ABSTRACT The macrolide antibiotic, macrolactin A (McA), has been known for its antimicrobial properties since the late 1980s, but the mechanism of its antibacterial activity is still unknown. In this study, we investigated the microbiological and molecular characteristics of McA antimicrobial activity. McA effect on bacteria was found to be both bacteriostatic and bactericidal, depending on species and strains. Regarding the mechanism of action of McA, the following important results were obtained: 1) using in vivo and in vitro systems, we showed that McA is an inhibitor of protein synthesis in bacteria; 2) the concentration of McA required to inhibit protein synthesis in the E. coli cell-free model was found to be 50 times lower than the concentration required in the S. aureus cell-free model; 3) the toe-printing assay revealed that McA inhibits the first step of elongation stage of protein synthesis; 4) we identified single and multiple nucleotide polymorphisms in the gene encoding the translation elongation factor Tu (EF-Tu) by annotating the genomes of McA-resistant Bacillus pumilus McA R and its parental strain. Molecular modeling showed that the McA molecule can form non-covalent bonds with amino acids at the interface of domains 1 and 2 of EF-Tu, characterized by a relatively high docking score. Overall, our study demonstrated that McA acts as an elfamycin-like antibiotic (targeting EF-Tu), addressing a substantial gap in our understanding of the mechanism of action of macrolactin A, a representative member of macrolides.

GTPase Era from Staphylococcus aureus belongs to the TRAFAC superfamily of the TrmE-Era-EngA-EngB-Septin-like GTPases class and plays a significant role in the vital activity of this pathogenic microorganism as a maturation factor of the 30S ribosome subunit. However, the functions of this protein are not fully understood, making it a promising object for further study. Here, the 2.76 Å resolution crystal structure of Staphylococcus aureus Era in complex with GDP is presented. Structural comparison with other GTP-bound and GDP-bound homologous proteins, GTPase domain and the KH domain revealed a mutual orientation in S. aureus which has not been described before. The GDP-bound Era structure presented here will facilitate efforts to elucidate its interactions with its regulators and lay the foundation for a structure-based search for specific inhibitors.

It has been shown that human seminal fluid is a major factor in enhancing HIV activity. The SEM2(49-107) peptide is a product of cleavage after ejaculation by internal prostheses of the semenogelin 2 protein, expressed in seminal vesicles. It is established that the peptide SEM2(49-107) forms amyloid fibrils, which increase probability of contracting HIV infection. In this nuclear magnetic resonance (NMR) study, we present almost complete (86%) resonance assignments for the 1H 15N and 13C atoms of the backbone and side-chain of the SEM2(49-107) peptide (BioMagResBank accession number 52356). The secondary structure of SEM2(49-107) peptide was estimated by using two approaches, secondary chemical shifts analysis (CSI) and TALOS-N prediction. Analysis of the secondary structure of the SEM2(49-107) peptide using both methods revealed that the peptide contains helical segments at the C-terminus. Also in this work, we used phase-sensitive 2D HSQC 1H- 15N experiments measuring longitudinal T1 and transverse T2 NMR relaxation times to report predicted secondary structure and backbone dynamics of the SEM2(49-107) peptide. This resonance assignment will form the basis of future NMR research, contributing to a better understanding of the peptide structure and internal dynamics of the molecule.

Protein crystal structure studies are an important tool for drug design. The growth of high-quality crystals suitable for X-ray diffraction is the limiting factor and the bottleneck in obtaining the structural data. Here we report the extraction, purification, and crystallization of the protein GTPase Era from the pathogenic bacterium Staphylococcus aureus . In bacterial cells, GTPase Era acts as a ribosome assembly factor. This enzyme is responsible for the cell growth and division. However, its structure is poorly understood. We obtained crystals of Staphylococcus aureus GTPase Era, which can be used in further structural studies by single-crystal X-ray diffraction analysis.

It is known that four peptide fragments of predominant protein in human semen Semenogelin 1 (SEM1) (SEM1(86–107), SEM1(68–107), SEM1(49–107) and SEM1(45–107)) are involved in fertilization and amyloid formation processes. In this work, the structure and dynamic behavior of SEM1(45–107) and SEM1(49–107) peptides and their N-domains were described. According to ThT fluorescence spectroscopy data, it was shown that the amyloid formation of SEM1(45–107) starts immediately after purification, which is not observed for SEM1(49–107). Seeing that the peptide amino acid sequence of SEM1(45–107) differs from SEM1(49–107) only by the presence of four additional amino acid residues in the N domain, these domains of both peptides were obtained via solid-phase synthesis and the difference in their dynamics and structure was investigated. SEM1(45–67) and SEM1(49–67) showed no principal difference in dynamic behavior in water solution. Furthermore, we obtained mostly disordered structures of SEM1(45–67) and SEM1(49–67). However, SEM1(45–67) contains a helix (E58-K60) and helix-like (S49-Q51) fragments. These helical fragments may rearrange into β-strands during amyloid formation process. Thus, the difference in full-length peptides’ (SEM1(45–107) and SEM1(49–107)) amyloid-forming behavior may be explained by the presence of a structured helix at the SEM1(45–107) N-terminus, which contributes to an increased rate of amyloid formation.

Ribosome biogenesis is a complex and highly accurate conservative process of ribosomal subunit maturation followed by association. Subunit maturation comprises sequential stages of ribosomal RNA and proteins’ folding, modification and binding, with the involvement of numerous RNAses, helicases, GTPases, chaperones, RNA, protein-modifying enzymes, and assembly factors. One such assembly factor involved in bacterial 30S subunit maturation is ribosomal binding factor A (RbfA). In this study, we present the crystal (determined at 2.2 Å resolution) and NMR structures of RbfA as well as the 2.9 Å resolution cryo-EM reconstruction of the 30S–RbfA complex from Staphylococcus aureus (S. aureus). Additionally, we show that the manner of RbfA action on the small ribosomal subunit during its maturation is shared between bacteria and mitochondria. The obtained results clarify the function of RbfA in the 30S maturation process and its role in ribosome functioning in general. Furthermore, given that S. aureus is a serious human pathogen, this study provides an additional prospect to develop antimicrobials targeting bacterial pathogens.

The ribosome maturation factor M (RimM) is a protein with a molecular mass of 19.072 kDa involved in assembling of the 30S ribosome subunit. The RimM is necessary for the efficient processing of 16S rRNA. However, the mechanism of interaction of the RimM N-terminal domain with 16S rRNA remains poorly studied. The synthesis of the N-terminal domain of RimM from Staphylococcus aureus enriched in 13C and 15N isotopes and subsequent analysis of chemical shifts of the 1H, 13C, and 15N signals from the backbone and side chains are described. An analysis of chemical shifts suggests that the N-terminal domain of RimM contains six β-chains and three α-helices with the topology β1-β2-α1-β3-β4-β5-α2-β6-β3. The secondary structure of the N-terminal domain of RimM contains a KH domain between the β1 and β2 fold with a strongly conserved segment with the GXXG sequence. The further structural studies by integrated structural biology approach (NMR spectroscopy, X-ray diffraction analysis, and cryoelectron microscopy) of RimM and its complex with ribosome will allow screening of highly selective inhibitors of Staphylococcus aureus translation.

Candida albicans (C. albicans) is a fungal pathogen that causes infections of the wet body surfaces and the blood in immunocompromised patients or individuals with imbalanced microflora. Since the cases of clinically meaningful candidosis are on the rise, efficient С. albicans therapy is in a high demand. Informed drug design requires well-characterized С. albicans targets, including these aimed at disrupting its post-translational modifications. C. albicans ORF19.2286 gene encodes an ortholog of human deoxyhypusine hydroxylase (DOHH). Here, this ORF was cloned from the SC5314 strain and re-expressed in Escherichia coli as sGB1 - CaDOHH construct with 6xHis tag on the N-terminus of the fusion protein, then purified, and GB1-tag was removed with Tobacco etch virus (TEV) protease. Several amino acid sequence differences between C. albicans and animal DOHHs were noted, and are useful for a selection of the binding sites for antimicrobials in CaDOHH. We present the protocol for the heterologous expression and purification of C. albicans DOHH, which is suitable for further crystallization.

This article gives an overview of Russian legislation and international recommendations on the safe management regulation of the accumulated very low level radioactive waste (RW), provides the comparison of Russian and international criteria for RW attribution to this or that class and also the existing radiation safety requirements. This article also deals with the difficulties in management of production waste containing radionuclides. Criteria and standards were selected from international (ICRP and IAEA publications) and Russian (Federal Law on Management of Radioactive Waste, Government Decree on Radioactive Waste Classification, The Basic Sanitary Rules of Radiation Safety) documents for comparative analysis. International and Russian criteria for the radioactive materials attribution to RW and production waste containing radionuclides were compared during the analysis. A number of radionuclides for which it is necessary to establish such criteria have been identified. It was found that Russian classification of radioactive materials is generally consistent with IAEA classification. Practices of managing production waste containing radionuclides (protection from contact with the environment, the placement in the long-term storage facilities or disposal) depend on the period of potential hazard of radionuclides. The management of production waste containing radionuclides requires a separate system which should be developed in accordance with the RW management system.

Purpose: The article covers issues related to the providing personal protection for the personnel of radiation hazardous facilities. Specific character of working conditions at the enterprises in the field of atomic energy use is caused by extremely high toxic level of radioactive substances and the fact that there is no human sense organ which can identify dangerous levels of radiation exposure. Establishment of mandatory requirements for manufacturing, identification and verification of personal protection equipment (PPE) in the field of atomic energy use is needed because using of low-quality PPE and PPE not complying with working conditions poses a risk for the health of workers. The article provides results of analysis of the regulatory frameworks, both current and under development, which set the requirements for PPE. Results: Since 2017 Rosatom is executing plans for development of series of industry standards specifying requirements for PPE in the field of atomic energy use and methods of their testing. More than 25 interstate and national standards on PPE were included in the Summary list of standardization documents in the field of atomic energy use. PPE was included in the list of products subject to mandatory certification and having requirements for safety assurance in the field of atomic energy use specified. Industry and national standards establishing requirements for PPE protecting from tritium, radioactive noble gases, radioactive iodine, high-toxic alpha-emitting radionuclides, as well as standards on methods of their testing are planned to be developed soon. Conclusion: The system of standardizing requirements for PPE in the field of atomic energy use being created, together with existing system of certification of PPE in the field of atomic energy use will help to increase effectiveness of personal protection of the personnel at radiation and chemical hazardous facilities in nuclear industry.

Purpose: Analysis of the current regulatory and methodological framework on control of doses from intake of 14C for the personnel and the public living in the control area of the nuclear power plant (NPP). Identifying the most informative methods of controlling radiation impact of 14C on a human being. Material and methods: Research literature on radiation impact of naturally occurring 14C; 14C entering the environment as a result of nuclear weapon tests; and 14C entering workplaces and the control area of NPP has been reviewed. Dose coefficients and other radiation characteristics of 14C provided in IAEA, ICRP and UNSCEAR publications have been summarized. Results: According to UNSCEAR, annual radiation burden caused by global 14C is the highest one (about 80 %) among radiation burdens associated with four critical naturally occurring cosmogenic radionuclides: 3H (0.01 µSv/year), 7Be (3.0 µSv/year), 14C (12 µSv/year) and 24Na (0.2µSv/year). The main way of 14C intake is the alimentary one when this isotope enters the human body with food. Dose from this kind of intake of global 14C can reach 40 µSv. The annual dose caused by aerogenic (inhalation) way of intake of global 14C does not exceed 1 µSv. The most informative methods of dose assessment for the personnel of NPP and the public living in the control area involve measurement of content of 14C in top soil, vegetation and food products. Conclusions: Significant amount of 14C enters the environment within the control area during operation of NPP, which causes the public radiation dose exceeding the dose from global 14C. The most informative objects characterizing content of technogenic 14C in the control area of NPP are top soil (humus) and vegetation. The liquid scintillation spectrometry involves sample preparation by burning of samples in oxygen with capturing of generated carbon dioxide and its transfer into organic solvent. This is the most technologically viable method for mass control of 14C content in samples of top soil and vegetation.

Background: An essential in bacteria GTPase Era is a multifunctional protein that is involved in cell cycle regulation and appears to play a significant role in ribosome biogenesis. It is required for the maturation of the 30S ribosomal subunit. Era consists of two domains: the GTPase N-terminal domain, conserved in the GTPase family, and a C-terminal RNA-binding KH domain. Era specifically binds to the 16S rRNA and stimulates processing of the small ribosomal subunit to its mature form. Precise determination of nucleotide and amino acid sequences in the active site of binding will help in finding specific ways to prevent this interaction. In this way, it will be possible to disrupt the biogenesis of the ribosome and, thereby, stop or slow down protein synthesis in the bacterial cell. It is very important in the fight against pathogenic bacteria, such as Staphylococcus aureus (S. aureus). Methods: The His-tagged Era (His–Era) protein from S. aureus was expressed in E. coli BL21 strain and purified by Ni-NTA and SEC. The 30S ribosomal subunits were collected after dissociation of the S. aureus 70S ribosomes in sucrose gradient (0 – 30%). Complex 30S-Era was obtained by mixing in vitro 30S subunits and His–Era, incubated for 15 min at 37°C and followed by Ni-NTA purification to remove unbound 30S subunits. The presence of a stable 30S-Era complex has been confirmed by SDS-PAGE and agarose gel electrophoresis. The final sample quality was analyzed by negative staining EM. Results: For the first time in vitro 30S-Era complex from S. aureus was assembled and a sample was prepared for further structural studies by cryo-electron microscopy.

Purpose: The article covers issues related to the providing personal protection for the personnel of radiation hazardous facilities. Specific character of working conditions at the enterprises in the field of atomic energy use is caused by extremely high toxic level of radioactive substances and the fact that there is no human sense organ which can identify dangerous levels of radiation exposure. Establishment of mandatory requirements for manufacturing, identification and verification of personal protection equipment (PPE) in the field of atomic energy use is needed because using of low-quality PPE and PPE not complying with working conditions poses a risk for the health of workers. The article provides results of analysis of the regulatory frameworks, both current and under development, which set the requirements for PPE. Results: Since 2017 Rosatom is executing plans for development of series of industry standards specifying requirements for PPE in the field of atomic energy use and methods of their testing. More than 25 interstate and national standards on PPE were included in the Summary list of standardization documents in the field of atomic energy use. PPE was included in the list of products subject to mandatory certification and having requirements for safety assurance in the field of atomic energy use specified. Industry and national standards establishing requirements for PPE protecting from tritium, radioactive noble gases, radioactive iodine, high-toxic alpha-emitting radionuclides, as well as standards on methods of their testing are planned to be developed soon. Conclusion: The system of standardizing requirements for PPE in the field of atomic energy use created by, together with existing system of certification of PPE in the field of atomic energy use will help to increase effectiveness of personal protection of the personnel at radiation and chemical hazardous facilities in nuclear industry.

The semenogelin 1 protein is secreted in the seminal vesicles. After ejaculation it is split into small peptide fragments using internal proteases. It was shown that the fragments SEM1(45-107), SEM1(49-107), SEM1(68-107) (SEM1(86-107) form amyloid fibrils, which increase the possibility of HIV infection. The article presents a protocol for the synthesis and purification of a 15N, 13C-labeled SEM1(68–107) peptide for further structural studies by high-resolution NMR spectroscopy. The work describes cloning, expression of fusion protein GB1-SEM1(68-107) in E.coli, its purification, removal of GB1 and purification of SEM1(68-107). The purity of SEM1(68-107) samples on each purification steps was evaluated by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) and tricine-SDS-PAGE. The developed protocol allows to obtain SEM1(68-107) peptide for NMR studies (using 3D experiments), instead of costly solid-phase synthesis.

Background: Ribosome biogenesis is a complex process of ribosomal RNA and protein binding. Bacterial ribosome maturation and components involved in it are especially interesting, because they are widespread targets for antibiotics. A number of special protein factors facilitating the maturation of the 30S small ribosomal subunit are known. One of them is a ribosome-binding factor A (RbfA). This is a small (~14 kDa) protein with KH-domain organization distinguishing RNA binding proteins. Recent cryo-EM reconstruction of E.coli 30S-RbfA complex indicates that RbfA binds to 30S subunit on the central decoding region and promotes the switch from the immature state of h28 (neck) to mature state. RbfA interacts with 3`-end of 16S rRNA on mRNA exit channel and stabilizes the conformation of the region between h28, h44/h45 linker and 3`-end. Methods: Pure S.aureus RbfA was obtained by homologous expression in E.coli BL21 strain followed by Ni-NTA and gel filtration. The 30S subunits were obtained by dissociation of the S.aureus 70S ribosomes in a sucrose gradient (0-30%). We performed 30S subunit and RbfA complex reconstitution, sample and grid preparation. Data was collected on Talos Arctica, Falcon 2 detector (FEI Company/Thermo Fisher). Results: The 30S-RbfA complex density map with average resolution ~ 3.5 Å (FSC=0.143) was obtained. In comparison with the free subunit map (EMD 23052) we observed an extra density on the neck region near the decoding center region. Conclusion: Obtained data is correlated with recent structural results of the homologous E.coli RbfA. We consider that S.aureus RbfA binds to the 30S subunit at the same region. The next step of our structural research is building the model of S.aureus 30S-RbfA complex.

Background: Staphylococcus aureus (S. aureus) is one of the main human pathogens causing numerous nosocomial soft tissue infections and is among the best-known causes of bacterial infections. The bacterial 70S ribosome consists of two subunits, designated the 30S (small) and 50S (large) subunits. The small subunit (30S) consists of 16S ribosomal RNA (rRNA), from which the assembly of 30S begins, and 21 ribosomal proteins (r-proteins). The ribosome maturation factor P (RimP protein) binds to the free 30S subunit. Strains lacking RimP accumulate immature 16S rRNA, and fewer polysomes and an increased amount of unassociated 30S and 50S subunits compared to wild-type strains are observed in the ribosomal profile. Structural studies of the 30S subunit complex and the ribosome maturation factor RimP will make it possible in the future to develop an antibiotic that slows down or completely stops the translation of Staphylococcus aureus, which will complicate the synthesis and isolation of its pathogenic factors. Here we present the protocol of the in vitro reconstruction of S. aureus 30S ribosome subunit in a complex with RimP for further structural studies by cryo-electron microscopy. Methods: Recombinant RimP protein from S. aureus was expressed in E. coli and purified by Ni-NTA chromatography and size exclusion chromatography. Reconstitution of the 30S–RimP complex was performed by mixing RimP protein with 30S ribosome. Unbound RimP protein was removed by Amicon Ultra Concentration (Merk KGaA, Darmstadt, Germany) with a cut-off limit of 100 kDa. The presence of RimP protein in the resulting 30S-RimP complex was confirmed by SDS-PAGE, and the quality of the final sample was analyzed by the negative staining EM. Results: Finally, by in vitro reconstruction, the 30S-RimP complex from S. aureus was obtained for further structural studies by cryo-electron microscopy.

Ribosome-binding factor A (RbfA) from Staphylococcus aureus is a cold adaptation protein that is required for the growth of pathogenic cells at low temperatures (10-15°C). RbfA is involved in the processing of 16S rRNA, as well as in the assembly and stabilization of the small 30S ribosomal subunit. Structural studies of the 30S–RbfA complex will help to better understand their interaction, the mechanism of such complexes, and the fundamental process such as 30S subunit assembly that determines and controls the overall level of protein biosynthesis. This article describes protocols for preparation of RbfA and the small 30S ribosomal subunits and reconstitution and optimization of the 30S–RbfA complex to obtain samples suitable for cryo-electron microscopy studies.