The genus Bifidobacterium is one of the most predominant bacterial populations in human-gut microbiota. Despite the increasing number of studies on the beneficial properties of bifidobacteria for human health, knowledge about their antioxidant potential is still insufficient. The role of the antioxidant potential of bifidobacteria in maintaining the homeostasis of the intestinal microbiota of the host organism as a whole is an important task that requires solutions. For the first time, this paper presents the data of genomic, transcriptome, and proteomic analyses of Bifidobacterium longum subsp. infantis ATCC 15697 strain after the action of oxidative stress. A growth culture of the strain is exposed to hydrogen peroxide for 2 hours and oxygen for 2 and 4 hours. Preliminary genome analysis of the strain shows the presence of 17 genes encoding a known protein with antioxidant function, as in other genomes of B. longum subsp. infantis available in the international database NCBI. Complete transcriptome analysis reveals an increase in the transcript levels by more than two times for 6 genes with a known antioxidant function. The data of quantitative proteomic analysis shows an increase in protein levels by more than two times for five enzymes with a known antioxidant function. Over 28 other proteins with levels increased by more than two times are identified in the cells of the growth culture in response to the long-term action of oxygen. These proteins can be involved in the processes of the cell’s response to stress, amino-acid- and nucleotide metabolism, and transport processes. Six proteins with unknown functions, which may play a significant role in the antioxidant response of anaerobic bifidobacteria, are found to have high levels in the cells after the action of stress. The obtained data are supposed to be used in the selection of B. longum subsp. infantis strains and the creation of pharmabiotics able to correct the composition of the microbiota.

Currently, the world is undergoing revolutionary changes in the development and use of pharmacological preparations based on bacteria and their biologically active components. The development of pharmabiotics, live biotherapeutic preparations and/or their metabolites and components with established pharmacological ingredients and mechanism of the action and aimed at the treatment of specific nosologies, becomes the most promising. When creating pharmabiotics, in addition to traditional microbiological and biotechnological approaches, a complex of omics technologies, genomic, transcriptomic, and proteomic, is used. In the presented work, these technologies were used to characterize the Limosilactobacillus fermentum U-21 strain previously selected for a number of unique antioxidant properties. Genomic analysis of the strain allowed us to identify 29 genes the products of which can exhibit antioxidant properties, including those toward the body of the studied animals. Genes of the thioredoxin complex and metabolism and transport of heavy metals may be the most important. Hydrogen peroxide was used as an inducer of oxidative stress. An increase in expression of 380 genes and a more than twofold decrease in expression of 370 genes were demonstrated. Genes of a putative urea carboxylase operon showed the greatest increase in expression (by 14–24 times). Changes in expression of genes of transport, including Fe2+ and Cu2+ metal ions, as well as of the synthesis and catabolism of some amino acids, are important for subsequent studies. Proteomic analysis of the exoproteome of the strain revealed the ClpB chaperone complex protein, which can play a key role in refolding of proteins misfolded as a result of oxidative stress in various tissues and organs of the animal body. The use of a complex of omics technologies to characterize the therapeutic properties and mechanism of the action of the L. fermentum U-21 strain is one of the first examples in this field of research.

The strain Streptomyces xinghaiensis ( fradiae ) ATCC19609 is sensitive to antibiotics of various chemical classes and is hypersensitive to the macrolide antibiotic oligomycin A. The spontaneous mutant S. fradiae -nitR + bld, resistant to nitron-oligomycin, carries a mutation in the gene of the PadR transcriptional regulator. A comparative proteomic analysis of the inverted membrane vesicles of the mutant strain and wild-type S. xinghaiensis ( fradiae ) ATCC19609 was performed. Using mass spectrometric analysis, quantitative changes in the protein fractions of two ABC transporters, leucyl aminopeptidase, alkaline phosphatase, and alanine dehydrogenase were detected in vesicles of the mutant strain compared with wild-type vesicles. As a result of transcriptional analysis, an increase in the expression levels of the genes of ABC transporters, alkaline phosphatase, and leucyl aminopeptidase and a decrease in the expression level of the alanine dehydrogenase gene in the mutant strain S. fradiae -nitR + bld compared with the wild-type strain were recorded. This suggested that the padR gene, in which the mutation was detected, may be involved in the regulation of antibiotic resistance and differentiation.

Streptomyces rimosus ATCC 10970 contains 14 genes annotated as aminoglycoside phosphotransferases in its genome: aphSR1–aphSR14. We have previously shown that the aphVIII (aphSR5) and aph(3'')-Id (aphSR3) genes, when cloning in E. coli, cause resistance to kanamycin, neomycin, paromomycin, and streptomycin. It was found for Aph(3')-VIII that antibiotic resistance increased after phosphorylation at the Ser146 motif in the active site of the enzyme by serine-threonine protein kinases (STPKs). The aphSR2 gene, when cloning in E. coli, causes resistance to neomycin and hygromycin. In this work, in order to assess the possibility of influence of STPK genes on increasing resistance to aminoglycoside antibiotics, we performed a combined cloning into E. coli at pET32a of the aphSR2 gene and the STPK genes (pkSR1 and pkSR2) localized in one cluster of the S. rimosus ATCC 10970 genome. We detected that, in the construction E. coli/aphSR2/pkSR1, there is a 2-fold increase in resistance to neomycin. The presented data are the second example of the STPK effect on the modulation of the level of resistance to aminoglycoside antibiotics in bacteria of the genus Streptomyces.

The objective of this study was to determine for phosphorylated substrates of the species-specific serinethreonine 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; ABCtransporters; molecular chaperone GroEL; and glutamine synthase, GInAl. All identified proteins were considered moonlighting proteins. Three out of 11 proteins (glutamine synthetase GInAl 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. (C) 2018 Elsevier Ltd. All rights reserved.

Previously, in the strain Streptomyces rimosus ATCC10970 (producer of oxytetracycline), the aminoglycoside phosphotransferase AphVIII, determining kanamycin, neomycin, and paromomycin resistance, was identified and characterized. Recently, the authors obtained the 3D structure of AphVIII. The 14 aph genes, including gene aphVIII, were annotated when the genome of S. rimosus ATCC10970 was sequenced. In the present study, a new aph(3'')-Id (aphSR3) gene encoding streptomycin phosphotansferase was first identified in the strain of S. rimosus ATCC10970 using bioinformatic and comparative phylogenetic analysis of the aphSR1-aphSR14 genes with the previously known aph genes from clinical isolates and producer strains of aminoglycoside antibiotics belonging to seven subfamilies. When cloning, it was found that the gene aphSR3 (aph(3'')-Id) in Escherichia coli causes resistance to streptomycin at a concentration of 150 μg/mL. The obtained data can be used in practical terms to study the distribution and features of the functions of genes that determine the natural resistance to aminoglycoside antibiotics in actinobacteria of the genus Streptomyces.

Туберкулез самая смертоносная бактериальная инфекция из известных человеку, при этом ее лечение осложнено появлением и быстрым распространением штаммов возбудителя, Mycobacterium tuberculosis, с множественной и широкой лекарственной устойчивостью (МЛУ и ШЛУ). В результате главным требованием к разрабатываемым противотуберкулезным препаратам является использование новых классов химических соединений, поражающих новые биомишени. Серин-треониновые протеинкиназы (СТПК) перспективные мишени, а аминопиридины и аминопири-мидины, ранее не применявшиеся в качестве противотуберкулезных препаратов, имеют предсказанную активность в отношении СТПК. В данной работе в тест-системе Mycobacterium smegmatis aphVIII+, предназначенной для отбора ингибиторов СТПК на клеточном уровне, был проведен скрининг 192 соединений двух указанных классов. Сначала отобрали 53 соединения с субингибирующей концентрацией до 100 нмоль/диск. Из них 22 соединения проявили активность в тест-системе как ингибиторы СТПК, которая была подтверждена in vitro на белке PknA М. tuberculosis (наивысшее значение показателя ингибирования 26,9 ± 6,1 %). Также отобранные соединения тестировали на токсичность in vitro на клетках фибробластов эмбриона человека с использованием МТТ-теста. В результате для дальнейших исследований в качестве новых препаратов для борьбы с МЛУ-туберкулезом были отобраны 3 ингибитора СТПК с относительно высокой активностью и относительно низкой токсичностью.

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.

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, ɛ, 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 ɛ-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.

Описано явление программированной гибели (ПГ) клеток мицелия Streptomyces lividans – отсроченного лизиса при действии производных индолилмалеимидов – ингибиторов серин-треониновых протеинкиназ (СТПК) актинобактерий. Отсроченный лизис мицелия сопровождается нарушением целостности ДНК, подобно феномену, установленному для ПГ дифференцирующегося мицелия S. lividans. Методами двумерного электрофореза и масс-спектрометрии идентифицированы белки, накапливающиеся в процессе ПГ. Большинство этих белков известны как стресс-индуцируемые. Модель отсроченного лизиса актинобактерий при ингибировании СТПК позволяет установить молекулярные механизмы особого вида ПГ прокариот и использовать эти механизмы в терапевтических целях.

Programmed death (PD) of the mycelium of Streptomyces lividans, namely, its delayed lysis in response to treatment with indolylmaleimide derivatives, which inhibit actinobacterial serine/threonine protein kinases (STPK), is described. Delayed lysis of mycelial cell was accompanied by DNA damage similar to PD in differentiating S. lividans mycelium. Two-dimensional electrophoresis and mass spectrometry were used to identify proteins up-regulated by a PD-inducing STPK inhibitor. Most of these proteins are known to be implicated in responses to various stress stimuli. Thus, our model of delayed cell lysis of actinobacteria upon STPK inhibition may serve for unveiling the molecular mechanisms of bacterial PD and for antimicrobial drug design.

Cells of a diploid line obtained from embryos with the Down’s syndrome, known to be unable to repair gamma-induced DNA damage, were treated with natural (garlic extract, retinol) and synthetic (crown compound) antimutagens and with adapting factors (heat shock, low CdCl2 concentrations, 10−8 M). The protective effect was evaluated by registering DNA breaks and cell survival, and the protection coefficients were calculated. The most effective results were obtained with the use of the garlic extract and retinol. No protection of the DNA structure was observed when cells were treated with low concentrations of cadmium chloride and then with high concentrations, i. e., no adaptive response (AR) was formed under these conditions. The spectrum of proteins in treated and control cells as well as detoxication genes (GSTM1, GSTT1, CYP1A1) were determined.