Structural mechanism of the Retron-Eco7 anti-phage defense system
Retrons are prokaryotic genetic elements involved in anti-phage defense and consist of a non-coding RNA, a reverse transcriptase (RT), and various effector proteins. Retron-Eco7 (previously known as Retron-Ec78) from Escherichia coli encodes two effector proteins (a PtuA ATPase and a PtuB nuclease) and degrades host tRNATyr upon phage infection, thereby protecting host cells against invading phages. However, its defense mechanism remains elusive. Here, we report the cryo-electron microscopy structures of the Retron-Eco7 complex, comprising the RT, multicopy single-stranded DNA (msDNA), PtuA, and PtuB. The Retron-Eco7 structure reveals that the RT–msDNA complex associates with two PtuA–PtuB complexes, potentially inhibiting their nuclease activity and suppressing bacterial growth arrest prior to phage infection. Furthermore, we found that a phage-encoded D15 nuclease acts as a trigger for the Retron-Eco7 system, cleaving the msDNA bound to the complex and facilitating the dissociation of PtuA–PtuB from RT–msDNA. Our data indicate that msDNA cleavage by D15 is the initial step required for the specific cleavage of host tRNATyr by the PtuA–PtuB nuclease, which leads to abortive infection. Overall, this study provides mechanistic insights into the Retron-Eco7 system and highlights the diversity of prokaryotic anti-phage defense mechanisms. Overall design: Overnight cultures of E. coli (DH10B) cells containing either the plasmid pBAD-PtuA–PtuB or the full set of retron-Eco7 (pLG008) were grown in 10 mL of LB medium until reaching an OD600 of 0.3. The bacteria carrying pBAD-PtuA–PtuB were induced with 0.2% arabinose to promote PtuAB expression, whereas those containing pLG008 were infected with phage SP15 and cultured at 37°C with shaking at 200 rpm for either 1 hour or 20 minutes, respectively. Following incubation, the cultures were centrifuged at 6,000 × g for 5 minutes, and the pellets were washed twice with PBS buffer. Total RNA was extracted using a miRNA isolation kit (Qiagen) following the manufacturer's instructions. Additionally, E. coli (DH10B) cells containing the Retron-Eco7 plasmid or an empty vector were infected with SP15 (MOI = 10) for 20 minutes, with uninfected cells serving as negative controls. The cells were then centrifuged at 10,000 × g for 10 minutes and washed twice with SM buffer. Total RNA was extracted using the miRNA isolation kit (Qiagen). The extracted RNAs were then submitted for tRNA-fragment sequencing.