48 research outputs found

    Size prediction for the <i>L</i>. <i>ruminis</i> pan- and core genomes.

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    <p>New gene numbers in the pan- and core genomes are plotted against the number of added <i>L</i>. <i>ruminis</i> genomes. Each fitted curve was generated using the median values for the number of new genes.</p

    Phylogenetic tree analysis of the <i>L</i>. <i>ruminis</i> genomes.

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    <p>Unrooted phylogenies of the <i>L</i>. <i>ruminis</i> genomes were based on the multiple sequence alignment of core proteins and constructed with the neighbor-joining tree-building algorithm (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175541#sec002" target="_blank">Materials and Methods</a> for details). Names of the <i>L</i>. <i>ruminis</i> strains from which each genome is derived are shown. Four distinct phyletic clades were identified based on host-gut source (human, bovine, porcine, or equine) and are indicated accordingly.</p

    Pan-genome of the nine <i>L</i>. <i>ruminis</i> strains.

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    <p>A flower-plot depicting the gene content in the pan- (4301), core (1234), and accessory (3067) genomes of <i>L</i>. <i>ruminis</i> is shown. The number of strain-specific genes per genome is shown in the flower petals. Names for each <i>L</i>. <i>ruminis</i> strain are indicated. Annotated core and accessory genes are provided in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175541#pone.0175541.s001" target="_blank">S1 Table</a>.</p

    Schematic outline of the putative homolactic and heterolactic fermentative pathways in <i>L</i>. <i>ruminis</i>.

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    <p>Depicted is the glycolytic metabolism that can occur during homolactic fermentation via the Embden-Meyerhof-Parnas (EMP) pathway <b>(A)</b> or heterolactic fermentation via the phosphoketolase pathway (PK) <b>(B)</b>. Included are the various pathway reactions with their precursor metabolites and enzymes. Shown in the insets are the names of the glycolytic enzymes, either numbered (1–11) or alphabetized (a-e) along with the locus tags of their putative genes in the nine <i>L</i>. <i>ruminis</i> genomes. Enzymes that overlap in both pathways are numbered similarly.</p

    Schematic representation of the fumarate reductase-catalyzed reaction.

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    <p>Shown is the reaction catalyzed by fumarate reductase, where the reduction of fumarate to succinate is coupled with the oxidation of menaquinol to menaquinone. Included are the nine locus tags for the putative fumarate reductase gene found present in the <i>L</i>. <i>ruminis</i> core genome.</p

    Binding of recombinant SpaFED-piliated lactococcal cells to ECM proteins.

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    <p><i>In vitro</i> binding specificities between the normalized (OD600 = 0.5) cultures of recombinant WT (GRS1189) and SpaF pilin-deleted (GRS1126) SpaFED-piliated lactococci (along with GRS71 and GRS1052 cells as controls) and the fibronectin (<b>A</b>), collagen I (<b>B</b>), and collagen IV (<b>C</b>) proteins were evaluated using the procedure described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113922#s4" target="_blank">Materials and Methods</a>. Triplicate measurements were made for each of the ECM protein experiments, each of which was performed independently twice. SEM is indicated as error bars. For all ECM proteins tested, differences between the GRS1189 and GRS1226 binding results from pairwise comparisons against the GRS71 data are regarded very significant (<i>P</i>≤0.005).</p

    Effect of cell-to-cell contacts on TLR2-induced NF-κB activation by recombinant SpaFED-piliated lactococci.

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    <p>Using a Transwell membrane-segregated system, HEK-TLR2 cells were treated with non-partitioned (−) and partitioned (+) normalized cultures of recombinant WT (GRS1189) and SpaF pilin-deleted (GRS1126) SpaFED-piliated lactococci (MOI 100). Monitoring of TLR2-dependent NF-κB activation was carried out as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113922#s4" target="_blank">Materials and Methods</a>. Included as controls were GRS71 and GRS1052 cells (MOI 100), DMEM cell-culture medium, and Pam3CSK4 (1 ng/ml). Triplicate measurements were taken for a single experiment. SEM is indicated as error bars.</p

    Stimulation of TLR2-dependent NF-κB activation by recombinant SpaFED-piliated lactococci.

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    <p>The HEK-TLR2 cell line was treated with live (−) or heat-treated (100°C for 10 minutes) (+) normalized cultures of recombinant WT (GRS1189) and SpaF pilin-deleted (GRS1126) SpaFED-piliated lactococci (MOI 100). Levels of TLR2-dependent NF-κB activation were assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113922#s4" target="_blank">Materials and Methods</a>. Testing of the GRS71 and GRS1052 control strains was as well conducted. DMEM cell-culture medium and a TLR2-agonist lipopeptide (Pam3CSK4; 1 ng/ml) served as negative and positive controls, respectively. Quadruplicate measurements were taken for two independent experiments. SEM is shown as error bars. Pairwise differences between the GRS1189 and GRS1226 data (without heat inactivation) are considered very significant (<i>P</i>≤0.005).</p

    Stimulation of DC-cytokine production by recombinant SpaFED-piliated lactococci.

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    <p>Human monocyte-derived dendritic cells (moDCs) were treated with normalized cultures of recombinant WT (GRS1189) and SpaF pilin-deleted (GRS1126) SpaFED-piliated lactococci (MOI 50). Stimulated TNF-α (<b>A</b>), IL-12 (<b>B</b>), and IL-10 (<b>C</b>) cytokine production was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113922#s4" target="_blank">Materials and Methods</a>. GRS71 and GRS1052 cells (MOI 50) and DMEM cell-culture medium were included as controls. Measurements were performed in triplicate using moDCs from four new and different donors each time. SEM is shown as error bars. For all tested cytokines, differences in a pairwise comparison between the GRS1189 and GRS1226 data are judged not significant (<i>P</i>≥0.05).</p

    Immunoblot analysis of recombinant SpaFED-piliated lactococcal cells.

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    <p>Immunoblots of lactococcal cells corresponding to the empty vector GRS1052 clone (lane 1) and those to the nisin-induced WT (GRS1189; lane 2) and SpaF pilin-deleted (GRS1126; lane 3) SpaFED-piliated clones were probed separately with polyclonal anti-SpaD (<b>A</b>), anti-SpaE (<b>B</b>), and anti-SpaF (<b>C</b>) sera as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113922#s4" target="_blank">Materials and Methods</a>. Apparent positioning of monomeric SpaD, SpaE, and SpaF proteins is indicated on the right of each immunoblot by an asterisk. A dense ladder-like smear of high-molecular-weight (HMW) protein bands represents the longest lengths of pili and these are indicated on the top left of the immunoblot. The positions and sizes of the molecular weight markers are shown along the left side of the immunoblot.</p
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