Bacterial Argonaute nucleases reveal different modes of DNA targeting in vitro and in vivo

Prokaryotic Argonaute proteins (pAgos) are homologs of eukaryotic Argonautes (eAgos) and are also thought to play a role in cell defense against invaders. However, pAgos are much more diverse than eAgos and little is known about their functional activities and target specificities in vivo. Here, we describe five pAgos from mesophilic bacteria that act as programmable DNA endonucleases and analyze their ability to target chromosomal and invader DNA. In vitro, the analyzed proteins use small guide DNAs for precise cleavage of single-stranded DNA at a wide range of temperatures. Upon their expression in Escherichia coli, all five pAgos are loaded with small DNAs preferentially produced from plasmids and chromosomal regions of replication termination. One of the tested pAgos, EmaAgo from Exiguobacterium marinum, can induce DNA interference between homologous sequences resulting in targeted processing of multicopy plasmid and genomic elements. EmaAgo also protects bacteria from bacteriophage infection, by loading phage-derived guide DNAs and decreasing phage DNA content and phage titers. Thus, the ability of pAgos to target multicopy elements may be crucial for their protective function. The wide spectrum of pAgo activities suggests that they may have diverse functions in vivo and paves the way for their use in biotechnology.

Automated summary

In this study, five Prokaryotic Argonaute proteins (pAgos) from mesophilic bacteria are found to function as programmable DNA endonucleases that can target chromosomal and invader DNA. These pAgos are able to precisely cleave single-stranded DNA using small guide DNAs at a wide range of temperatures in vitro. They are loaded with small DNAs preferentially produced from plasmids and chromosomal regions upon expression in Escherichia coli. One particular pAgo, EmaAgo, can induce DNA interference between homologous sequences and protect bacteria from bacteriophage infection by loading phage-derived guide DNAs. The diverse activities of pAgos suggest potential diverse functions in vivo and potential applications in biotechnology.