Translocation of viral double-stranded DNA (dsDNA) into the icosahedral prohead shell is catalyzed by TerL, a motor protein that has ATPase, endonuclease, and translocase activities. TerL, following endonucleolytic cleavage of immature viral DNA concatemer recognized by TerS, assembles into a pentameric ring motor on the prohead's portal vertex and uses ATP hydrolysis energy for DNA translocation. TerL's N-terminal ATPase is connected by a hinge to the C-terminal endonuclease. Inchworm models propose that modest domain motions accompanying ATP hydrolysis are amplified, through changes in electrostatic interactions, into larger movements of the C-terminal domain bound to DNA. In phage ϕ29, four of the five TerL subunits sequentially hydrolyze ATP, each powering translocation of 2.5 bp. After one viral genome is encapsidated, the internal pressure signals termination of packaging and ejection of the motor. Current focus is on the structures of packaging complexes and the dynamics of TerL during DNA packaging, endonuclease regulation, and motor mechanics.

β-Toxin is a sphingomyelinase hemolysin that significantly contributes to Staphylococcus aureus pathogenesis. In most S. aureus isolates the prophage ϕSa3int inserts into the β-toxin gene hlb , inactivating it, but human and experimental infections give rise to β-toxin-producing variants. However, it remained to be established whether ϕSa3mw excises in response to specific environmental cues, restoring the β-toxin gene sequence. This is not only of fundamental interest but also critical when designing intervention strategies and therapeutics. We provide evidence that ϕSa3mw actively excises, allowing the conditional expression of β-toxin. ϕSa3int prophages may play a novel and largely uncharacterized role in S. aureus pathogenesis as molecular regulatory switches that promote bacterial fitness and adaptation to the challenges presented by the mammalian host.

ASCE ATPases include ring-translocases such as cellular helicases and viral DNA packaging motors (terminases). These motors have conserved Walker A and B motifs that bind Mg²⁺-ATP and a catalytic carboxylate that activates water for hydrolysis. Here we demonstrate that Glu179 serves as the catalytic carboxylate in bacteriophage λ terminase and probe its mechanistic role. All changes of Glu179 are lethal: non-conservative changes abrogate ATP hydrolysis and DNA translocation, while the conservative E179D change attenuates ATP hydrolysis and alters single molecule translocation dynamics, consistent with a slowed chemical hydrolysis step. Molecular dynamics simulations of several homologous terminases suggest a novel mechanism, supported by experiments, wherein the conserved Walker A arginine ‘toggles’ between interacting with a glutamate residue in the ‘lid’ subdomain and the catalytic glutamate upon ATP binding; this switch helps mediate a transition from an ‘open’ state to a ‘closed’ state that tightly binds nucleotide and DNA, and also positions the catalytic glutamate next to the γ-phosphate to align the hydrolysis transition state. Concomitant reorientation of the lid subdomain may mediate mechanochemical coupling of ATP hydrolysis and DNA translocation. Given the strong conservation of these structural elements in terminase enzymes, this mechanism may be universal for viral packaging motors.