Journal: Cell Rep / Year: 2023 Title: Phage T3 overcomes the BREX defense through SAM cleavage and inhibition of SAM synthesis by SAM lyase. Authors: Aleksandr Andriianov / Silvia Trigüis / Alena Drobiazko / Nicolas Sierro / Nikolai V Ivanov / Maria Selmer / Konstantin Severinov / Artem Isaev / Abstract: 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 ...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.
Average exposure time: 3 sec. / Electron dose: 47.75 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5447
EM imaging optics
Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scans
Sampling size: 5 µm / Width: 5760 / Height: 4092
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Processing
EM software
ID
Name
Version
Category
1
cryoSPARC
3
particleselection
2
EPU
2.11
imageacquisition
4
cryoSPARC
3
CTFcorrection
7
Coot
0.9.3
modelfitting
9
PHENIX
1.20.1-4487
modelrefinement
10
cryoSPARC
3
initialEulerassignment
11
cryoSPARC
3
finalEulerassignment
12
cryoSPARC
3
classification
13
cryoSPARC
3
3Dreconstruction
CTF correction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
Num. of particles selected: 1445186
Symmetry
Point symmetry: D2 (2x2 fold dihedral)
3D reconstruction
Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 244912 / Num. of class averages: 1 / Symmetry type: POINT
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