11 research outputs found

    Effects of <i>Tt</i>Ago on plasmid DNA and plasmid encoded RNA.

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    <p>A, Schematic representation of the <i>Escherichia coli-T</i>. <i>thermophilus</i> shuttle vector pMKPnqosGFP. Ori/MCS indicates the <i>E</i>. <i>coli</i> origin of replication (Ori) and a multiple cloning site (MCS). Note that cloning of this plasmid resulted in insertion of (incomplete) TTC1921 and TTHV050 genes. B, Relative plasmid content of <i>T</i>. <i>thermophilus</i> strains HB27 and HB27Δ<i>ago</i> transformed with pMKPnqosGFP. Plasmid content was calculated from the complete DNA isolated from biological triplicates at an OD<sub>600 nm</sub> of 0.5. C, Gene expression of plasmid encoded genes. Expression values are given in Fragments Per Kilobase of exon per Million fragments mapped (FPKM).</p

    Scatterplot of A = 1/a versus the promiscuity π for every domain.

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    <p>Domains with smaller slope <i>a</i>, and thus larger <i>A</i>, tend to be absolutely promiscuous. The intercept gives a measure of the degree of promiscuity: the larger the <i>p</i>, the smaller the promiscuity.</p

    Genes differentially expressed in HB27+P compared to HB27.

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    <p>*: Fold-change increase in RNA levels in HB27+P compared to HB27. For all changes <i>P</i><0.02.</p><p>**: Function predicted based on domains and similarity to other genes.</p><p>Genes differentially expressed in HB27+P compared to HB27.</p

    Assessing the Metabolic Diversity of <i>Streptococcus - Fig 3 </i> from a Protein Domain Point of View

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    <p>A) Distribution of functional pathways across different strains B) Score plot of principal component analysis performed on the metabolic diversity matrix. C) Hierarchical clustering performed on the metabolic diversity matrix. Strains cluster on the basis of their capability of performing certain metabolic functions.</p

    Δ<i>ago</i> result in stochastic changes in gene expression in <i>T</i>. <i>thermophilus</i> strains.

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    <p>A, Schematic representation of the gene regions encoding <i>Tt</i>Ago (TT_P0026) of <i>T</i>. <i>thermophilus</i> strain HB27 and HB27Δ<i>ago</i>. B, Schematic representation of the gene regions encoding TTB068 in <i>T</i>. <i>thermophilus</i> strain HB8. As no information on how the HB8 <i>ago</i> knockout was generated is available [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124880#pone.0124880.ref021" target="_blank">21</a>], HB8Δ<i>ago</i> is not displayed. HB8 genes colored grey and white are homologous to the HB27 genes indicated in Fig 1A. C, Expression of genes located near <i>ago</i> (TT_P0026) on the genome. Expression values are given in Fragments Per Kilobase of exon per Million fragments mapped (FPKM). D, Overlap in >2-fold up-regulated (▲) and >2-fold down-regulated (▼) homologous genes in HB27Δ<i>ago</i> relative to HB27, and HB8Δ<i>ago</i> relative to HB8.</p

    CRISPR loci and cas genes encoded by <i>T</i>. <i>thermophilus</i> HB27.

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    <p>A, Schematic representation of CRISPR loci and <i>cas</i> genes encoded on mega-plasmid pTT27 and the <i>T</i>. <i>thermophilus</i> HB27 chromosome. Encoded protein and KEGG annotation are given below each gene. Note that size of illustrated genes do not correspond to their actual size. CRISPR loci with type I and III repeats are colored gray and black, respectively. Repeat types are based on [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124880#pone.0124880.ref028" target="_blank">28</a>] and should not be confused with CRISPR-Cas Types [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124880#pone.0124880.ref029" target="_blank">29</a>]. Transp: Transposase. Hyp: Hypothetical protein. B, Characteristics of CRISPR loci encoded by <i>T</i>. <i>thermophilus</i> HB27. Fold change in CRISPR RNA levels is shown for HB27+P compared to HB27.</p

    Effects of Argonaute on Gene Expression in <i>Thermus thermophilus</i>

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    <div><p>Background</p><p>Eukaryotic Argonaute proteins mediate RNA-guided RNA interference, allowing both regulation of host gene expression and defense against invading mobile genetic elements. Recently, it has become evident that prokaryotic Argonaute homologs mediate DNA-guided DNA interference, and play a role in host defense. Argonaute of the bacterium <i>Thermus thermophilus</i> (<i>Tt</i>Ago) targets invading plasmid DNA during and after transformation. Using small interfering DNA guides, <i>Tt</i>Ago can cleave single and double stranded DNAs. Although <i>Tt</i>Ago additionally has been demonstrated to cleave RNA targets complementary to its DNA guide <i>in vitro</i>, RNA targeting by <i>Tt</i>Ago has not been demonstrated <i>in vivo</i>.</p><p>Methods</p><p>To investigate if <i>Tt</i>Ago also has the potential to control RNA levels, we analyzed RNA-seq data derived from cultures of four <i>T</i>. <i>thermophilus</i> strain HB27 variants: wild type, <i>Tt</i>Ago knockout (Δ<i>ago</i>), and either strain transformed with a plasmid. Additionally we determined the effect of <i>Tt</i>Ago on expression of plasmid-encoded RNA and plasmid DNA levels.</p><p>Results</p><p>In the absence of exogenous DNA (plasmid), <i>Tt</i>Ago presence or absence had no effect on gene expression levels. When plasmid DNA is present, <i>Tt</i>Ago reduces plasmid DNA levels 4-fold, and a corresponding reduction of plasmid gene transcript levels was observed. We therefore conclude that <i>Tt</i>Ago interferes with plasmid DNA, but not with plasmid-encoded RNA. Interestingly, <i>Tt</i>Ago presence stimulates expression of specific endogenous genes, but only when exogenous plasmid DNA was present. Specifically, the presence of <i>Tt</i>Ago directly or indirectly stimulates expression of CRISPR loci and associated genes, some of which are involved in CRISPR adaptation. This suggests that <i>Tt</i>Ago-mediated interference with plasmid DNA stimulates CRISPR adaptation.</p></div

    Two domains IPR000421 and IPR000115 (Panels A and B, respectively) exemplify the effect of sample size on the domain promiscuity definition.

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    <p>Domain A shows a decrease in promiscuity depending on the number of strains used for calculation. Domain B has a stable promiscuity, independent of the number of strains used for calculation. Panel C shows a graphical illustration of the concept of absolute promiscuity. In this cartoon, domain A is absolutely promiscuous as it has always the same neighbouring partners, which is not the case of domain B.</p
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