Elena Dudkina

Three‐step procedure for preparation of pure Bacillus altitudinis ribonuclease

Ribonucleases are considered as promising tools for anticancer treatment due to their selective cytotoxicity against tumor cells. We investigated a new RNase from Bacillus altitudinis termed BALNASE (B. altitudinisRNase). 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 RNase might be changed by A106T substitution. Here, we have developed a chromatography‐based rapid and modern technique for the purification of this new RNase which allowed us to get a protein sample of high quality with specific activity of 1.2 × 106 units in preparative amounts, suitable for further investigation of its biological properties.

New insight into secreted ribonuclease structure: binase is a natural dimer.

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.

Draft Whole-Genome Sequence of Bacillus altitudinis Strain B-388, a Producer of Extracellular RNase

ABSTRACT Here, we present a draft genome sequence of Bacillus altitudinis strain B-388, including a putative plasmid. The strain was isolated from the intestine of Indian meal moth, a common pest of stored grains, and it is characterized by the production of extracellular RNase, similar to binase, which is of interest for comparative studies and biotechnology.

Direct inhibition of oncogenic KRAS by Bacillus pumilus ribonuclease (binase).

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.

Draft Whole Genome Sequence of Bacillus pumilus Strain 3-19, a Chemical Mutant Overproducing Extracellular Ribonuclease

ABSTRACT Here, we present a draft genome sequence of Bacillus pumilus strain 3-19. It was derived from soil-isolated B. pumilus 7P using chemical mutagenesis and is characterized by elevated production of extracellular ribonuclease which is known to possess different biological activities with potential of applications in experimental research, medicine, and biotechnology.

Preparations of Bacillus pumilus secreted RNase: One enzyme or two?

Immunochemical analysis of the following purified preparations of Bacillus pumilus RNase (binase) was carried out: industrially manufactured enzyme (Institute of Organic Synthesis, Riga, Latvia) and the enzymes isolated from the culture liquid of the native B. pumilus producer and from the Escherichia coli BL21 recombinant strain bearing the pGEMGX1/ent/Bi plasmid. Electrophoresis of all three samples of purified binase revealed two protein fractions with ribonuclease activity possessing molecular masses of ∼12 and 25 kDa. The possible presence of binase II, a second secreted RNase, was ruled out. Both high- and low-molecular mass proteins interacted with binase-specific antibodies in the immunoblotting reaction, which indicated their antigenic identity. The difference in molecular mass between these proteins indicated the possible presence of two forms of binase in solution, a monomer and a dimer.

The Native Monomer of Bacillus Pumilus Ribonuclease Does Not Exist Extracellularly

Supported by crystallography studies, secreted ribonuclease of Bacillus pumilus (binase) has long been considered to be monomeric in form. Recent evidence obtained using native polyacrylamide gel electrophoresis and size-exclusion chromatography suggests that binase is in fact dimeric. To eliminate ambiguity and contradictions in the data we have measured conformational changes, hypochromic effect, and hydrodynamic radius of binase. The immutability of binase secondary structure upon transition from low to high protein concentration was registered, suggesting the binase dimerization immediately after translocation through the cell membrane and leading to detection of binase dimers only in the culture fluid regardless of ribonuclease concentration. Our results made it necessary to take a fresh look at the binase stability and cytotoxicity towards virus-infected or tumor cells.

Bacteriophages of soil bacilli: A new multivalent phage of Bacillus altitudinis

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.