Abstract—Production of extracellular membrane vesicles plays an important role in communication in bacterial populations and in bacteria–host interactions. Vesicles as carriers of various regulatory and signaling molecules may be potentially used as disease biomarkers and promising therapeutic agents, including vaccine preparations. The composition of membrane vesicles has been deciphered for a limited number of Gram-negative and Gram-positive bacteria. In this work, for the first time, extracellular membrane vesicles of a streptomycin-resistant strain Bacillus pumilus 3-19, a producer of extracellular guanyl-preferring ribonuclease binase, are isolated, visualized, and characterized by their genome and proteome composition. It has been established that there is no genetic material in the vesicles and the spectrum of the proteins differs depending on the phosphate content in the culture medium of the strain. Vesicles from a phosphate-deficient medium carry 49 unique proteins in comparison with 101 from a medium with the high phosphate content. The two types of vesicles had 140 mutual proteins. Flagellar proteins, RNase J, which is the main enzyme of RNA degradosomes, phosphatases, peptidases, iron transporters, signal peptides, were identified in vesicles. Antibiotic resistance proteins and amyloid-like proteins whose genes are present in B. pumilus 3-19 cells are absent. Phosphate deficiency-induced binase was found only in vesicles from a phosphate-deficient medium.

Therapeutic muds (peloids), which are widely used for body healing, improve metabolism and have antibacterial, anti-inflammatory and analgesic effects due to enrichment with necessary microelements and biological active substances. However, the microbiological component of these effects is not well studied.OBJECTIVE:To characterize the microbiome of therapeutic muds, used in the Tatarstan Republic, by identifying spectrum of cultivated microorganisms, using molecular analysis of bacterial communities, and by determining their biodiversity and functional potential based on revealed genetic determinants.MATERIAL AND METHODS:The study design of 5 peloids samples (local sapropels and peat deposits of swamp; 3 samples of Crimean sulfide muds) included three main techniques: isolation and taxonomic determination of cultivated microorganisms by time-of-flight mass-spectrometry; molecular analysis of peloids bacterial communities by 16S RNA high-throughput sequencing; identification of functional profiles of communities by their genetic determinant using Global Mapper tool on iVikodak platform.RESULTS:Experimental studies have confirmed the safety of examined peloids, where non-pathogenic cultivated bacteria belonging mainly to Bacillus and Rhodococcus genera were dominant. Metagenomic analysis showed that Firmicutes, Proteobacteria and Actinobacteria were predominant in all samples in different ratios. It has been established, that there is both the internal biodiversity of each sample and difference between them. The functional profile of microbial communities was determined based on the identification of bacterial genes. It has been revealed that all communities have an ability to synthesize antibiotics, as well as to decompose dangerous xenobiotics - polyaromatic hydrocarbons, cyclic compounds, and dioxins.CONCLUSION:Various microbial communities, which were identified in the therapeutic muds, contribute both to the clearance of toxicants in the peloids and to the antibacterial properties of the latter. The obtained priority results create a fundamental basis for the subsequent study of the role of peloids' microbiome of different origin in their healing action.

— Probiotic preparations are popular means for restoring the intestinal microbiota; their effectiveness requires long-term preservation of bacteria viability, induction of their proliferation, and manifestation of their functional properties in the body. Carriers of diverse nature are designed to ensure a prolonged release of the probiotic during their transit through the gastrointestinal tract. In the present work, a natural zeolite-containing rock with 25% clinoptilolite, which has sorption, detoxifying, and ion-exchange properties, was used as a carrier. Using transmission microscopy methods, the structure of the carrier was characterized, and the distribution of pores depending on their equivalent diameter was determined. It was shown that the size of the cavities was sufficient for adsorption of bacteria in the macropores and on the surface of the particles. Three Lactobacillus strains loaded onto a carrier and freeze-dried remained viable for 10 months.

Aim. To conduct a comparative analysis of the microbiome of biopsies of a tumor and normal intestinal epithelium of patients diagnosed with colorectal cancer and to identify of functional activities of the obtained bacterial isolates that affect the development of the tumor. Methods. The study included 50 patients with malignant neoplasms of the colon: 36 men and 24 women. The mean age of the patients was 64.110.2 years. To analyze the microbiota of the biopsies, DNA samples were obtained from the tissue of the unaffected colon mucosa and tumor of the patients. Bacterial 16S rRNA genes fragments were amplified using bar-coded primer bakt_341f. Metagenomic next-generation sequencing was performed using the MiSeq platform (Illumina, USA). The obtained data were processed by bioinformatic methods using the QIIME package. Recognition of microorganisms depending on the morphotype and gram staining of the microflora was carried out using combination differential media and biochemical tests. Statistical analysis was carried out using Microsoft Excel, Service Pack 2 for Office XP, Statistica 6.0 (StatSoft). A comparative analysis was performed with the Student's t-test and the MannWhitney test in case of unmet conditions of validity. Alpha diversity of bacterial communities was quantified by the Shannon diversity index and the UniFrac distance for beta diversity analysis. Results. In patients with colorectal cancer, 5 bacterial phyla were isolated, the predominant of which were Firmicutes and Bacteroidetes, while the content of Actinobacteria was low. In addition, a higher number of representatives of Fusobacteria was observed in the tumor tissue compared to the tissue of a healthy mucosa, at a distance of 5 centimeters proximal to the tumor. The results of this study indicate that the microbiome of a tumor and a healthy mucosa fundamentally differ from each other not only in morphotype and gram staining but also in antagonistic, hemolytic and ribonucleolytic activities. Conclusion. Colonization of the tumor by dominant aggressive Gram-negative bacteria leads to significant changes in the tumor microbiome composition compared with normal mucosa, whose representatives are displaced from the damaged epithelium by more aggressive strains.

Cytotoxic ribonucleases (RNases) of the T1 family, including binase, the secreted guanyl-preferring RNase of Bacillus pumilus, are considered as promising agents of alternative anticancer chemotherapy. Binase has a selective apoptosis-inducing action against cells expressing oncogenes ras, kit, AML-ETO. The crystal structure of the binase mutant with two-point amino acid substitutions at positions 43 and 81 (Glu43Ala/Phe81Ala) indicates the absence of dimeric forms, which are characteristic for the wild-type binase. We studied the native structural organization of the Glu43Ala/Phe81Ala mutant. It was found that the mutant enzyme, similarly to the wild-type binase, possesses catalytically active dimers of different stability levels identified not only under denaturing gel electrophoresis, but also under native conditions. The results of the study show that binase exists predominantly in the dimeric form, whereas the Glu43Ala/Phe81Ala mutant approximately equally represented by both dimers and monomers formed as a result of the decomposition of unstable dimers. Although the catalytic activity of the mutant with respect to the natural substrate, RNA, was lower, as compared to the wild-type enzyme, it exhibited a 32-35% higher cytotoxicity against human adenocarcinoma cells. The data obtained indicate the contribution of the structural organization of RNases to their cytotoxicity and confirm the significance of the analysis of the native conformation of cytotoxic proteins.

Unlike bacterial signal molecules, secreted yeast ones, which ensure their coordinated behavior as a single system, have been little studied. We used the image processing technique to evaluate the environmental conditions. Communication through quorum sensing molecules (QSM) is the dominant signaling in prokaryotic populations. In eukaryotic yeast cells, stress, caused primarily by the nutrient limit, causes a phenotypic manifestation of the mechanism of dimorphic switching, encompassing the repression of certain groups of genes and the activation of others, determining adhesion and virulence. Analysis of literature data and the results of the authors' own research emphasize the importance of signaling studies involving autoinducer molecules to elucidate the fundamental laws governing the regulation of yeast physiology, including growth parameters, morphogenesis and pathogenicity.

Herpes simplex virus type 1 is a highly contagious worldwide prevalent infection, which, once it enters the organism, persists during its whole lifetime. According to World Health Organization estimates, about 3.7 billion people under the age of 50 years (90% of the human population) are infected with by HSV-1 worldwide. The important role played by ribonucleases (RNases) in protection of the cell and whole organism from viruses has been confirmed by a considerable body of data that allow considering RNases not only as immune system components, but also as a basis for development of the new antiviral drugs. The purpose of the present work was to demonstrate the antiviral activity of the RNase from Bacillus pumilus (binase) toward HSV-1 at the cell entrance and reproduction stages. Virus treatment with binase in the concentration of 100 μg/mL for 60 min reduced virus replication in the bovine kidney epithelial cell culture MDBK by 100 times compared with untreated virus. Development of cytopathic effects produced by untreated virus in the cells grown on the medium with binase was retarded by 7 h compared with the growth of virus-infected cells on the medium without binase. It may be suggested that HSV-1 treatment with RNases reduces its ability to enter the cell; antiviral action of RNases toward the intracellular virus is realized at the initial stage of virus reproduction.

The experimental study identified the antiviral activity of Bacillus pumilus RNase (binase) against the reovirus of serotype 1/strain Lang. For the first time, it has been found that 50 μg/mL of binase effectively reduced the hemagglutinin and cytocidal activity of reovirus in Vero cell line. The preincubation of the enzyme with reovirus before infection of the cells inhibited the viral replication. To determine the stagedependent effect of reovirus reproduction upon binase inhibition, the infected cells were treated with binase or RNase A at different phases of the infectious cycle. The treatment of virus-infected cells has revealed that both enzymes have a maximal antiviral effect on the reovirus propagation during early phases of the reovirus reproduction cycle, with binase being more effective than RNase A. It has been hypothesized that the combined action of the oncolytic reovirus and binase is promising for the elimination of tumor cells carrying mutated RAS gene.

Besides the dominant well-studied low-molecular weight ribonuclease (RNase) binase I, Bacillus pumilus secretes a high-molecular weight binase II which differs from binase I by the absence of substrate specificity and the mode of RNA cleavage. Similar to many other RNases, binase II was proposed to have therapeutic potential. Deciphering molecular mechanisms of binase II biosynthesis regulation is required to boost expression of this RNase. Here, we have shown that increase of salinity in growth medium leads to elevated biosynthesis level of binase II. We detected in the binase II promoter the gene sequences homologous to the recognition sites of response regulator DegU. DegS-DegU signal transduction system is stimulated by high salt concentrations. Using the Bacillus subtilis strains with various mutations in DegU gene, we have shown that the DegS-DegU system is responsible for the increase of binase II gene expression under salt stress, indeed.

The experimental study identified the antiviral activity of Bacillus pumilus RNase (binase) against the reovirus of serotype 1/strain Lang. For the first time, it has been found that 50 μg/mL of binase effectively reduced the hemagglutinin and cytocidal activity of reovirus in Vero cell line. The preincubation of the enzyme with reovirus before infection of the cells inhibited the viral replication. To determine the stagedependent effect of reovirus reproduction upon binase inhibition, the infected cells were treated with binase or RNase A at different phases of the infectious cycle. The treatment of virus-infected cells has revealed that both enzymes have a maximal antiviral effect on the reovirus propagation during early phases of the reovirus reproduction cycle, with binase being more effective than RNase A. It has been hypothesized that the combined action of the oncolytic reovirus and binase is promising for the elimination of tumor cells carrying mutated RAS gene.

Bacillus are soil saprophytes, facultative anaerobes developing in the temperature range of 28–37°С. 16S rRNA cataloging shows that these bacteria form a coherent class with broad variability of virulence. Bacillus phages can be extensively used for phagotyping bacteria in the process of soil, water, and food monitoring. Bacillus phages can also be used as vectors in horizontal gene transfer and potential therapeutic agents. Thus, description of the biological diversity of the Bacillus phages is useful for further development of tools used in molecular biology and biomedicine. In this work, the scheme for isolation of soil bacteriophages was unified, which allowed ten bacillus phages to be isolated from different types of soil. It was shown that the number of phages depended on the soil fertility, decreasing as the soil changed from black soil to chestnut soil to gray forest soil to uncontaminated urban soil to oil-contaminated urban soil. A new polyvalent DNA-containing bacteriophage SRT01hs of B. altitudinis (it is also able to infect B. subtilis, B. cereus, and B. pumilus, but not B. licheniformis and B. atrophaeus) was described in detail. It has a typical structure: a total length of 360 nm and an icosahedron-shaped head 100 nm in diameter. Several phages simultaneously attack a B. altitudinis cell by increasing the level of intracellular low-molecular RNA. Infection with the phage virtually eliminates the stationary growth phase of infected bacilli and leads to a permanent increase in the number of phages in cultural liquor, with the exception of the time period of high activity of the secreted ribonuclease.

Members of the genus Bacillus can successfully counteract a sudden increase in salinity. In addition to the accumulation of osmolytes, saline stress also affects other aspects of bacterial physiology such as exoenzymes synthesis and motility. Here, we have shown that increase of salinity in growth medium leads to elevated biosynthesis level of low-molecular weight ribonuclease (RNase) binase I from Bacillus pumilus . The same effect was established previously for high-molecular weight binase II. Transmission electron microscopy revealed the absence of flagella and some other changes in salt-stressed cells of B. pumilis . We also detected the gene sequences homologous to the recognition sites of response regulator DegU in the binase I and binase II promoters. Using the B. subtilis strains with various mutations in DegU gene, we found that the two-component signal transduction system DegS-DegU which regulates the motility under salt stress participates in the control of biosynthesis for both secreted RNase of B. pumilis (binase I and binase II).

The facultative aerobic bacteria isolated from the mucosa of rectum in the zone of malignant tumor and neighboring normal mucosa in patients with colorectal cancer were studied using molecular genetic methods. The species attribution of bacteria was implemented using the cultural morphological analysis and sequencing of the 16S rRNA locus. The microorganisms with intraepithelial invasion to rectal mucosa were identified as the representatives of adherent-invasive (AIEC) subgroup of Escherichia coli and species Klebsiella pneumonia. The molecular analysis by genetic determinants controlling adhesive, hemolytic, and toxigenic activity revealed that some bacterial isolates were able to produce toxins with potential cancerogenic activity (e.g., colibactin and cytotoxic necrotizing factor 1). Certain bacterial species isolated from malignant and normal rectal epithelium of the same patient demonstrated no difference between analyzed factors of toxigenicity.

A dose-dependent effect of budding suppression in yeasts, depending on their concentration (1.0, 10, and 100 mu M) and the growth phase of the culture at the time of exposure to phenylethanol was observed during the study of growth peculiarities of the S. cerevisiae culture in the presence of exogenous factors determining the intercellular communication of bacteria, namely acylated homoserine lactone derivatives (N-hexanoyl-and N-octanoyl-DL-homoserine lactones). The antagonists of homoserine lactones are chemically synthesized furanones, which were added to the growing culture in microdoses (0.1, 1.0, and 10 mu M) and stimulated budding in yeasts. The culture cells in the early lag phase of growth (G1 phase), i.e., preparing for budding, were most sensitive to exogenous furanones.

Binase, the RNAse from Bacillus pumilus, is an endonuclease that cleaves the phosphodiester bond between the 3′-guanyl residue and 5′-OH residue of an adjacent nucleotide, with the formation of a corresponding intermediate, 2′,3′-cGMP. Subsequent hydrolysis of 2′,3′-cGMP into a 3′-phosphate is highly specific and proceeds slowly. Thus, a question arises in respect to the time interval that this positional isomer exists during catalytic cleavage of RNA by binase, and whether it may contribute to antitumor activity of the enzyme. In this study, we found, by implementing an enzyme-linked immunosorbent assay, that during catalytic cleavage of RNA by binase, 2′,3′-cGMP is maintained in the reaction mixture for about one hour. Activation of phosphodiesterases did not lead to a complete elimination of 2′,3′-cGMP. The highest amount of 2′,3′-cGMP was detected at pH 8.5, underat which conditions, it reached nanomolar levels. The Iinitial RNA concentration in the reactions was in the range of 100–1000 μg/mL. We found that exogenous 2′,3′-cGMP, as well as its positional isomer, 3′,5′-cGMP, do not induce apoptosis of human lung adenocarcinoma A549 cells, which are sensitive to binase apoptogenic activity. Taking into consideration the data on binase internalization, and on the role of 2′,3′-cGMP in the activation of opening of mitochondrial pores, we propose that 2′,3′-cyclic guanosine phosphates contributes to apoptotic processes, induced by binase, only when generated intracellularly.