Viruses compete with each other for limited cellular resources, and some deliver defence mechanisms that protect the host from competing genetic parasites¹. The phage antirestriction induced system (PARIS) is a defence system, often encoded in viral genomes, that is composed of a 55 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB)². However, the mechanism by which AriA and AriB function in phage defence is unknown. Here we show that AriA and AriB assemble into a 425 kDa supramolecular immune complex. We use cryo-electron microscopy to determine the structure of this complex, thereby explaining how six molecules of AriA assemble into a propeller-shaped scaffold that coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving host lysine transfer RNA. Phage T5 subverts PARIS immunity through expression of a lysine transfer RNA variant that is not cleaved by PARIS, thereby restoring viral infection. Collectively, these data explain how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes for the recognition of foreign proteins, rather than foreign nucleic acids³.

Bacteriophages or phages are viruses that infect bacterial cells (for the scope of this review we will also consider viruses that infect Archaea). Constant threat of phage infection is a major force that shapes evolution of the microbial genomes. To withstand infection, bacteria had evolved numerous strategies to avoid recognition by phages or to directly interfere with phage propagation inside the cell. Classical molecular biology and genetic engineering have been deeply intertwined with the study of phages and host defenses. Nowadays, owing to the rise of phage therapy, broad application of CRISPR-Cas technologies, and development of bioinformatics approaches that facilitate discovery of new systems, phage biology experiences a revival. This review describes variety of strategies employed by microbes to counter phage infection, with a focus on novel systems discovered in recent years. First chapter covers defense associated with cell surface, role of small molecules, and innate immunity systems relying on DNA modification.

Bacteriophages or phages are viruses that infect bacterial cells (for the scope of this review we will also consider viruses that infect Archaea). The constant threat of phage infection is a major force that shapes evolution of microbial genomes. To withstand infection, bacteria had evolved numerous strategies to avoid recognition by phages or to directly interfere with phage propagation inside the cell. Classical molecular biology and genetic engineering had been deeply intertwined with the study of phages and host defenses. Nowadays, owing to the rise of phage therapy, broad application of CRISPR-Cas technologies, and development of bioinformatics approaches that facilitate discovery of new systems, phage biology experiences a revival. This review describes variety of strategies employed by microbes to counter phage infection. In the first part defense associated with cell surface, roles of small molecules, and innate immunity systems relying on DNA modification were discussed. The second part focuses on adaptive immunity systems, abortive infection mechanisms, defenses associated with mobile genetic elements, and novel systems discovered in recent years through metagenomic mining.

Metagenomics is a powerful tool to study marine microbial communities. However, obtaining high-quality environmental DNA suitable for downstream sequencing applications is a challenging task. The quality and quantity of isolated DNA heavily depend on the choice of purification procedure and the type of sample. Selection of an appropriate DNA isolation method for a new type of material often entails a lengthy trial and error process. Further, each DNA purification approach introduces biases and thus affects the composition of the studied community. To account for these problems and biases, we systematically investigated efficiency of DNA purification from three types of samples (water, sea sediment, and digestive tract of a model invertebrate Magallana gigas) with eight commercially available DNA isolation kits. For each kit-sample combination we measured the quantity of purified DNA, extent of DNA fragmentation, the presence of PCR-inhibiting contaminants, admixture of eukaryotic DNA, alpha-diversity, and reproducibility of the resulting community composition based on 16S rRNA amplicons sequencing. Additionally, we determined a “kitome”, e.g., a set of contaminating taxa inherent for each type of purification kit used. The resulting matrix of evaluated parameters allows one to select the best DNA purification procedure for a given type of sample.

Bacteriophage T3 encodes a SAMase that, through cleavage of S-adenosyl methionine (SAM), circumvents the SAM-dependent type I restriction-modification (R-M) defense. We show that SAMase also allows T3 to evade the BREX defense. Although SAM depletion weakly affects BREX methylation, it completely inhibits the defensive function of BREX, suggesting that SAM could be a co-factor for BREX-mediated exclusion of phage DNA, similar to its anti-defense role in type I R-M. The anti-BREX activity of T3 SAMase is mediated not just by enzymatic degradation of SAM but also by direct inhibition of MetK, the host SAM synthase. We present a 2.8 Å cryoelectron microscopy (cryo-EM) structure of the eight-subunit T3 SAMase-MetK complex. Structure-guided mutagenesis reveals that this interaction stabilizes T3 SAMase in vivo, further stimulating its anti-BREX activity. This work provides insights in the versatility of bacteriophage counterdefense mechanisms and highlights the role of SAM as a co-factor of diverse bacterial immunity systems.

Anti-phage systems of the BREX (BacteRiophage EXclusion) superfamily rely on site-specific epigenetic DNA methylation to discriminate between the host and invading DNA. We demonstrate that in Type I BREX systems, defense and methylation require BREX site DNA binding by the BrxX (PglX) methyltransferase employing S-adenosyl methionine as a cofactor. We determined 2.2-Å cryoEM structure of Escherichia coli BrxX bound to target dsDNA revealing molecular details of BREX DNA recognition. Structure-guided engineering of BrxX expands its DNA specificity and dramatically enhances phage defense. We show that BrxX alone does not methylate DNA, and BREX activity requires an assembly of a supramolecular BrxBCXZ immune complex. Finally, we present a cryoEM structure of BrxX bound to a phage-encoded inhibitor Ocr that sequesters BrxX in an inactive dimeric form. We propose that BrxX-mediated foreign DNA sensing is a necessary first step in activation of BREX defense.