pAgo-induced DNA interference protects bacteria from invader DNA

Abstract

Members of the conservedArgonaute protein family use small RNA guides to find their mRNA targets to regulate gene expression and suppress mobile genetic elements in eukaryotes. Argonautes are also present in many bacterial and archaeal species 1-3 . Unlike eukaryotic proteins, several studied prokaryotic Argonautes use small DNA guides to cleave DNA, a process dubbed DNA interference 4-8 . However, the natural functions and targets of DNA interference are poorly understood and the mechanisms of DNA guide generation and target discrimination remain unknown. Here, we studied the in vivo activities of a bacterial Argonaute nuclease CbAgo and demonstrated that it induces cleavage of multicopy genetic elements, including plasmids, transposons and repetitive chromosomal loci. Generation of small DNA guides employed by CbAgo requires cooperation between its intrinsic endonuclease activity and the cellular double-strand break repair machinery. The mechanism of guide generation ensures that small DNA guides are enriched in sequences that target foreign DNA and endows CbAgo with capacity to eliminate plasmids and fight phage infection. Similar principles may underlie the specificity of self-nonself discrimination by diverse defense systems in prokaryotes.Argonaute (Ago) proteins are a ubiquitous family of guide-dependent nucleases found in all three domains of life 1,3,9,10 . Eukaryotic Ago proteins (eAgos) are RNA-dependent RNA nucleases that suppress gene expression by binding to their RNA targets in the cytoplasm or in the nucleus 11,12 . Prokaryotic Agos (pAgos) are extremely diverse in comparison with eAgos and are randomly distributed among bacterial and archaeal lineages suggesting their active spread by horizontal gene transfer 1-3 . In contrast to their eukaryotic counterparts, several studied pAgos were shown to act in vitro as nucleases with distinct specificity toward DNA targets [4][5][6]8,[13][14][15][16][17] . The majority of studied pAgos also prefer to use short single-stranded DNA molecules as guides for their endonuclease activity, however, the molecular mechanism of DNA guide generation in bacterial cells has remained poorly understood. Two pAgo proteins studied in vivo were shown to decrease plasmid content and transformation efficiency in bacterial cells 7,18 , leading to the hypothesis that pAgos use DNA interference to protect the cells against foreign DNA. However, the ability of pAgos to fight genuine

Bacterial Retrons Function In Anti-Phage Defense

“…RecBCD rapidly degrades linear dsDNA and hence phage DNA, which is usually injected into the infected cell in a linear form, is susceptible to RecBCD-mediated degradation (Dillingham and Kowalczykowski, 2008). RecBCD also plays an important role in acquisition of new CRISPR spacers from phage genomes (Levy et al, 2015) and in the formation of guide DNAs for prokaryotic argonaute proteins that defend against foreign nucleic acids (Kuzmenko et al, 2020). Because RecBCD is a central immunity hub in the bacterial cell, numerous phages Ec48, gp5.9 induced Ec48, gp5.9 uninduced NC, gp5.9 induced NC, gp5.9 uninduced Ec48, gp5.9 induced Ec48, gp5.9 uninduced NC, gp5.9 induced NC, gp5.9 uninduced…”

Retrons: Complementing CRISPR in Phage Defense

“…The RecBCD complex is known to participate in anti-phage activities through multiple mechanisms, including destroying the linear double-stranded DNA phage genome as it enters the cell, 7 assisting in the acquisition of new CRISPR spacers, 8 and promoting the formation of guide DNAs for bacterial argonaute proteins. 9 It appears that the Ec48 retron defense system characterized in this study acts as a guardian of the RecBCD complex, with the phage inhibitors triggering the retron system and causing cell suicide through an abortive infection mechanism (Fig. 1).…”

A thermostable DNA primase‐polymerase from a mobile genetic element involved in defence against environmental DNA

“…In any case, it is relevant to note that this putative activity of Ppol as guide‐provider needed for ThAgo could be a recruited property, and not the product of coevolved genes, since Ppol distribution does not fully match that of ThAgo proteins across the few sequenced Thermus spp genomes (Supporting Information Table S1), being more common the presence of TthAgo (specifically in megaplasmids) than that of Ppol. Actually, a more likely mechanism than DNA chopping for the generation of ssDNA guides in collaboration with recombination proteins has been shown recently for the Clostridium butyricum Ago protein in E. coli (Kuzmenko et al ., 2020). Also, the role of ThAgo in the physiology of Thermus seems to be much more relevant than DNA invasion defence as it has been involved in chromosomal segregation (Jolly et al ., 2019), and Δ ago mutants show a somehow stressed‐like phenotype differences respect to the wild type like an increased number of pili an motility (Blesa, 2016).…”