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.

Structure and mechanism of a phage-encoded SAM lyase revises catalytic function of enzyme family.

The first S-adenosyl methionine (SAM) degrading enzyme (SAMase) was discovered in bacteriophage T3, as a counter-defense against the bacterial restriction-modification system, and annotated as a SAM hydrolase forming 5'-methyl-thioadenosine (MTA) and L-homoserine. From environmental phages, we recently discovered three SAMases with barely detectable sequence similarity to T3 SAMase and without homology to proteins of known structure. Here, we present the very first phage SAMase structures, in complex with a substrate analogue and the product MTA. The structure shows a trimer of alpha-beta sandwiches similar to the GlnB-like superfamily, with active sites formed at the trimer interfaces. Quantum-mechanical calculations, thin-layer chromatography, and nuclear magnetic resonance spectroscopy demonstrate that this family of enzymes are not hydrolases but lyases forming MTA and L-homoserine lactone in a unimolecular reaction mechanism. Sequence analysis and in vitro and in vivo mutagenesis support that T3 SAMase belongs to the same structural family and utilizes the same reaction mechanism.

Bacteria can be infected by viruses known as bacteriophages. These viruses inject their genetic material into bacterial cells and use the bacteria's own machinery to build the proteins they need to survive and infect other cells. To protect themselves, bacteria produce a molecule called S-adenosyl methionine, or SAM for short, which deposits marks on the bacteria's DNA. These marks help the bacteria distinguish their own genetic material from the genetic material of foreign invaders: any DNA not bearing the mark from SAM will be immediately broken down by the bacterial cell. This system helps to block many types of bacteriophage infections, but not all. Some bacteriophages carry genes that code for enzymes called SAMases, which can break down SAM, switching off the bacteria's defenses. The most well-known SAMase was first discovered in the 1960s in a bacteriophage called T3. Chemical studies of this SAMase suggested that it works as a 'hydrolase', meaning that it uses water to break SAM apart. New SAMases have since been discovered in bacteriophages from environmental water samples, which, despite being able to degrade SAM, are genetically dissimilar to one another and the SAMase in T3. This brings into question whether these enzymes all use the same mechanism to break SAM down. To gain a better understanding of how these SAMases work, Guo, Söderholm, Kanchugal, Isaksen et al. solved the crystal structure of one of the newly discovered enzymes called Svi3-3. This revealed three copies of the Svi3-3 enzyme join together to form a unit that SAM binds to at the border between two of the enzymes. Computer simulations of this structure suggested that Svi3-3 holds SAM in a position where it cannot interact with water, and that once in the grip of the SAMase, SAM instead reacts with itself and splits into two. Experiments confirmed these predictions for Svi3-3 and the other tested SAMases. Furthermore, the SAMase from bacteriophage T3 was also found to degrade SAM using the same mechanism. This shows that this group of SAMases are not hydrolases as originally thought, but in fact 'lyases': enzymes that break molecules apart without using water. These findings form a starting point for further investigations into how SAM lyases help bacteriophages evade detection. SAM has various different functions in other living organisms, and these lyases could be used to modulate the levels of SAM in future studies investigating its role.

ATP-Driven Contraction of Phage T3 Capsids with DNA Incompletely Packaged In Vivo.

Adenosine triphosphate (ATP) cleavage powers packaging of a double-stranded DNA (dsDNA) molecule in a pre-assembled capsid of phages that include T3. Several observations constitute a challenge to the conventional view that the shell of the capsid is energetically inert during packaging. Here, we test this challenge by analyzing the in vitro effects of ATP on the shells of capsids generated by DNA packaging in vivo. These capsids retain incompletely packaged DNA (ipDNA) and are called ipDNA-capsids; the ipDNA-capsids are assumed to be products of premature genome maturation-cleavage. They were isolated via preparative Nycodenz buoyant density centrifugation. For some ipDNA-capsids, Nycodenz impermeability increases hydration and generates density so low that shell hyper-expansion must exist to accommodate associated water. Electron microscopy (EM) confirmed hyper-expansion and low permeability and revealed that 3.0 mM magnesium ATP (physiological concentration) causes contraction of hyper-expanded, lowpermeability ipDNA-capsids to less than mature size; 5.0 mM magnesium ATP (border of supraphysiological concentration) or more disrupts them. Additionally, excess sodium ADP reverses 3.0 mM magnesium ATP-induced contraction and re-generates hyper-expansion. The Nycodenz impermeability implies assembly perfection that suggests selection for function in DNA packaging. These findings support the above challenge and can be explained via the assumption that T3 DNA packaging includes a back-up cycle of ATP-driven capsid contraction and hyper-expansion.

Rotary DNA motors.

Many molecular motors move unidirectionally along a DNA strand powered by nucleotide hydrolysis. These motors are multimeric ATPases with more than one hydrolysis site. We present here a model for how these motors generate the requisite force to process along their DNA track. This novel mechanism for force generation is based on a fluctuating electrostatic field driven by nucleotide hydrolysis. We apply the principle to explain the motion of certain DNA helicases and the portal protein, the motor that bacteriophages use to pump the genome into their capsids. The motor can reverse its direction without reversing the polarity of its electrostatic field, that is, without major structural modifications of the protein. We also show that the motor can be driven by an ion gradient; thus the mechanism may apply as well to the bacterial flagellar motor and to ATP synthase.

Analysis of functional domains of the packaging proteins of bacteriophage T3 by site-directed mutagenesis.

Intracellular phage T3 DNA is synthesized as a concatemer in which unit-length molecules are jointed together in head-to-tail fashion through terminally redundant sequences. The concatemeric DNA is processed and packaged into the prohead with the aid of non-capsid proteins, gp18 and gp19. We have developed a defined system, composed of purified gp18, gp19 and proheads, and a crude system, composed of lysates of T3 infected cells, for in vitro packaging of T3 DNA. The defined system displays an ATPase activity which is composed of DNA packaging-dependent and -independent ATPases (pac- and nonpac-ATPases, respectively). In the crude system, DNA is packaged by a way of concatemer as an intermediate. gp19 has ATP binding activity and three ATP binding and two Mg2+ binding consensus motifs in its amino acid sequence. We have expanded the previous studies on the roles of these domains in the DNA packaging reaction by more extensive analysis by site-directed mutagenesis. gp19 mutants, including the previously isolated four mutants, were divided into four groups according to the DNA packaging activity in the defined and crude systems: group 1 mutants were defective in both systems (gp19-G61D, which is a gp19 mutant with Gly to Asp at amino acid 61 and so on, and gp19-H344D); the group 2 mutant had decreased activity in both systems (gp19-G429R); group 3 mutants were active in the defined system but defective in the crude system (gp19-G63D, gp19-H347R, gp19-G367D, gp19-G369D, gp19-G424E); group 4 mutants had almost the same activity as gp19-wt (gp19-K64T, gp19-K370I, gp19-G429L, gp19-K430T and gp19-H553L). Group 1 mutants had an altered conformation, resulting in defective interaction with ATP and in abortive binding to the prohead, and lost specifically the pac-ATPase activity. The group 2 mutant had an increased pac-ATPase activity in spite of the decreased DNA packaging activity, indicating that this mutant is inefficient in coupling of ATP hydrolysis to DNA translocation. The inability of the group 3 mutants except gp19-H347R to package DNA in the crude system would be due to a defect in processing of concatemer DNA. gp19-H347R would be a mutant defective in the initiation event(s) of DNA packaging.