Current studies of human gut microbiome usually do not consider the special functional role of transient microbiota, although some of its members remain in the host for a long time and produce broad spectrum of biologically active substances. Getting into the gastrointestinal tract (GIT) with food, water and probiotic preparations, two representatives of Bacilli class, genera Bacillus and Lactobacillus, colonize epithelium blurring the boundaries between resident and transient microbiota. Despite their minor proportion in the microbiome composition, these bacteria can significantly affect both the intestinal microbiota and the entire body thanks to a wide range of secreted compounds. Recently, insufficiency and limitations of pure genome-based analysis of gut microbiota became known. Thus, the need for intense functional studies is evident. This review aims to characterize the Bacillus and Lactobacillus in GIT, as well as the functional roles of the components released by these members of microbial intestinal community. Complex of their secreted compounds is referred by us as the “bacillary secretome.” The composition of the bacillary secretome, its biological effects in GIT and role in counteraction to infectious diseases and oncological pathologies in human organism is the subject of the review.

RNases are enzymes that cleave RNAs, resulting in remarkably diverse biological consequences. Many RNases are cytotoxic. In some cases, they attack selectively malignant cells triggering an apoptotic response. A number of eukaryotic and bacterial RNase‐based strategies are being developed for use in anticancer and antiviral therapy. However, the physiological functions of these RNases are often poorly understood. This review focuses on the properties of the extracellular RNases from Bacillus amyloliquefaciens (barnase) and Bacillus intermedius (binase), the characteristics of their biosynthesis regulation and their physiological role, with an emphasis on the similarities and differences. Barnase and binase can be regarded as molecular twins according to their highly similar structure, physical–chemical and catalytic properties. Nevertheless, the ‘life paths’ of these enzymes are not the same, as their expression in bacteria is controlled by diverse signals. Binase is predominantly synthesized under phosphate starvation, whereas barnase production is strictly dependent on the multifunctional Spo0A regulator controlling sporulation, biofilm formation and cannibalism. Barnase and binase also have some distinctions in practical applications. Barnase was initially suggested to be useful in research and biotechnology as a tool for studying protein–protein interactions, for RNA elimination from biological samples, for affinity purification of RNase fusion proteins, for the development of cloning vectors and for sterility acquisition by transgenic plants. Binase, as later barnase, was tested for antiviral, antitumour and immunogenic effects. Both RNases have found their own niche in cancer research as a result of success in targeted delivery and selectivity towards tumour cells.

RAS proteins function as molecular switches that transmit signals from cell surface receptors into specific cellular responses via activation of defined signaling pathways (Fang, 2015). Aberrant constitutive RAS activation occurs with high incidence in different types of cancer (Bos, 1989). Thus, inhibition of RAS-mediated signaling is extremely important for therapeutic approaches against cancer. Here we showed that the ribonuclease (RNase) binase, directly interacts with endogenous KRAS. Further, molecular structure models suggested an inhibitory nature of binase-RAS interaction involving regions of RAS that are important for different aspects of its function. Consistent with these models, phosphorylation analysis of effectors of RAS-mediated signaling revealed that binase inhibits the MAPK/ERK signaling pathway. Interestingly, RAS activation assays using a non-hydrolysable GTP analog (GTPγS) demonstrated that binase interferes with the exchange of GDP by GTP. Furthermore, we showed that binase reduced the interaction of RAS with the guanine nucleotide exchange factor (GEF), SOS1. Our data support a model in which binase-KRAS interaction interferes with the function of GEFs and stabilizes the inactive GDP-bound conformation of RAS thereby inhibiting MAPK/ERK signaling. This model plausibly explains the previously reported, antitumor-effect of binase specific towards RAS-transformed cells and suggests the development of anticancer therapies based on this ribonuclease.

The biological effects of ribonucleases (RNases), such as the control of the blood vessels growth, the toxicity towards tumour cells and antiviral activity, require a detailed explanation. One of the most intriguing properties of RNases which can contribute to their biological effects is the ability to form dimers, which facilitates efficient RNA hydrolysis and the evasion of ribonuclease inhibitor. Dimeric forms of microbial RNase binase secreted by Bacillus pumilus (former B. intermedius) have only been found in crystals to date. Our study is the first report directly confirming the existence of binase dimers in solution and under natural conditions of enzyme biosynthesis and secretion by bacilli. Using different variants of gel electrophoresis, immunoblotting, size-exclusion chromatography and mass-spectrometry, we revealed that binase is a stable natural dimer with high catalytic activity.

Ribonucleases are considered as promising tools for anticancer treatment due to their selective cytotoxicity against tumor cells. We investigated a new RN ase from Bacillus altitudinis termed BALNASE ( B . altitudinis RN ase). Balnase is a close homolog of the well‐known cytotoxic binase, differing by only one amino acid residue: nonpolar hydrophobic alanine at position 106 in the balnase molecule is replaced by a polar uncharged threonine in binase. The most exciting question is how the physico‐chemical properties and biological effects of RN ase might be changed by A106T substitution. Here, we have developed a chromatography‐based rapid and modern technique for the purification of this new RN ase which allowed us to get a protein sample of high quality with specific activity of 1.2 × 10 6 units in preparative amounts, suitable for further investigation of its biological properties.