Effects of Argonaute on Gene Expression in Thermus thermophilus

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