PAM and repeat-end correlation.

<p>(A): PAMs of observed spacers and the co-occurring trinucleotide repeat-ends associated with these spacers. Notice that the spacer-proximal nucleotide of the repeat end is identical to the protospacer-proximal nucleotide of the PAM. (B): Schematic of the proposed mechanism for spacer acquisition during CRISPR adaptation. A protospacer with specific PAM is selected after which it is processed into the pre-spacer (at least 33–34 bp), which contains the last nucleotide of the PAM (the pre-spacer could be single-stranded or double-stranded). The pre-spacer is than integrated at the leader proximal end of the CRISPR locus. The nucleotide derived from the PAM forms the last nucleotide of the repeat. (C): R-loop formation by mature crRNA (61 nucleotides) during CRISPR interference. Notice that the last nucleotide of the repeat (the nucleotide derived from the PAM) is complementary to the target DNA sequence. It remains unknown whether base-pairing between these nucleotides is important for interference.</p

Graphical representation of AG and GC contents of each observed and possible spacer.

<p>Observed spacers (⧫) are spacers integrated in CRISPR loci 2.1 and 2.3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035888#pone.0035888.s001" target="_blank">Table S1</a>). These spacers are 32 or 33-mers with various PAMs. Possible spacers (Ο) are all 32-mers found on pRSF-1b directly downstream of an AAG PAM.</p

Linear display of pRSF-1b and locations of protospacers.

<p>The (+) and (−) strands and corresponding protospacers are coloured red and black, respectively. Kanamycin marker (Kan), Origin of replication (Ori) and <i>lacI</i> (LacI) are shown as arrows. Protospacers have an AAG PAM unless indicated otherwise.</p

Model of the strand specific positive feedback loop.

<p>Cells with a spacer against a known and actively present invader DNA produce targeting Cascade complexes in the expression stage. In the interference stage, Cascade binds the target dsDNA after which the target is cleaved and degraded by Cas3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035888#pone.0035888-Westra1" target="_blank">[15]</a>. DNA degradation products generated by Cascade and Cas3 (which could be ssDNA or dsDNA) act as precursors for new spacers in the adaptation phase in a strand-specific manner. By integration of these strand-specific precursors, the spacer repertoire against an actively present invader is expanded, completing the positive feedback loop.</p

Effect of integrated spacers on retransformation efficiency.

<p>Transformation efficiencies of various PIMs and the control (Wild type <i>E. coli</i> K12 W3110) are given in a logarithmic scale as colony forming units (CFU) per µg of pRSF-1b plasmid DNA. For each PIM, the number of spacers integrated in either CRISPR locus 2.1 or 2.3 is given. All spacers have an AAG PAM, unless indicated otherwise. The exact spacer composition of each PIM is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035888#pone.0035888.s001" target="_blank">Table S1</a>.</p

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

<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_{600 nm} 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.

<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

Genes differentially expressed in HB27+P compared to HB27.

<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

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

<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>

<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