Random guide-independent DNA cleavage from the Argonaute of Exiguobacterium sp. AB2.

BACKGROUND

Bacteria and bacteriophages (phages) are locked in a coevolutionary "arms race" to outcompete one another with novel systems and strategies. Regularly outnumbered tenfold by phages, bacteria have responded to the constant threat of phage predation by evolving a vast array of sophisticated defense systems. Among these, prokaryotic Argonautes (pAgos) are nucleic acid-guided endonucleases that target complementary sequences of invading mobile genetic elements (MGEs). However, as the preference for targeting MGE sequences has been demonstrated in only a limited number of pAgos, their precise physiological functions remain elusive. Here, we discovered a pAgo in Exiguobacterium sp. AB2, EsAgo, encoded in close proximity to other putative defense systems on the E. AB2 genome. Such clustering into genomic "defense islands" is a common phenomenon among prokaryotic defense systems, further implicating pAgos with a role in host defense. Accordingly, we had sought to characterize EsAgo as a nucleic acid-guided nucleic acid-targeting nuclease against MGEs for bacterial defense in this study.

RESULTS

Using sequence to structure homology tools, we show that the predicted model of EsAgo exhibits the structural characteristics typical of a full-length, catalytically active, DNA-guided pAgo. Akin to other pAgos, EsAgo uses a divalent cation cofactor to indiscriminately "chop" plasmids in vitro. Furthermore, a site-directed double mutant of EsAgo bearing two missense mutations at the catalytic site exhibited significantly reduced levels of this random plasmid-degrading activity. Lastly, when EsAgo was supplied with synthetic 5'-P ssDNA guides, random nuclease activity was attenuated.

CONCLUSIONS

These findings suggest that EsAgo functions as a DNA-interfering nuclease with or without DNA guides. Within the cell, it is possible that EsAgo utilizes this mechanism to screen and destroy foreign genetic elements. Moreover, the potential capacity for specific dsDNA cleavage at moderate temperatures gives rise to intriguing possibilities of repurposing EsAgo as a programmable nuclease for future biotechnological use.