Bacteriophage T3 encodes a SAMase that through cleavage of S-adenosyl-methionine (SAM) circumvents the SAM-dependent Type I Restriction-Modification defence of the host bacterium Escherichia coli. Here, we show that the SAMase also allows T3 to evade BREX defence. SAM degradation weakly affects BREX methylation of host DNA, but completely inhibits the defensive function of BREX, suggesting that SAM is required as a co-factor for BREX-mediated exclusion of phage DNA. The anti-BREX activity of the T3 SAMase is mediated by two independent mechanisms: enzymatic degradation of SAM and downregulation of SAM synthesis through direct inhibition of the host SAM synthase MetK. We determined a 2.8 Angstrom cryo-EM structure of the eight-subunit T3 SAMase-MetK complex. Structure guided mutagenesis of the SAMase-MetK interface revealed that the interaction with MetK stabilizes the T3 SAMase in vivo, thus further stimulating its anti-BREX activity. This work provides insights in the versatility and intricacy of bacteriophage counter-defence mechanisms and highlights the role of SAM as an important co-factor of diverse phage-defence systems.

No abstract

Bacteriophages—viruses that infect bacterial cells—are the most abundant biological entities on Earth. The use of phages in fundamental research and industry requires tools for precise manipulation of their genomes. Yet, compared to bacterial genome engineering, modification of phage genomes is challenging because of the lack of selective markers and thus requires laborious screenings of recombinant/mutated phage variants. The development of the CRISPR-Cas technologies allowed to solve this issue by the implementation of negative selection that eliminates the parental phage genomes. In this manuscript, we summarize current methods of phage genome engineering and their coupling with CRISPR-Cas technologies. We also provide examples of our successful application of these methods for introduction of specific insertions, deletions, and point mutations in the genomes of model Escherichia coli lytic phages T7, T5, and T3.

No abstract