18 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

    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

    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

    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

    Additional file 1: of Engineering Geobacillus thermodenitrificans to introduce cellulolytic activity; expression of native and heterologous cellulase genes

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    Table S1. Codon harmonization of celS from C. thermocellum. An overview of the codon harmonization method used to adapt the C. thermocellum derived exoglucanase encoding gene celS for expression in G. thermodenitrificans. (XLSX 78 kb

    Additional file 4: of Engineering Geobacillus thermodenitrificans to introduce cellulolytic activity; expression of native and heterologous cellulase genes

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    Figure S2. Congo red assays of cellulase expressing G. thermodenitrificans cultures. Congo red assays of G. thermodenitrificans cultures grown on LB2 medium with 1% carboxymethylcellulose. Each culture produces a different cellulase. Ø: empty plasmid (pNW33n) control; CelA: GH5 endoglucanase CelA (Geobacillus 70PC53); CelK: GH9 exoglucanase (C. thermocellum); GE40wt: GH5 endoglucanase (Geobacillus metagenome derived) containing its native signal peptide; GE40: GH5 endoglucanase (Geobacillus metagenome derived). (PDF 48 kb

    Additional file 2: Figure S2. of Biochemical characterization of the xylan hydrolysis profile of the extracellular endo-xylanase from Geobacillus thermodenitrificans T12

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    10% SDS-PAGE of purified endo-xylanase from G. thermodenitrificans T12 followed by PageBlue staining. Lane 1: Protein marker; Lane 2: Pellet fraction; Lane 3: cell-free extract; Lane 4: non-binding protein fraction from FPLC; Lane 5: Purified recombinant GtXynA1; Lane 6: Purified and desalted recombinant GtXynA1; Lane 7: Protein marker. (DOCX 614 kb

    Additional file 1: Figure S1. of Biochemical characterization of the xylan hydrolysis profile of the extracellular endo-xylanase from Geobacillus thermodenitrificans T12

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    FPLC purification of GtXynA1. Protein fraction not bound to the nickel column eluted with the first 65 mL eluent. Bound GtXynA1 protein was removed from the nickel column using a imidazole gradient (yellow line) which increased from 0 mM to 500 mM over a time span of 20 min (A). Fractions 22–25 were pooled and used for desalting the purified GtXynA1 (B). Fractions 8–10 of the desalting column were pooled and then used for further experiments. (DOCX 311 kb
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