 8p-a3-Clr-Amx strain was obtained, showing increased resistance simultaneously to amoxicillin-clavulanic acid and clarithromycin (antibiotics, the combined use of which is widely used for Helicobacter pylori eradication) compared to the parent strain (MIC8p-a3-Clr-Amx of 20 µg/mL and 10 µg/mL, and MIC8p-a3 of 0.5 µg/mL and 0.05 µg/mL, respectively). The results of a comparative analysis of antibiotic-resistant and parental strains indicate that the development of resistance to the corresponding antimicrobial drugs in L. plantarum in vitro is accompanied by the following: (i) significant changes in the genomic profile (point mutations as well as deletions, insertions, duplications, and displacement of DNA sequences) associated in part with the resistome and mobilome; (ii) changes in phenotypic sensitivity to a number of antimicrobial drugs; and (iii) an increase in the level of virulence against Drosophila melanogaster, a model organism for which L. plantarum is considered to be a symbiont. The data obtained by us indicate that the mechanisms of adaptation to antimicrobial drugs in L. plantarum are not limited to those described earlier and determine the need for comprehensive studies of antibiotic resistance scenarios as well as the trajectories of virulence evolution in probiotic bacteria in vivo and in vitro to develop a standardized system for detecting virulent strains of the corresponding microorganisms. IMPORTANCE Ensuring the safety of the use of probiotics is a top priority. We found that increased resistance to popular antimicrobial drugs in Lactiplantibacillus plantarum is accompanied by significant changes in the genomic profile and phenotypic sensitivity to a number of antimicrobial drugs as well as in the level of virulence of this bacterium against Drosophila. The data obtained in our work indicate that the mechanisms of antibiotic resistance in this bacterium are not limited to those described earlier and determine the need for comprehensive studies of the potential for the evolution of virulence in lactic acid bacteria in vivo and in vitro and to develop a reliable control system to detect virulent strains among probiotics.

Markers of dysbiosis in patients with ulcerative colitis and Crohn's disease.

AIM: The aim of the study was to study the taxonomic and functional composition of the gut microbiota in ulcerative colitis (UC) and Crohn's disease (CD) patients to identify key markers of dysbiosis in IBD. MATERIALS AND METHODS: Fecal samples obtained from 95 IBD patients (78 UC and 17 CD) as well as 96 healthy volunteers were used for whole-genome sequencing carried out on the SOLiD 5500 W platform. Taxonomic profiling was performed by aligning the reeds, not maped on hg19, on MetaPhlAn2 reference database. Reeds were mapped using the HUNAnN2 algorithm to the ChocoPhlAn database to assess the representation of microbial metabolic pathways. Short-chain fatty acids (SCFA) level were measured in fecal samples by gas-liquid chromatographic analysis. RESULTS: Changes in IBD patients gut microbiota were characterized by an increase in the representation of Proteobacteria and Bacteroidetes phyla bacteria and decrease in the number of Firmicutes phylum bacteria and Euryarchaeota phylum archaea; a decrease in the alpha-diversity index, relative representation of butyrate-producing, hydrogen-utilizing bacteria, and Methanobrevibacter smithii; increase in the relative representation of Ruminococcus gnavus in UC and CD patients and Akkermansia muciniphila in CD patients. Reduction of Butyryl-CoA: acetate CoA transferase gene relative representation in CD patients, decrease of absolute content of SCFA total number as well as particular SCFAs and main SCFAs ratio in IBD patients may indicate inhibition of functional activity and number of anaerobic microflora and/or an change in SCFA utilization by colonocytes. CONCLUSION: the revealed changes can be considered as typical signs of dysbiosis in IBD patients and can be used as potential targets for IBD patients personalized treatment development.

Gene Expression in the Three-Spined Stickleback (Gasterosteus aculeatus) of Marine and Freshwater Ecotypes.

Three-spine stickleback (Gasterosteus aculeatus) is a well-known model organism that is routinely used to explore microevolution processes and speciation, and the number of studies related to this fish has been growing recently. The main reason for the increased interest is the processes of freshwater adaptation taking place in natural populations of this species. Freshwater three-spined stickleback populations form when marine water three-spined sticklebacks fish start spending their entire lifecycle in freshwater lakes and streams. To boot, these freshwater populations acquire novel biological traits during their adaptation to a freshwater environment. The processes taking place in these populations are of great interest to evolutionary biologists. Here, we present differential gene expression profiling in G. aculeatus gills, which was performed in marine and freshwater populations of sticklebacks. In total, 2,982 differentially expressed genes between marine and freshwater populations were discovered. We assumed that differentially expressed genes were distributed not randomly along stickleback chromosomes and that they are regularly observed in the "divergence islands" that are responsible for stickleback freshwater adaptation.

Differential miRNA expression in the three-spined stickleback, response to environmental changes.

miRNAs play important role in the various physiological and evolutionary processes, however, there is no data allowing comparison of evolutionary differences between various ecotypes adapted to different environmental conditions and specimen demonstrating immediate physiological response to the environmental changes. We compared miRNA expression profiles between marine and freshwater stickleback populations of the three-spined stickleback to identify the evolutionary differences. To study the immediate physiological response to foreign environment, we explored the changes induced by transfer of marine sticklebacks into freshwater environment and vice versa. Comparative analysis of changes in miRNA expression suggested that they are driven by three independent factors: (1) non-specific changes in miRNA expression under different environmental conditions; (2) specific response to freshwater conditions in the marine stickleback ecotype; (3) specific response to extreme osmotic conditions for both marine and freshwater ecotypes during the contact with non-native environment. Gene Ontology enrichment analysis of differential expressed miRNA targets supports our current hypothesis.

Metagenomic analysis of microbial community of a parasitoid wasp Megaphragma amalphitanum.

The vast majority of multicellular organisms coexist with bacterial symbionts that may play various roles during their life cycle. Parasitoid wasp Megaphragma amalphitanum (Hymenoptera: Trichogrammatidae) belongs to the smallest known insects whose size is comparable with some bacteria. Using 16S rRNA gene sequencing and Whole Genome Sequencing (WGS), we described microbiota diversity for this arthropod and its potential impact on their lifecycle. Metagenomic sequences were deposited to SRA database which is available at NCBI with accession number SRX2363723 and SRX2363724. We found that small body size and limited lifespan do not lead to a significant reduction of bacterial symbionts diversity. At the same time, we show here a specific feature of microbiota composition in M. amalphitanum - the absence of the Rickettsiaceae family representatives that are known to cause sex-ratio distortion in arthropods and well represented in other populations of parasitoid wasps.

Identification of novel microRNA genes in freshwater and marine ecotypes of the three-spined stickleback (Gasterosteus aculeatus).

The three-spined stickleback (Gasterosteus aculeatus L.) is an important model organism for studying the molecular mechanisms of speciation and adaptation to salinity. Despite increased interest to microRNA discovery and recent publication on microRNA prediction in the three-spined stickleback using bioinformatics approaches, there is still a lack of experimental support for these data. In this paper, high-throughput sequencing technology was applied to identify microRNA genes in gills of the three-spined stickleback. In total, 595 miRNA genes were discovered; half of them were predicted in previous computational studies and were confirmed here as microRNAs expressed in gill tissue. Moreover, 298 novel microRNA genes were identified. The presence of miRNA genes in selected 'divergence islands' was analysed and 10 miRNA genes were identified as not randomly located in 'divergence islands'. Regulatory regions of miRNA genes were found enriched with selective SNPs that may play a role in freshwater adaptation.

Genomic and proteomic profiles of Acholeplasma laidlawii strains differing in sensitivity to ciprofloxacin.

As a result of comparative analysis of complete genomes as well as cell and vesicular proteomes of A. laidlawii strains differing in sensitivity to ciprofloxacin, it was first shown that the mycoplasma resistance to the antibiotic is associated with the reorganization of genomic and proteomic profiles, which concerns many genes and proteins involved in fundamental cellular processes and realization of bacterial virulence.

Analysis of the Mitochondrial Genome of a Novosvobodnaya Culture Representative using Next-Generation Sequencing and Its Relation to the Funnel Beaker Culture.

The Novosvobodnaya culture is known as a Bronze Age archaeological culture in the North Caucasus region of Southern Russia. It dates back to the middle of the 4th millennium B.C. and seems to have occurred during the time of the Maikop culture. There are now two hypotheses about the emergence of the Novosvobodnaya culture. One hypothesis suggests that the Novosvobodnaya culture was a phase of the Maikop culture, whereas the other one classifies it as an independent event based on the material culture items found in graves. Comparison between Novosvobodnaya pottery and Funnelbeaker (TRB) pottery from Germany has allowed researchers to suggest that the Novosvobodnaya culture developed under the influence of Indo-European culture. Nevertheless, the origin of the Novosvobodnaya culture remains a matter of debate. We applied next-generation sequencing to study ~5000-year-old human remains from the Klady kurgan grave in Novosvobodnaya stanitsa (now the Republic of Adygea, Russia). A total of 58,771,105 reads were generated using Illumina GAIIx with a coverage depth of 13.4x over the mitochondrial (mt) DNA genome. The mtDNA haplogroup affiliation was determined as V7, suggesting a role of the TRB culture in the development of the Novosvobodnaya culture and supporting the model of sharing between Novosvobodnaya and early Indo-European cultures.

Individual Genome of the Russian Male: SNP Calling and a de novo Assembly of Unmapped Reads.

A somatic cell genome was recently resequenced for a patient with renal cancer. The data were submitted to the NCBI Sequence Read Archive under the accession number SRA012240. Here, we have performed SNP calling for the genome and compared it with several published genomes. We have found 2, 921, 724 SNPs, including 1, 472, 679 newly described ones. Among them, 63, 462 SNPs have been mapped to the Y chromosome and, based on 18 markers, the genome has been ascribed to the R1a1a haplogroup predominant in Russian males. The mitochondrial haplogroup has been determined as U5a, which is also common in the European part of Russia. Short reads unmapped to the human genome were used for thede novoassembly of DNA sequences. This resulted in genome-specific contigs (more than 100 bp in length) with an overall length of 154 kbp (for GAII) and 4.7 kbp (for SOLiD).

Combining two technologies for full genome sequencing of human.

At present, the new technologies of DNA sequencing are rapidly developing allowing quick and efficient characterisation of organisms at the level of the genome structure. In this study, the whole genome sequencing of a human (Russian man) was performed using two technologies currently present on the market - Sequencing by Oligonucleotide Ligation and Detection (SOLiD™) (Applied Biosystems) and sequencing technologies of molecular clusters using fluorescently labeled precursors (Illumina). The total number of generated data resulted in 108.3 billion base pairs (60.2 billion from Illumina technology and 48.1 billion from SOLiD technology). Statistics performed on reads generated by GAII and SOLiD showed that they covered 75% and 96% of the genome respectively. Short polymorphic regions were detected with comparable accuracy however, the absolute amount of them revealed by SOLiD was several times less than by GAII. Optimal algorithm for using the latest methods of sequencing was established for the analysis of individual human genomes. The study is the first Russian effort towards whole human genome sequencing.

Natronobacillus azotifigens gen. nov., sp. nov., an anaerobic diazotrophic haloalkaliphile from soda-rich habitats.

Gram-positive bacteria capable of nitrogen fixation were obtained in microoxic enrichments from soda soils in south-western Siberia, north-eastern Mongolia, and the Lybian desert (Egypt). The same organisms were obtained in anoxic enrichments with glucose from soda lake sediments in the Kulunda Steppe (Altai, Russia) using nitrogen-free alkaline medium of pH 10. The isolates were represented by thin motile rods forming terminal round endospores. They are strictly fermentative saccharolytic anaerobes but tolerate high oxygen concentrations, probably due to a high catalase activity. All of the strains are obligately alkaliphilic and highly salt-tolerant natronophiles (chloride-independent sodaphiles). Growth was possible within a pH range from 7.5 to 10.6, with an optimum at 9.5-10, and within a salt range from 0.2 to 4 M Na(+), with an optimum at 0.5-1.5 M for the different strains. The nitrogenase activity in the whole cells also had an alkaline pH optimum but was much more sensitive to high salt concentrations compared to the growing cells. The isolates formed a compact genetic group with a high level of DNA similarity. Phylogenetic analysis based on 16S-rRNA gene sequences placed the isolates into Bacillus rRNA group 1 as a separate lineage with Amphibacillus tropicus as the nearest relative. In all isolates the key functional nitrogenase gene nifH was detected. A new genus and species, Natronobacillus azotifigens gen. nov., sp. nov., is proposed to accommodate the novel diazotrophic haloalkaliphiles.

Methylopila capsulata gen. nov., sp. nov., a novel non-pigmented aerobic facultatively methylotrophic bacterium.

A new genus, Methylopila, and one new species are described for a group of seven strains of facultatively methylotrophic bacteria with the serine pathway of C1 assimilation. These bacteria are aerobic, Gram-negative, non-spore--forming, motile, colourless rods that multiply by binary fission. Their DNA base content ranges from 66 to 70 mol % G + C. Their cellular fatty acid profile consists primarily of C18:1 omega 7 cis-vaccenic and C19:0 cyclopropane acids. The major hydroxy acid is 3-OH C14:0. The main ubiquinone is Q-10. The dominant cellular phospholipids are phosphatidylethanolamine and phosphatidylcholine. The new isolates have a low level of DNA-DNA homology (5-10%) with the type strains of the serine pathway methylobacteria belonging to the genera Methylobacterium, Aminobacter, Hyphomicrobium and Methylorhabdus. Another approach, involving 16S rRNA gene sequence analysis of strain IM1T, has shown that the new isolates represent a separate branch within the alpha-2 subclass of the Proteobacteria. The type species of the new genus is Methylopila capsulata sp. nov., with the type strain IM1T (= VKM B-1606T).

Characterization of a methane-utilizing bacterium from a bacterial consortium that rapidly degrades trichloroethylene and chloroform.

A mixed culture of bacteria grown in a bioreactor with methane as a carbon and energy source rapidly oxidized trichloroethylene and chloroform. The most abundant organism was a crescent-shaped bacterium that bound the fluorescent oligonucleotide signature probes that specifically hybridize to serine pathway methylotrophs. The 5S rRNA from this bacterium was found to be 93.5% homologous to the Methylosinus trichosporium OB3b 5S RNA sequence. A type II methanotrophic bacterium, isolated in pure culture from the bioreactor, synthesized soluble methane monooxygenase during growth in a copper-limited medium and was also capable of rapid trichloroethylene oxidation. The bacterium contained the gene that encodes the soluble methane monooxygenase B component on an AseI restriction fragment identical in size to a restriction fragment present in AseI digests of DNA from bacteria in the mixed culture. The sequence of the 16S rRNA from the pure culture was found to be 92 and 94% homologous to the 16S rRNAs of M. trichosporium OB3b and M. sporium, respectively. Both the pure and mixed cultures oxidized naphthalene to naphthol, indicating the presence of soluble methane monooxygenase. The mixed culture also synthesized soluble methane monooxygenase, as evidenced by the presence of proteins that cross-reacted with antibodies prepared against purified soluble methane monooxygenase components from M. trichosporium OB3b on Western blots (immunoblots). It was concluded that a type II methanotrophic bacterium phylogenetically related to Methylosinus species synthesizes soluble methane monooxygenase and is responsible for trichloroethylene oxidation in the bioreactor.