T5-like phage BF23 evades host-mediated DNA restriction and methylation.

Bacteriophage BF23 is a close relative of phage T5, a prototypical Tequintavirus that infects Escherichia coli. BF23 was isolated in the middle of the XXth century and was extensively studied as a model object. Like T5, BF23 carries long ∼9.7 kb terminal repeats, injects its genome into infected cell in a two-stage process, and carries multiple specific nicks in its double-stranded genomic DNA. The two phages rely on different host secondary receptors-FhuA (T5) and BtuB (BF23). Only short fragments of the BF23 genome, including the region encoding receptor interacting proteins, have been determined. Here, we report the full genomic sequence of BF23 and describe the protein content of its virion. T5-like phages represent a unique group that resist restriction by most nuclease-based host immunity systems. We show that BF23, like other Tequintavirus phages, resist Types I/II/III restriction-modification host immunity systems if their recognition sites are located outside the terminal repeats. We also demonstrate that the BF23 avoids host-mediated methylation. We propose that inhibition of methylation is a common feature of Tequintavirus and Epseptimavirus genera phages, that is not, however, associated with their antirestriction activity.

Phage T3 overcomes the BREX defense through SAM cleavage and inhibition of SAM synthesis by SAM lyase.

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.