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

Abstract

<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

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