Single nucleotide polymorphisms of Beijing lineage Mycobacterium tuberculosis toxin-antitoxin system genes: Their role in the changes of protein activity and evolution.

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 mc2 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.

Species-specific serine-threonine protein kinase Pkb2 of Bifidobacterium longum subsp. longum: Genetic environment and substrate specificity.

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.

Isolation and Purification of Recombinant Serine/Threonine Protein Kinases of the Strain Bifidobacterium longum B379M and Investigation of Their Activity.

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.

FoF1-ATP synthase of Streptomyces fradiae ATCC 19609: structural, biochemical, and functional characterization.

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 ß- 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, ß, 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-, ß-, α- and ε-subunits. The ß-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.

[Distribution of genes of toxin-antitoxin systems of mazEF and relBE families in bifidobacteria from human intestinal microbiota].

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.

Identification of phosphorylation sites in aminoglycoside phosphotransferase VIII from Streptomyces rimosus.

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.

[Genetic instability of probiotic characteristics in the Bifidobacterium longum subsp. longum B379M strain during cultivation and maintenance].

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.

[The Pim family of protein kinases: structure, functions and roles in hematopoietic malignancies].

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.

New Test System for Serine/Threonine Protein Kinase Inhibitors Screening: E. coli APHVIII/Pk25 design.

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.