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Genetic tools for targeted modification of the mycobacterial genome contribute to the understanding of the physiology and virulence mechanisms of mycobacteria. Human and animal pathogens, such as the Mycobacterium tuberculosis complex, which causes tuberculosis, and M. leprae, which causes leprosy, are of particular importance. Genetic research opens up novel opportunities to identify and validate new targets for antibacterial drugs and to develop improved vaccines. Although mycobacteria are difficult to work with due to their slow growth rate and a limited possibility to transfer genetic information, significant progress has been made in developing genetic engineering methods for mycobacteria. The review considers the main approaches to changing the mycobacterial genome in a targeted manner, including homologous and site-specific recombination and use of the CRISPR/Cas system.

The review is devoted to the use of the CRISPR/Cas system for obtaining knockdowns of target bacterial genes by CRISPR-mediated interference (CRISPRi). CRISPRi is based on the preservation of the ability of the inactivated dCas nuclease in complex with guide RNA to bind a target, which leads to reversible repression of the selected genes. The review describes the principle of operation of CRISPR/Cas and CRIS-PRi/dCas and provides examples of various approaches to the use of CRISPRi with the most popular inactivated nucleases dCas9 and dCas12a. Also, attention is paid to the use of CRISPRi screening for genome-wide studies and the modular system for identifying many important patterns at the Mobile-CRISPRi genome level. In addition, we discuss the use of CRISPRi to optimize biotechnological production, such as the synthesis of malonyl-CoA, L-lysine, L-glutamate, and other significant products.

Bacillus subtilis bacteria play an important role in veterinary medicine, medicine, and biotechnology, and the permanently growing demand for biotechnological products fuels the improvement of the properties of biotechnological strains. B. subtilis strains with improved characteristics maybe obtained by rational design and the directed evolution technologies, or be found among newly described strains. In the course of the long-term microbiome composition studies in the Russian segment of the International Space Station, the B. subtilis 20 strain was isolated, this strain shows the capacity for rapid growth and considerable biomass accumulation, as well as increased resistance to acidification of the environment in comparison to the "terrestrial" B. subtilis 168 strain. What is more, B. subtilis 20 is hyperresistant to the DNA and protein damaging factors that are linked to the overexpression of the genes controlling DNA repair, hydrogen sulfide production, and reactive oxygen species neutralization. The described properties of B. subtilis 20 are indicative of its considerable potential as a promising producer of biologically active compounds.

It has been shown that inactivation of the VapC toxin from the VapВС toxin-antitoxin system prevents mycobacterial cells from transitioning to an ovoid state that meets the criteria of dormancy. The results indicate a potential target for medicines that prevent the development of latent tuberculosis infection and provide a basis to obtain bacterial cells for the testing of compounds that are active towards dormant forms of mycobacteria.

Aim: To assess whether silk fibroin-based microvehicles (MVs) may be used to grow fibroblasts (FBs) and keratinocytes (KCs), key cellular components in skin regeneration after injury., Subjects and Methods: Cryogrinding was applied to derive MVs from fibroin-based and fibroin- and 30% gelatin-containing composite matrices. To examine the structure of the matrices and MVs, confocal microscopy was used to conjugate the polymer with the dye tetramethylrhodamine isothiocyanate. Microparticle size distribution was estimated by granulometric analysis. 3T3 mouse FBs and cultured primary mouse KCs expressing green fluorescent protein (GFP) were used to study whether fibroin-based MVs might be suitable for growing the cells involved in skin regeneration. KC growth was analyzed by confocal laser scanning microscopy from cellular GFP expression. The proliferation rate of FBs and KCs was estimated by a MTT assay., Results: There were two derived MV types: fibroin-based and fibroin and 30% gelatin-containing composite ones. On day 1, 3T3 mouse FBs on the fibroin-based gelatin-free MVs actively proliferated and the presence of gelatin in MVs diminished the proliferation of these cells. Fibroin-based MVs were shown to be suitable for the effective in vitro growth of KCs expressing cytokeratins 5 and 14, the major markers of KCs in the basal layer. Gelatin did not give rise to accelerated KC growth. The investigation has demonstrated that is possible to regulate FB proliferation on MVs, which is of great importance in delivering the cells into the site of injury since intensive proliferation of FBs may lead to the development of fibrosis and the formation of scar tissue. Balanced FB growth is essential to the creation of optimal conditions for KC growth in composite tissue-engineering constructions., Conclusion: The use of fibroin-based MVs is promising for the design of novel therapeutic materials and injectable cell therapy for different diseases.

The structure of various toxin-antitoxin (TA) families and the principles of their action are reviewed. TA loci are widely distributed in the genomes of eubacteria and archaea. Most TA systems are two-component and function in a similar way: a stable toxin alters vitally important cell functions and can be inactivated by a labile antitoxin.

Four major families of bacterial histone-like proteins (HU, IHF, H-NS, FIS), united on the basis of structural similarity and performing specific structural and regulatory functions in the cell, are discussed. Histone-like proteins perform topological modification of the chromosome (twisting, bending, and folding) and directly regulate the functioning of promoters of individual operons. Histone-like proteins are critical for the regulation of cell metabolism, are involved in the response to environmental changes, and play a key role in the transition to and maintenance of the quiescent state of bacteria.

The new model of obtaining of ovoid resting forms Mycobacterium smegmatis, which are morphologically different from vegetative (rod-like) cells, was developed. Ovoid forms were characterized by a drastically decreased level of metabolic activity, an increased stability to heat processing and antibiotics action, and also by prolonged (more than 2 months) storage time preserving colony-forming ability. Obtained resting forms of mycobacteria may be used in test-systems for checking efficiency of new medical agents against latent forms of tuberculosis and determination of role of these of those genes in entering rest state.

To work out a test system for studying the inhibition of isoprenoid biosynthesis through non-mevalonate pathway, the conversion of 2-(14)C-methylerythritol 2,4-cyclodiphosphate (14C-MEC) into isoprenoids of three bacteria species and into plastids of 11 higher plant species was studied. The highest efficiency (up to 63%) of the process was observed with chromoplasts from narcissus (Narcissus pseudonarcissus L.) and celandine (Chelidonium majus L.). Twenty five percent of added 14C-MEC was recovered in an isoprenoid fraction of red pepper (Capsicum annuum L.) chromoplasts. Thus, these three models can be used to test antibiotic inhibitors of isoprenoid biosynthesis.

2-C-Methyl-D-erythritol-2,4-cyclopyrophosphate (MEC), an intermediate of the biosynthesis of isoprenoid compounds in bacteria, was found to be capable of exerting a resuscitating effect on resting Mycobacterium smegmatis cells. The introduction of an additional copy of the ispE gene encoding cytidyl-methylerythritol kinase, an enzyme involved in MEC synthesis in M. smegmatis, resulted in the emergence of a capacity for spontaneous reactivation of "nonculturable" M. smegmatis cells, which is not characteristic of the wild-type cells of this species. The involvement of MEC in the transition from the "nonculturable" state to the state of active growth is indicative of a previously unknown function of MEC, assumed to consist in regulation of the bacterial genome activity.

Extraction and purification from the biomass of Corynebacterium ammoniagenes of 2-C-methyl-D-erhythritol 2,4-cyclopyrophosphate (MEC) was associated with its spontaneous transformation into a number of derivatives (which was due to pyrophosphate bond lability and the formation of complexes with metals). These derivatives included 1,2-cyclophospho-4-phosphate, 2,4-diphosphate, 2,3-cyclophosphate, 1,4-diphosphate, and 3,5-diphosphate (identified by 1H, 31P, and 13C NMR spectroscopy) and accounted for about 10% MEC. When added to a solution of DNA in the presence of the Fenton reagent, MEC prevented DNA decomposition. In addition, MEC slowed down the interaction of the reagent with tempol radicals, which indicates that complexation of ferrous ions by MEC attenuates their ability to catalyze the formation of hydroxyl radicals from hydrogen peroxide. In the presence of 0.23 mM MEC, the rate of respiration of rat liver mitochondria increased 1.8 times. At 0.1-1.0 mM, MEC activated in vitro proliferation of human Vgamma9 T-cells. It is suggested that MEC acts as an endogenous stabilizing agent for bacterial cells subjected to oxidative stress and as an immunomodulator for eukaryotic hosts.

Cross reactions between N. meningitidis and M. catarrhalis proteins were studied with the use of a panel of monoclonal antibodies to M. catarrhalis protein antigens. All antigenic preparations under study were shown to give cross reactions between N. meningitidis serotype porin of 39 kD (strain B125) and M. catarrhalis proteins of 40-41 kD. These M. catarrhalis proteins belonged to main proteins of class F and had the function of porins in the cell. In addition, the epitope of 41-kD antigen, detected by monoclonal antibodies 3E10, is common for both N. meningitidis porin and N. meningitidis iron-regulated proteins of 70 and 50 kD. The epitope of M. catarrhalis protein of 67 kD, detected by monoclonal antibodies 1G6, is common for N. meningitidis porin and N. meningitidis iron-regulated proteins of 50 and 55 kD.