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The article investigates SNP in genes of toxin-antitoxin systems type II in Mycobacterium tuberculosis Beijing lineage strains and their possible role in the development and formation of new sublineages. We established the catalog of SNPs in 142 TA systems genes in 1349 sequenced genomes of the M. tuberculosis Beijing lineage. Based on the catalog, 15 new sublineages were identified as part of Beijing lineages by non-synonymous SNP in 21 genes of TA systems. We discovered three toxin genes with mutations specific for epidemiologically dangerous sublineages Beijing-modern (vapC37 A46G, vapC38 T143C) and Beijing-B0/W148 (vapC12 A95G). We proved the functional significance of these polymorphisms by cloning these genes wild-type and with marker mutations for the Beijing lineage vapC12 (A95G), vapC37 (A46G), vapC38 (T143C). In vitro study of their activities revealed effect of mutations on the RNase activity of toxin proteins. Mutations in vapC37 and vapC38 decreased toxin activity, and mutation in the vapC12 increased it. We cloned the toxin vapC37 gene of Mycobacterium smegmatis mc 2 155 in both allelic variants: without mutation and with A46G mutation, specific for the Beijing-modern lineage. It was shown that this mutation leads to a loss of toxicity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We describe Streptomyces fradiae mechanisms of sensitivity to nitrone-oligomycin A, a derivative of oligomycin A. We obtained S. fradiae-nitR + bld, a nitrone-oligomycin A resistant mutant with a «bald» phenotype. Comparative genomic analysis of the wild-type S. fradiae ATCC19609 and S. fradiae-nitR + bld revealed a mutation in padR - a gene encoding a multifunction transcription regulator, which resulted in the amino acid replacement in a highly conserved DNA-binding domain. Bioinformatics genome analysis of S. fradiae ATCC19609 discovered a PadR binding site 13 bp upstream the start codon of the marR transcription factor gene. Induction of S. fradiaenitR + bld and w.t. strains with nitrone-oligomycin A lead to a significant increase in expression level of the marR gene in the w.t. strain, but no change observed in mutant strain. We identified differences between DNA-protein interactions of the mutant and native PadR proteins with its putative binding site in S. fradiae ATCC19609. This allowed us to suggest that the padR gene, that harbored a single nucleotide mutation in the S. fradiaenitR + bld strain, might be involved in the mechanism of resistance to nitrone-oligomycin A. We assume the participation of the transcriptional factorpadR in the formation of the bald phenotype., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The objective of this study was to determine for phosphorylated substrates of the species-specific serine-threonine protein kinase (STPK) Pkb2 from Bifidobacterium longum subsp. longum GT15. Two approaches were employed: analyses of phosphorylated membrane vesicles protein spectra following kinase reactions and analyses of the genes surrounding pkb2. A bioinformatics analysis of the genes surrounding pkb2 found a species-specific gene cluster PFNA in the genomes of 34 different bifidobacterial species. The identified cluster consisted of 5-8 genes depending on the species. The first five genes are characteristic for all considered species. These are the following genes encoding serine-threonine protein kinase (pkb2), fibronectin type III domain-containing protein (fn3), AAA-ATPase (aaa-atp), hypothetical protein with DUF58 domain (duf58) and transglutaminase (tgm). The sixth (protein phosphatase, prpC), seventh (hypothetical protein, BLGT_RS02790), and eighth (FHA domain-containing protein, fha) genes are included in this cluster, but they are not found in all species. The operon organization of the PFNA gene cluster was confirmed with transcriptional analysis. AAA-ATPase, which is encoded by a gene of the PFNA gene cluster, was found to be a substrate of the STPK Pkb2. Fourteen AAA-ATPase sites (seven serine, six threonine, and one tyrosine) phosphorylated by STPK Pkb2 were revealed. Analysis of the spectra of phosphorylated membrane vesicles proteins allowed us to identify eleven proteins that were considered as possible Pkb2 substrates. They belong to several functional classes: proteins involved in transcription and translation; proteins of the F1-domain of the FoF1-ATPase; ABC-transporters; molecular chaperone GroEL; and glutamine synthase, GlnA1. All identified proteins were considered moonlighting proteins. Three out of 11 proteins (glutamine synthetase GlnA1 and FoF1-ATPase alpha and beta subunits) were selected for further in vitro phosphorylation assays and were shown to be phosphorylated by Pkb2. Four phosphorylated substrates of the species-specific STPK Pkb2 from B. longum subsp. longum GT15 were identified for the first time. They included the moonlighting protein glutamine synthase GlnA, FoF1-ATPase alpha and beta subunits, and the chaperone MoxR family of AAA-ATPase. The ability of bifidobacterial STPK to phosphorylate the substrate on serine, threonine, and tyrosine residues was shown for the first time., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The diversity of Lb. rhamnosus and Lb. fermentum strains isolated from feces, saliva, and the vaginal cavity of 18-22-year-old healthy women residing in central regions of the Russian Federation has been characterized. The results obtained using multilocus sequence typing were identical to those obtained with the analysis of genetic and genomic polymorphism in TA systems. Different as well as identical Lb. rhamnosus and Lb. fermentum sequence types (ST) were isolated from various parts of the body of the same person. Identical ST were also isolated from different women, suggesting that such strains belong to a common pool of strains circulating among the population members. Our results demonstrate that TAs are suitable for characterizing intra-specific diversity of Lb. rhamnosus and Lb. fermentum strains. The advantage of using polymorphisms in TA systems for genotyping is based on the weak number of genes used, and consequently, less time is required for the analysis.

Although, the structure of oligomycin A (1) was confirmed by spectroscopic and chemical evaluations, some crystallographic data cast doubt on the originally adopted structure of the side 2-hydroxypropyl moiety of this antibiotic. It was suggested that the side chain of the oligomycin is enol-related (2-hydroxy-1-propenyl). To clarify this matter we synthesized and evaluated 33-dehydrooligomycin A (2) prepared by the Kornblum oxidation of 33-O-mesyloligomycin A (3) by dimethyl sulfoxide. NMR data for 33-dehydrooligomycin (2) and results of quantum chemical calculations have shown that this derivative exists in the keto rather than in the enol tautomer 2a. The in vitro antimicrobial activity of 2 was approximately two times weaker in comparison with oligomycin A against Streptomyces fradiae ATCC-19609 and reference Candida spp. strains and similar activity against certain filamentous fungi. The docking binding estimate of 2 with F O F 1 ATP synthase showed a slight decrease in binding affinity for 2 when compared with oligomycin A; that correlated with its activity against S. fradiae ATCC 19609 that is supersensitive to oligomycin A. The in vitro antiproliferative activities of 2 are also discussed.

Mycobacterium tuberculosis is divided into several distinct lineages, and various genetic markers such as IS-elements, VNTR, and SNPs are used for lineage identification. We propose an M. tuberculosis classification approach based on functional polymorphisms in virulence genes. An M. tuberculosis virulence genes catalog has been established, including 319 genes from various protein groups, such as proteases, cell wall proteins, fatty acid and lipid metabolism proteins, sigma factors, toxin-antitoxin systems. Another catalog of 1,573 M. tuberculosis isolates of different lineages has been developed. The developed SNP-calling program has identified 3,563 nonsynonymous SNPs. The constructed SNP-based phylogeny reflected the evolutionary relationship between lineages and detected new sublineages. SNP analysis of sublineage F15/LAM4/KZN revealed four lineage-specific mutations in cyp125, mce3B, vapC25, and vapB34. The Ural lineage has been divided into two geographical clusters based on different SNPs in virulence genes. A new sublineage, B0/N-90, was detected inside the Beijing-B0/W-148 by SNPs in irtB, mce3F and vapC46. We have found 27 members of B0/N-90 among the 227 available genomes of the Beijing-B0/W-148 sublineage. Whole-genome sequencing of strain B9741, isolated from an HIV-positive patient, was demonstrated to belong to the new B0/N-90 group. A primer set for PCR detection of B0/N-90 lineage-specific mutations has been developed. The prospective use of mce3 mutant genes as genetically engineered vaccine is discussed., (© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Recent results related to investigation of the role of intestinal microbiota (IM) in development and functioning of the human nervous system are discussed. The role of the microbiota in bidirectional communication between the gastrointestinal tract and the central nervous system is considered. Special attention is paid to the primary IM of infants, which is actively involved in formation of immune and other physiological mechanisms, including the nervous system, and is responsible for the subsequent general and psychical health of a human. The results of research on ability of the commensal intestinal microflora to produce neuroactive compounds, including neurotransmitters, short- and long-chain fatty acids, γ-aminobutyric acid, etc., are summarized. These compounds may have a considerable effect on development and functioning of the central nervous system, including the brain. Research on various animal models is discussed, including investigation of IM effect on behavior, learning abilities and memory, anxiety and depression levels, reaction to emotional stimuli, and stress resistance. A special section deals with probiotic bacteria, which are presently considered as psychobiotics with preventive and therapeutic potential for treatment of neurological and neurophysiological disorders. Development of new paradigms and concepts, rejection of some classical concepts of neurobiology is presently the key condition for the future breakthrough in investigation of human nervous activity.

Gamma-amino butyric acid (GABA) is an active biogenic substance synthesized in plants, fungi, vertebrate animals and bacteria. Lactic acid bacteria are considered the main producers of GABA among bacteria. GABA-producing lactobacilli are isolated from food products such as cheese, yogurt, sourdough, etc. and are the source of bioactive properties assigned to those foods. The ability of human-derived lactobacilli and bifidobacteria to synthesize GABA remains poorly characterized. In this paper, we screened our collection of 135 human-derived Lactobacillus and Bifidobacterium strains for their ability to produce GABA from its precursor monosodium glutamate. Fifty eight strains were able to produce GABA. The most efficient GABA-producers were Bifidobacterium strains (up to 6 g/L). Time profiles of cell growth and GABA production as well as the influence of pyridoxal phosphate on GABA production were studied for L. plantarum 90sk, L. brevis 15f, B. adolescentis 150 and B. angulatum GT102. DNA of these strains was sequenced; the gadB and gadC genes were identified. The presence of these genes was analyzed in 14 metagenomes of healthy individuals. The genes were found in the following genera of bacteria: Bacteroidetes (Bacteroides, Parabacteroides, Alistipes, Odoribacter, Prevotella), Proteobacterium (Esherichia), Firmicutes (Enterococcus), Actinobacteria (Bifidobacterium). These data indicate that gad genes as well as the ability to produce GABA are widely distributed among lactobacilli and bifidobacteria (mainly in L. plantarum, L. brevis, B. adolescentis, B. angulatum, B. dentium) and other gut-derived bacterial species. Perhaps, GABA is involved in the interaction of gut microbiota with the macroorganism and the ability to synthesize GABA may be an important feature in the selection of bacterial strains - psychobiotics., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Aminoglycoside phosphotransferases represent a broad class of enzymes that promote bacterial resistance to aminoglycoside antibiotics via the phosphorylation of hydroxyl groups in the latter. Here we report the spatial structure of the 3'-aminoglycoside phosphotransferase of novel VIII class (AphVIII) solved by X-ray diffraction method with a resolution of 2.15 Å. Deep analysis of APHVIII structure and its comparison with known structures of aminoglycoside phosphotransferases of various types reveals that AphVIII has a typical two-domain fold and, however, possesses some unique characteristics that distinguish the enzyme from its known homologues. The most important difference is the presence of the activation loop with unique Ser146 residue. We demonstrate that in the apo-state of the enzyme the activation loop does not interact with other parts of the enzyme and seems to adopt catalytically competent state only after substrate binding., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We report a draft genome sequence of Mycobacterium tuberculosis strain B9741 belonging to Beijing B0/W lineage isolated from a HIV patient from Siberia, Russia. This clinical isolate showed MDR phenotype and resistance to isoniazid, rifampin, streptomycin and pyrazinamide. We analyzed SNPs associated with virulence and resistance. The draft genome sequence and annotation have been deposited at GenBank under the accession NZ_LVJJ00000000.

Synthetic routes to novel N-(purin-6-yl)- and N-(2-aminopurin-6-yl) conjugates with amino acids and glycine-containing dipeptides were developed. In vitro testing of 42 new and known compounds made it possible to reveal a series of N-(purin-6-yl)- and N-(2-aminopurin-6-yl) conjugates exhibiting significant antimycobacterial activity against Mycobacterium tuberculosis H37Rv, Mycobacterium avium, Mycobacterium terrae, and multidrug-resistant M. tuberculosis strain isolated from tuberculosis patients in the Ural region (Russia). N-(2-Aminopurin-6-yl)- and N-(purin-6-yl)-glycyl-(S)-glutamic acids were the most active compounds., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The paper provides the annotation and data on sequencing the antibiotic resistance genes in Streptomyces fradiae strain ATCC19609, highly sensitive to different antibiotics. Genome analysis revealed four groups of genes that determined the resistome of the tested strain. These included classical antibiotic resistance genes (nine aminoglycoside phosphotransferase genes, two beta-lactamase genes, and the genes of puromycin N-acetyltransferase, phosphinothricin N-acetyltransferase, and aminoglycoside acetyltransferase); the genes of ATP-dependent ABC transporters, involved in the efflux of antibiotics from the cell (MacB-2, BcrA, two-subunit MDR1); the genes of positive and negative regulation of transcription (whiB and padR families); and the genes of post-translational modification (serine-threonine protein kinases). A comparative characteristic of aminoglycoside phosphotransferase genes in S. fradiae ATCC19609, S. lividans TK24, and S. albus J1074, the causative agent of actinomycosis, is provided. The possibility of using the S. fradiae strain ATCC19609 as the test system for selection of the macrolide antibiotic oligomycin A derivatives with different levels of activity is demonstrated. Analysis of more than 20 semisynthetic oligomycin A derivatives made it possible to divide them into three groups according to the level of activity: inactive (>1 nmol/disk), 10 substances; with medium activity level (0.05–1 nmol/disk), 12 substances; and more active (0.01–0.05 nmol/disk), 2 substances. Important for the activity of semisynthetic derivatives is the change in the position of the 33rd carbon atom in the oligomycin A molecule.

Various genetic markers such as IS-elements, DR-elements, variable number tandem repeats (VNTR), single nucleotide polymorphisms (SNPs) in housekeeping genes and other groups of genes are being used for genotyping. We propose a different approach. We suggest the type II toxin-antitoxin (TA) systems, which play a significant role in the formation of pathogenicity, tolerance and persistence phenotypes, and thus in the survival of Mycobacterium tuberculosis in the host organism at various developmental stages (colonization, infection of macrophages, etc.), as the marker genes. Most genes of TA systems function together, forming a single network: an antitoxin from one pair may interact with toxins from other pairs and even from other families. In this work a bioinformatics analysis of genes of the type II TA systems from 173 sequenced genomes of M. tuberculosis was performed. A number of genes of type II TA systems were found to carry SNPs that correlate with specific genotypes. We propose a minimally sufficient set of genes of TA systems for separation of M. tuberculosis strains at nine basic genotype and for further division into subtypes. Using this set of genes, we genotyped a collection consisting of 62 clinical isolates of M. tuberculosis. The possibility of using our set of genes for genotyping using PCR is also demonstrated.

We report a draft genome sequence of Streptomyces fradiae olg1-1, a mutant strain derived from the model object S. fradiae ATCC 19609, which is resistant to nitrone-oligomycin and has a mutation in the DNA-binding domain of a transcriptional regulator PadR., (Copyright © 2015 Vatlin et al.)

Previously, we identified six serine/threonine protein kinases (STPK) of Bifidobacterium and named them Pkb1-Pkb6. In the present study, we optimized methods for isolation of the six STPK catalytic domains proteins of B. longum B379M: a method for isolation of Pkb3 and Pkb4 in native conditions, a method for isolation of Pkb5 in denaturing conditions, and a method for isolation of Pkb1, Pkb2, and Pkb6 from inclusion bodies. The dialysis conditions for the renaturation of the proteins were optimized. All of the enzymes were isolated in quantities sufficient for study of the protein activity. The proteins were homogeneous according to SDS-PAGE. The autophosphorylation ability of Pkb1, Pkb3, Pkb4, and Pkb6 was investigated for the first time. Autophosphorylation was detected only for the Pkb3 catalytic domain.

In this study, we report the first completely annotated genome sequence of the Russia origin Bifidobacterium longum subsp. longum strain GT15. Comparative genomic analysis of this genome with other available completely annotated genome sequences of B. longum strains isolated from other countries has revealed a high degree of conservation and synteny across the entire genomes. However, it was discovered that the open reading frames to 35 genes were detected only from the B. longum GT15 genome and absent from other genomes B. longum strains (not of Russian origin). These so-called unique genes (UGs) represent a total length of 39,066 bp, with G + C content ranging from 37 to 65 %. Interestingly, certain genes were detected in other B. longum strains of Russian origin. In our analysis, we examined genes for global regulatory systems: proteins of toxin-antitoxin (TA) systems type II, serine/threonine protein kinases (STPKs) of eukaryotic type, and genes of the WhiB-like family proteins. In addition, we have made in silico analysis of all the most significant probiotic genes and considered genes involved in epigenetic regulation and genes responsible for producing various neuromediators. This genome sequence may elucidate the biology of this probiotic strain as a promising candidate for practical (pharmaceutical) applications.

Resistance to pyrazinamide (PZA) may impact clinical outcome of anti-tuberculosis chemotherapy. PZA susceptibility testing using MGIT 960 is not reliable and little information is available on the prevalence of PZA resistance in Russia. A collection of 64 clinical isolates of Mycobacterium tuberculosis, including 35 multidrug resistant and extensively drug-resistant (MDR/XDR), was analyzed for PZA resistance using MGIT 960, Wayne test, and sequencing of PZA resistance genes pncA, rpsA and panD. In addition, we analyzed 519 MDR-TB strains for susceptibility to PZA by MGIT 960. Sequencing of pncA revealed 17 of 25 (68%) MDR strains and all 10 XDR strains harboring pncA mutations. A correlation of φ = 0.81 between MGIT 960 and pncA sequencing was observed. Mutations in rpsA and panD not associated with PZA resistance as defined by MGIT 960 were identified. We found 1 PZA-resistant strain without mutations in known PZA resistance genes. Almost 73% of MDR-TB strains isolated in Moscow, Russia, were PZA-resistant by MGIT 960 testing of 519 MDR-TB clinical isolates. Further studies are needed to determine the role of rpsA and panD mutations in possible low-level PZA resistance and to identify the molecular basis of new PZA resistance in the isolate without known PZA resistance mutations., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Lactobacilli are important microorganisms in various activities, for example, diary products, meat ripening, bread and pickles, but, moreover, are associated directly with human skin and cavities (e.g., mouth, gut, or vagina). Some of them are used as probiotics. Therefore, the molecular biological investigation of these bacteria is important. Earlier we described several toxin antitoxin systems (type II) in lactobacilli. Here, we describe the structure and transcriptional regulation of genes, encoding TA system YefM-YoeB(Lrh) in three strains of Lactobacillus rhamnosus comparing stationary and exponential growth phases, the influence of stress factors and mRNA stability. The same TA system is responding to physiological and stress conditions differently in related strains. Using primer extension and RLM-RACE methods we determined three transcription start sites of RNAs in the operon. The promoter region of the operon is preceded by a conserved BOX element occurring at multiple positions in the genomes of L. rhamnosus strains. Downstream of and partially overlapping with the 3' end of the yoeB(Lrh) toxin gene, a divergently transcribed unexpected RNA was detected., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report draft genome sequences of two pyrazinamide (PZA)-resistant isolates, Mycobacterium tuberculosis 13-4152 and 13-2459. Isolate 13-4152 is PZA resistant, though it lacks mutations in known genes of PZA resistance. The comparative analysis of these genomes with those stored in GenBank revealed unique mutations, which may elucidate new mechanisms of PZA resistance., (Copyright © 2015 Maslov et al.)

The draft genome sequences of Bifidobacterium angulatum GT102 and Bifidobacterium adolescentis 150 strains isolated from the human intestinal microbiota are reported. Both strains are able to produce gamma-aminobutyric acid (GABA). Detailed genomes analysis will help to understand the role of GABA in the functioning of gut-brain axis., (Copyright © 2015 Dyachkova et al.)

We report a draft genome sequence of Mycobacterium tuberculosis strain E186hv, belonging to the Beijing B0/W lineage and isolated from a patient from Kurgan, Russia. This clinical isolate showed a reduced virulence phenotype unusual for this lineage and resistance to isoniazid, rifampin, ethambutol, pyrazinamide, and ofloxacin. We analyzed single nucleotide polymorphisms (SNPs) associated with virulence., (Copyright © 2015 Shur et al.)

The genomes of Lactobacillus plantarum strain 90sk and Lactobacillus brevis strain 15f were isolated from human intestinal microbiota. Both strains synthesize gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter. Detailed genome analyses will help to understand the role of GABA in the interaction of bacteria with human intestinal cells., (Copyright © 2015 Yunes et al.)

The patterns of protein phosphorylation in inverted membrane vesicles from the strain Streptomyces fradiae ATCC 19609 were investigated to elucidate the mechanisms of regulation of bacterial membrane bound FoF1-ATP synthase. We found for the first time by two-dimensional gel electrophoresis and mass spectrometry that the β- and b-subunits of the FoF1-ATP synthase complex undergo phosphorylation; 20 proteins with known functions were identified. All eight subunits of FoF1-ATP synthase, i.e. α, β, γ, δ, ε, a, b, and c, were cloned into Escherichia coli and expressed as recombinant proteins. Using a crude preparation of serine/threonine protein kinases, we demonstrated the phosphorylation of recombinant γ-, β-, α- and ε-subunits. The β-subunit was phosphorylated both as a recombinant protein and in vesicles. Differential phosphorylation of membrane-bound and recombinant proteins can be attributed to different pools of protein kinases in each preparation; in addition, certain steps of FoF1-ATP synthase assembly and function might be accompanied by individual phosphorylation patterns. The structure of the operon containing all subunits and regulatory protein I was identified. The phylogenetic similarity of FoF1-ATP synthase from Streptomyces fradiae ATCC 19609 with the respective proteins in saprophytic and pathogenic (including Mycobacterium tuberculosis) bacteria was investigated. Thus, bacterial serine/threonine protein kinases are important for the regulation of FoF1-ATP synthase. From the practical standpoint, our results provide a basis for designing targeted antibacterial drugs.

The 156 samples of feces separated from healthy residents of the Central region of Russia were used to compose collection of 87 strains of Bifidobacterium out of which 5 strains with wide antagonistic activity related to pathogenic and opportunistic microorganisms were selected. The selected strains are characterized by high probiotic potentials. They have adhesive properties and sensitivity to antibiotics and preparations corresponding to main requirements of common pharmacopoeia articles to strains of microorganisms used in production of probiotics for medicinal application. The given strains of Bifidobacterium can be recommended for development of effective probiotic pharmaceuticals directed to residents of the Central region of Russia.

In this study, we report the first completely annotated genome sequence of the Russian-origin Bifidobacterium longum subsp. longum strain GT15. We discovered 35 unique genes (UGs) which were detected from only the B. longum GT15 genome and were absent from other B. longum strain genomes (not of Russian origin)., (Copyright © 2014 Zakharevich et al.)

We report here a sequence of the genome of the Streptomyces fradiae ATCC 19609 strain, initially isolated from the soil, which produces tylosin. S. fradiae is highly sensitive to different classes of antibiotics, compared to the sensitivities of other bacteria. We have identified 9 groups of genes directly or indirectly involved in the resistome formation., (Copyright © 2014 Bekker et al.)

The problem of Mycobacterium tuberculosis virulence, together with drug resistance, is becoming key for the design of drugs with a new mechanism of action and the production of modern concepts and tuberculosis treatment schemes. The review describes gene complexes and their products, including mycolic acids and global regulatory systems at the level of transcriptional, translational, and post-translational modification, etc. The criteria for selection of virulence/pathogenicity factors that might be used for comparative genomic analysis of strains differing in the degree of virulence were recommended. The experimental approaches and test systems for an adequate estimation of the virulence degree of different strains of M. tuberculosis were analyzed.

Studies of reactivity of antibiotic oligomycin A in various alkaline conditions showed that the compound easily undergoes retroaldol degradation in β-hydroxy ketone fragments positioned in the C7-C13 moiety of the antibiotic molecule. Depending on reaction conditions, the retroaldol fragmentation of the 8,9 or 12,13 bonds or formation of a product through double retroaldol degradation, when the fragment C9-C12 was detached, took place followed by further transformations of the intermediate aldehydes formed. The structures of the obtained non-cyclic derivatives of oligomycin A were supported by NMR and MS methods. NMR parameters demonstrate the striking similarity of the geometry (conformation) of the fragment C20-C34 in the non-cyclic products of retroaldol degradation and the starting antibiotic 1. The compounds obtained had lower cytototoxic properties than oligomycin A for human leukemia cells K-562 and colon cancer cells HCT-116 and lower activity against growth inhibition of model object Streptomyces fradiae. It cannot be excluded that the products of retroaldol degradation participate in the biological effects of antibiotic oligomycin A.

Six genes encoding the bifidobacterial Hanks-type (eukaryote-like) serine/threonine protein kinases (STPK) were identified and classified. The genome of each bifidobacterial strain contains four conserved genes and one species-specific gene. Bifidobacterium longum and Bifidobacterium bifidum possess the unique gene found only in these species. The STPK genes of Russian industrial probiotic strain B. longum B379M were cloned and sequenced. The expression of these genes in Escherichia coli and bifidobacteria was observed. Autophosphorylation of the conserved STPK Pkb5 and species-specific STPK Pkb2 was demonstrated. This is the first report on Hanks-type STPK in bifidobacteria.

It was found by virtual screening that 3-amino-1H-pyrazolo[3,4-b]quinolines could have wide protein kinase inhibitory activity. Amides of titled amines and thioureas were synthesized regioselectively. 3-Amino-7-methoxy-1H-pyrazolo[3,4-b]quinoline demonstrated in vitro significant inhibitory activity on bacterial serine/threonine protein kinases (inhibition of resistance to kanamycin in Streptomyces lividans regulated by protein kinases). The studies of Structure Activity Relationship (SAR) showed that the substitution of the NH2 group and 1-NH of pyrazole ring or aromatic ring at the position 6 decreased or removed inhibitory activity.

The toxin-antitoxin gene systems (TASs) are present in the genomes of the overwhelming majority of bacteria and archaea. These systems are involved in various cellular regulatory processes (including stress response), and have not been previously investigated in Lactobacilli. We identified 6 putative TASs with toxins belonging to the MazE and RelE superfamilies (PemK1-А1Lrh, PemK2-А2Lrh, PemK3-RelB2Lrh, RelE1Lrh, RelB3-RelE3Lrh, and YefM-YoeBLrh) in the genomes of annotated strains of Lactobacillus rhamnosus. PCR analyses revealed that all systems were found in the genomes of 15 strains of L. rhamnosus isolated from humans in central Russia. These strains were highly heterogeneous with respect to the presence of TASs, as well as their nucleotide and amino acid sequences. In three cases, the relE1 genes contained IS3 elements. TAS heterogeneity may be used to reveal inter-genus differences between strains. Cloning of the toxin genes of 3 TASs inhibited Escherichia coli growth, thus confirming their functionality. Cell growth arrest caused by expression of the toxin genes could be reverted by the expression of a cognate antitoxins. Transcription of toxin-antitoxin loci in L. rhamnosus was shown by RT-PCR., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A novel way of chemical modification of the macrolide antibiotic oligomycin A (1) at the side chain was developed. Mesylation of 1 with methane sulfonyl chloride in the presence of 4-dimethylaminopyridine produced 33-O-mesyl oligomycin in 56% yield. Reactions of this intermediate with sodium azide produced the key derivative 33-azido-33-deoxy-oligomycin A in 60% yield. 1,3-Dipolar cycloaddition reaction with propiolic acid, methyl ester of propiolic acid, and phenyl acetylene resulted in 33-deoxy-33-(1,2,3-triazol-1-yl)oligomycin A derivatives substituted at N4 of the triazole cycle. The mesylated oligomycin A and 33-deoxy-33-azidooligomycin A did not inhibit F0F1 ATFase ATPase; however, 33-azido-33-deoxy-oligomycin A and the derivatives containing 4-phenyltriazole, 4-methoxycarbonyl-triazole and 3-dimethylaminoethyl amide of carboxyltriazole substituents demonstrated a high cytotoxicity against K562 leukemia and HCT116 human colon carcinoma cell lines whereas non-malignant skin fibroblasts were less sensitive to these compounds. Novel series of oligomycin A derivatives allow for the search of intracellular molecules beyond F0F1 ATP synthase relevant to the cytotoxic properties of this perspective chemical class., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The in silico analysis of 36 sequenced genomes of bacteria of the Bifidobacterium genus determined the presence of 19 genes of toxin-antitoxin (TA) systems that belong to the MazEF and RelBE families, including five mazF and two relE genes that encode toxins and 12 relB genes that encode antitoxins. A high level ofgene (at the level of nucleotide changes) and genomic (presence or absence of genes in distinct genomes) polymorphism in the investigated genes was revealed. The highest level of polymorphism was observed in strains of the Bifidobacterium longum species, primarily in relB1-10 genes. Gene and genomic polymorphism might be used to identify the strain of B. longum species. PCR analysis ofgenomic DNA of 30 bifidobacteria strains belonging to the three species, B. longum, B. adolscentis, and B. bifidum, isolated from the intestinal microbiota of astronauts demonstrated the presence of mazF and relB genes. The observed polymorphism of TA genes indicates the presence of differences in bifidobacteria strains isolated from the intestinal microbiota of astronauts before and after space flight and the control group.

This review describes and summarizes the data of ESX secretory system peculiarities characteristic of mainly mycobacteria. This system is involved in the secretion of small proteins of the WXG100 family. Some of these proteins represent virulence factors of Mycobacterium tuberculosis and other pathogenic mycobacteria. The role of these proteins in pathogenesis apparently consists of protecting mycobacteria from lysis in the macrophages that absorb them; the cytolysis of macrophages; and, hence, mycobacterium output into the surrounding tissue. A number of proteins that make up this secretory system are homologs of proteins involved in the conjugation process in saprophytic Mycobacterium smegmatis.

We demonstrate for the first time the role of phosphorylation in the regulation of activities of enzymes responsible for inactivation of aminoglycoside antibiotics. The aminoglycoside phosphotransferase VIII (APHVIII) from the actinobacterial strain Streptomyces rimosus ATCC 10970 is an enzyme regulated by protein kinases. Two serine residues in APHVIII are shown to be phosphorylated by protein kinases from extracts of the kanamycin-resistant strain S. rimosus 683 (a derivative of strain ATCC 10970). Using site-directed mutagenesis and molecular modeling, we have identified the Ser146 residue in the activation loop of the enzyme as the key site for Ca2+-dependent phosphorylation of APHVIII. Comparison of the kanamycin kinase activities of the unphosphorylated and phosphorylated forms of the initial and mutant APHVIII shows that the Ser146 modification leads to a 6-7-fold increase in the kanamycin kinase activity of APHVIII. Thus, Ser146 in the activation loop of APHVIII is crucial for the enzyme activity. The resistance of bacterial cells to kanamycin increases proportionally. From the practical viewpoint, our results increase prospects for creation of highly effective test systems for selecting inhibitors of human and bacterial serine/threonine protein kinases based on APHVIII constructs and corresponding human and bacterial serine/threonine protein kinases.

The stability of inheriting several genes in the Russian commercial strain Bifidobacterium longum subsp. longum B379M during cultivation and maintenance under laboratory conditions has been studied. The examined genes code for probiotic characteristics, such as utilization of several sugars (lacA2 gene, encoding beta-galactosidase; ara gene, encoding arabinosidase; and galA gene, encoding arabinogalactan endo-beta-galactosidase); synthesis of bacteriocins (lans gene, encoding lanthionine synthetase); and mobile gene tet(W), conferring resistance to the antibiotic tetracycline. The other gene families studied include the genes responsible for signal transduction and adaptation to stress conditions in the majority of bacteria (serine/threonine protein kinases and the toxin-antitoxin systems of MazEF and RelBE types) and transcription regulators (genes encoding WhiB family proteins). Genomic DNA was analyzed by PCR using specially selected primers. A loss of the genes galA and tet(W) has been shown. It is proposed to expand the requirements on probiotic strains, namely, to control retention of the key probiotic genes using molecular biological methods.

Bifidobacteria are strictly anaerobic bacteria, that are an important component of human microbiote due to their probiotic characteristics. They are frequently exposed to a variety of stresses, therefore, identification of genes implicated in stress responses in bifidobacteria is critical for biomedicine and maintenance of industrial strains. The WhiB-like family proteins unique for Actinobacteria are transcriptional regulators involved in major cellular processes, including stress responses. The aim of this study was the identification of WhiB-like family proteins of the Bifidobacterium genus of the Actinobacteria class and functional characterization of conservative whiB-like genes. The DNA sequence database of 36 strains revealed a family of WhiB-encoding genes. It were identified the wblE orthologs in all Bifidobacteria species and the whiB2 orthologs in all bifidobacterial strains except of all strains of Bifidobacterium animalis subsp. lactis and Bifidobacterium gallicum. Some strains, in particular, those of the Bifidobacterium longum group, contain additional whiB-like genes of different length and a low degree of similarity in sequences. The wblE and whiB2 genes of the Bifidobacterium genus are evolutionary conservative and ancient genes. The real-time PCR analysis showed that transcription of wblE is induced by a variety of stress conditions such as heat shock, osmotic, oxidative stresses, by antibiotic tetracycline and bile salt treatment, the nutrient starvation and entry into late stationary phase. The wblE gene may play a significant role in general stress responses in bifidobacteria., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The antibiotic oligomycin A in the presence of K(2)CO(3) and n-Bu(4)NHSO(4) in chloroform in phase-transfer conditions afforded a novel derivative through the initial retro-aldol fragmentation of the 8,9 bond, followed by further transformation of the intermediate aldehyde. NMR, MS and quantum chemical calculations showed that the novel compound is the acyclic oligomycin A derivative, in which the 8,9 carbon bond is disrupted and two polyfunctional branches are connected with spiroketal moiety in positions C-23 and C-25. The tri-O-acetyl derivative of the novel derivative was prepared. The acyclic oligomycin A derivative retained the ability to induce apoptosis in tumor cells at low micromolar concentrations, whereas its antimicrobial potencies decreased substantially. The derivative virtually lost the inhibitory activity against F(0)F(1) ATP synthase-containing proteoliposomes, strongly suggesting the existence of the target(s) beyond F(0)F(1) ATP synthase that is important for the antitumor potency of oligomycin A.

Eukaryotic-like serine/threonine protein kinases (ESTPKs) are widely spread throughout the bacterial genomes. These enzymes can be potential targets of new antibacterial drugs useful for the treatment of socially important diseases such as tuberculosis. In this study, ESTPKs of pathogenic, probiotic, and antibiotic-producing Gram-positive bacteria were classified according to the physicochemical properties of amino acid residues in the ATP-binding site of the enzyme. Nine residues were identified that line the surface of the adenine-binding pocket, and ESTPKs were classified based on these signatures. Twenty groups were discovered, five of them containing >10 representatives. The two most abundant groups contained >150 protein kinases that belong to the various branches of the phylogenetic tree, whereas certain groups are genus- or even species-specific. Homology modeling of the typical representatives of each group revealed that the classification is reliable, and the differences between the protein kinase ATP-binding pockets predicted based on their signatures are apparent in their structure. The classification is expected to be useful for the selection of targets for new anti-infective drugs., (Copyright © 2012 Wiley Periodicals, Inc.)

The review summarizes the data on the Mycobacterium tuberculosis mutations that lead to multidrug resistance (MDR) to various antibiotics. MDR strains arose over the past 30 years as a variety of antituberculosis drugs were introduced in medicine, and they largely discount the results of chemotherapy for tuberculosis. The most dangerous of them are strains with extensive drug resistance (XDR), which are resistant to four or five different drugs on average. The molecular mechanisms that make a strain resistant are considered. XDR and MDR strains result from successive and usually independent resistance mutations, which arise in various regions of the mycobacterial genome. In addition, the formation of resistant strains is affected by the phenomenon of tolerance and mycobacterial latency in infected tissues.

Phosphorylation is the universal regulatory mechanism in key physiological processes such as development, cell differentiation, proliferation, survival and malignant transformation. In this review we analyze serine/threonine protein kinases of the Pim (proviral integration of Moloney virus) family that have been initially discovered in experimental lymphomas. We provide data on gene structure, evolution, functions and substrates of Pim protein kinases. Focusing on Pim-1 as the major isoform, we analyze its role in the biology of hematopoietic malignancies. Pim-1 is a pro-proliferative and pro-survival protein kinase. It is constitutively active due to autophosphorylation, and its downstream partners positively regulate the cell cycle. Pim-1 cooperates with c-Myc oncoprotein in leukemogenesis; furthermore, Pim-1, like the Akt protein kinase, prevents cell death. Thus, Pim kinases are regarded as new therapeutic targets. Finally, we present an original test system f or screening of Pim inhibitors. In this test system the growth of a genetically engineered Escherichia coli strain in the presence of kanamycin is dependent on the phosphorylation of aminoglycoside-3' phosphotransferase VIII by Pim-1: pharmacological inhibition of this phosphorylation increases the bacterial cell lysis.

Objective: Glycogen synthase kinase 3 (GSK-3) is a promising target for the treatment of various human diseases such as type 2 diabetes, Alzheimer's disease and inflammation. Successful inhibition of the homologues of this kinase in Plasmodium falciparum, Trypanosoma brucei and Leishmania donovani makes the kinase an attractive target for the treatment of malaria, trypanosomiasis and leishmaniasis, respectively. The aim of this work was to compare the binding sites of the GSK-3 kinases of different parasites and to analyse them as possible targets for therapeutic compounds., Methods: Both a sequence alignment and homology models of the structure of 21 different GSK-3 homologues belonging to mammals, insects, pathogenic fungi, nematodes, trematodes and protozoa have been analysed, 17 of them being studied for the first time., Results: The structure of the kinases and, in particular, their binding sites, were found to be rather conserved, possessing small insertions or deletions and conserved amino acid substitutions. Nevertheless, the kinases of most species of parasite did have some amino acid differences from the human kinase, which could be exploited for the design of selective drugs., Conclusion: Comparison of the human and parasite GSK-3 ATP binding site models has shown that the development of selective drugs affecting parasite GSK-3 is possible. Known inhibitors of human GSK-3 can also be used as starting scaffolds for the search for drugs acting against parasitic diseases.

Signaling through protein kinases is an evolutionary conserved, widespread language of biological regulation. The eukaryotic type serine-threonine protein kinases (STPKs) found in normal human microbiote and in pathogenic bacteria play a key role in regulation of microbial survival, virulence and pathogenicity. Therefore, down-regulation of bacterial STPKs emerges as an attractive approach to cure infections. In this review we focused on actinobacterial STPKs to demonstrate that these enzymes can be used for crystal structure studies, modeling of 3D structure, construction of test systems and design of novel chemical libraries of low molecule as weight inhibitors. In particular, the prototypic pharmacological antagonists of Mycobacterium tuberculosis STPKs are perspective for development of a novel generation of drugs to combat the socially important disease. These inhibitors may modulate both actinobacterial and host STPKs and trigger programmed death of pathogenic bacteria.

The species and strain genetic diversity of bacterial cultures belonging to the genus Lactobacillus, which were isolated from the gastrointestinal microbiome of the human population living in the former Soviet Union in the years 1960-1980, was studied. The bacteria demonstrated probiotic characteristics. Phylogenetic analysis of sequences of the gene coding for 16S rRNA detected earlier by us, showed that the gene found in bacteria isolated from the intestinal content of healthy adults and represented by species L. plantarum, L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum has high homology (97-100%) with this gene in type representatives of the species. The genotypic and strain diversity of cultures was studied using RAPD-PCR and nonspecific primers. This method with the use of the ERIC-1 primer gave reliable and reproducible results as compared that using with M13 and MSP primers and allowed the identification of examined bacteria belonging to the genus Lactobacillus at the level of species and certification at the strain level.

Thirteen strains of industrial bacterial cultures of the genus Lactobacillus (from a collection of Gabrichevsky Research Institute of Epidemiology and Microbiology) were studied. These strains were used for decades in Russian Federation for food and drug production, as ferments for lactic acid products, for production of probiotics, biologically active and veterinary preparations. Complex analysis of data on cultures obtained using microbiological and molecular-genetic methods was conducted for the first time. Biochemical characteristics of these cultures were studied and the sequence of the proximal region of 16S ribosomal RNA gene was determined. The employment of the test system API-50CHL was shown to broaden the opportunities of a more accurate biochemical identification of bacteria belonging to the genus Lactobacillus, in comparison with the set ANAEROTEST-23. According to the results obtained in a comparative analysis of nucleotide sequences of 16S rRNA gene, all strains examined show 97-99% homology of the proximal region of this gene with that of the type representatives of studied species. These data allowed taxonomic reclassification of the species position of cultures with consideration of the more advanced level of systematics. Nucleotide sequences of gene fragments of examined lactobacilli strains were recorded in NCBI database (accession numbers of deposits GU560031, GU560032, GU560033, GU560034, GU560035, GU560036, GU560037, GU560038, GU560039, GU560040, GU560041, GU560042, GU560043).

Aim: To study strains of bacteria from Lactobacillus genus using combination of microbiological and molecular biological methods in order to define more accurately their systematic position and biochemical characteristics., Materials and Methods: Thirteen cultures of Lactobacillus bacteria isolated from stool of healthy persons were studied: L. plantarum CS 396, L. plantarum 8-PA-3, L. plantarum 421-2, L. fermentum 90-TC-4, L. delbrueckii gKNM 101, L. delbrueckii gKNM 526, L. acidophilus Er 317/402 NARINE, L. acidophilus 100 ash, L. acidophilus NK-1, L. acidophilus NNIE, L. acidophilus K3sh24, L. brevis gKNM 23 11, L. casei gKNM 577. Their enzymatic activity relative to 50 sugars was studied using API-50 system. Structure of proximal region of 16S rRNA gene was studied also., Results: According to results of 16S rRNA gene sequence analysis strains were divided on 2 groups: 1) L. casei gKNM 577, L. plantarum 8-PA-3, L. plantarum CS 396, which species belonging corresponded to stated description. Comparison of nucleotide sequence of 16S rRNA gene of group 2 strains with nucleotide sequences database revealed that cultures NK-1, Er315/402 NARINE, 100 ash, NNIE identified early as L. acidophilus belong to species L. helveticus; L. brevis gKNM 23 and L. acidophilus K3sh24--to group L. casei/paracasei, L. delbrueckii gKNM 101 and L. fermentum 90-TC-4--to L. plantarum, L. delbrueckii gKNM 526--to L. fermentum, and L. plantarum 421-2--to L. rhamnosus., Conclusion: Obtained data allowed to perform taxonomic reclassification of species belonging of studied probiotic cultures of lactobacilli according to modem level of systematic of bacteria.

An efficient test system for serine/threonine protein kinase inhibitors screening has been developed based on theE. coliprotein system APHVIII/Pk25. Phosphorylation of aminoglycoside phosphotransferase VIII (APHVIII) by protein kinases enhances resistance of the bacterial cell to aminoglycoside antibiotics, e.g. kanamycin. Addition of protein kinase inhibitors prevents phosphorylation and increases cell sensitivity to kanamycin. We have obtained modifications of APHVIII in which phosphorylatable Ser146 was encompassed into the canonical autophosphorylation sequence ofStreptomyces coelicolorPk25 protein kinase. Mutant and wild-typeaphVIII were cloned intoE. coliwith the catalytic domain ofpk25. As a result of the expression of these genes, accumulation of corresponding proteins was clearly observed. Extracted from bacterial lysates, Pk25 demonstrated its ability to autophosphorylate. It was shown that variants ofE. colicontaining bothaphVIIIand рк25were more resistant to kanamycin than those carrying onlyaphVIII. Protein kinase inhibitors of the indolylmaleimide class actively inhibited Pk25 and reduced cell resistance to kanamycin. Modeling of APHVIII and Pk25 3D structures showed that pSer146 is an analog of phosphoserine in the ribose pocket of protein kinase A. Pk25 conformation was similar to that of РknB ofMycobacterium tuberculosis. Potential indolylmaleimide inhibitors were docked into the ATP-binding pocket of Pk25. The designed test system can be used for the primary selection of ATP-competitive small molecule protein kinase inhibitors.