Disassembly activates Retron-Septu for antiphage defense

Abstract

Retrons are antiphage defense systems that produce multicopy single-stranded DNA (msDNA) and hold promise for genome engineering. However, the mechanisms of defense remain unclear. The Retron-Septu system integrates retron and Septu antiphage defenses. Cryo-electron microscopy structures reveal asymmetric nucleoprotein complexes comprising a reverse transcriptase, msDNA (a hybrid of msdDNA and msrRNA), and two PtuAB copies. msdDNA and msrRNA are essential for assembling this complex, with msrRNA adopting a conserved lariat-like structure that regulates reverse transcription. Notably, the assembled Retron-Septu complex is inactive, with msdDNA occupying the PtuA DNA binding site. Activation occurs upon disassembly, releasing PtuAB, which degrades single-stranded DNA to restrict phage replication. This "arrest-and-release" mechanism underscores the dynamic regulatory roles of msDNA, advancing our understanding of antiphage defense strategies.

Fig. 1.. Retron and Septu assemble into a supramolecular complex.

(A) Schematic depiction of the retron msdDNA synthesis process using Ec83 as an example. (B) Schematic of the retron Ec83-Septu system (the operon containing retron Ec83 and its cognate Septu effectors) in the E. coli genome. (C) Schematic of the proteins in the retron Ec83-Septu system. (D) Cryo-EM map of retron Ec78-Septu complex. (E) Cryo-EM map of retron Ec83-Septu complex. (F) Cartoon representation showing the atomic model of the Retron Ec83-Septu complex. (G) Surface representation of protein elements in the retron Ec83-Septu complex with nucleic acids represented as sticks. (H) Schematic representation showing an overview of the interaction between the retron and Septu components mediated by retron msDNA.

Fig. 2.. msrRNA and msdDNA interact with reverse transcriptase to regulate reverse transcription.

(A) Schematic representation of the retron Ec83 ncRNA gene that encodes msrRNA and msdDNA. (B) Retron Ec83 msDNA tertiary structure. (C) Secondary structure of Ec83 msDNA based on the solved structure. Regions not observed in the solved structure are colored gray. (D) Schematic representation showing the domain architectures of Ec83 reverse transcriptase (top panel) and its atomic model (cartoon) in matching colors (bottom panel). (E) Overlayed structures of retron Ec83 RT (green) with msDNA and HIV-1 RT (PDB: 1RTD, pink) with an incoming dTTP. (F) Close-up view of the RT active site shown in (E). (G) Sequence alignment of different ncRNA genes that encode msDNA in various Retron-Septu systems.

Fig. 3.. PtuA asymmetrically engages msDNA to stabilize the autoinhibited complex.

(A) Schematic representation of the msDNA showing an overview of its interaction with RT and PtuAs. (B) Intermolecular interactions between retron Ec83 msDNA and PtuAs. Proteins are shown in surface representation. (C-F) Close-up views of the interaction interfaces indicated in (B). (G) Serial dilution plaque assays shown for the T5n phage on E. coli MG1655 strain transformed with plasmids encoding wildtype or mutated retron Ec83-Septu systems. Images are representative of three replicates.

Fig. 4.. Retron Ec83-Septu suppresses phage replication through PtuAB-mediated single-stranded DNA cleavage.

(A) Nuclease activity assays of different components of the retron Ec83-Septu system against a Cy3-labeled dual-fork DNA substrate. Retron-Septu, the RT-msDNA-PtuAB complex fraction. PtuA-PtuB^H73A, the PtuAB subcomplex carrying a PtuB active-site mutation (Histidine 73 to Alanine). (B) Nuclease activity assays of different components of the retron Ec83-Septu system against a single-stranded DNA (ssDNA) substrate. (C) Nuclease activity of the PtuAB (PtuA-PtuB) from the retron Ec83-Septu system against a double-stranded DNA (dsDNA) substrate. (D) Nuclease activity of the PtuAB (PtuA-PtuB) from the retron Ec83-Septu system against a single-stranded RNA (ssRNA) substrate. (E) Time-course nuclease assays testing the nuclease activity of PtuAB (PtuA-PtuB) and the PtuAB mutant (PtuA-PtuB^H73A). (F) Representative fluorescence microscopy images from a T5n infection time-course in host strains of MG1655 expressing the wildtype retron Ec83-Septu operon or an inactive mutant (PtuB^H73A or PtuB^H73A/N93A) or the empty vector at 30°C. Magenta = outer membrane (FM4–64), cyan = DNA (DAPI). White arrowheads = intracellular DNA foci. Scale bar = 3 μm. Imaging was performed in biological triplicate. This assay was representative of two replicate experiments. (G) Cell viability assays of the E. coli MG1655 strain carrying retron Ec83-Septu PtuAB on plates in the presence and absence of ampicillin to select for the plasmid. (H) Cell viability assays of the E. coli MG1655 strain carrying retron Ec78-Septu PtuAB on plates in the presence and absence of ampicillin to select for the plasmid. Time course imaging of phage infection assay was representative of two replicate experiments. All other assays in this figure are representative of three replicates.

Fig. 5.. Disassembly of the retron Ec83-Septu system liberates PtuAB to initiate antiphage defense.

(A) Cryo-EM density map and atomic structural model (cartoon representation) of the PtuAB complex dissociated from the retron Ec83-Septu complex. (B) Cryo-EM density map and atomic structural model (cartoon representation) of the PtuA dimer. PtuA dimer was obtained through expression of C-terminally tagged PtuA. (C) Cryo-EM density map and atomic structural model (cartoon representation) of the PtuA*B* PtuA^D399A/E400A-PtuB^H73A) mutant complex. PtuA*B* (PtuA^D399A/E400A-PtuB^H73A) mutant was obtained through co-expression of C-terminally tagged PtuA^D399A/E400A and PtuB^H73A. (D) Conformational changes in the msDNA-binding region of PtuA from the dissociated PtuAB complex (rosy brown), the purified PtuA dimer (thistle), and the PtuAB mutant complex (tan) compared to the msDNA-bound PtuA in the Retron-Septu complex (gray). (E) Top panel, schematic illustrating the design of a disulfide-linked version of the retron Ec83-Septu complex, engineered at the PtuA tetramerization interface I. bottom panel, serial dilution plaque assays shown for the T5n phage on E. coli MG1655 strain transformed with plasmids encoding wildtype or mutated retron Ec83-Septu systems. The assay is representative of three replicates. (F) Proposed model for retron Ec83-Septu system mediated antiphage defense. In bacterial cells, the reverse transcriptase (RT), msDNA, PtuA, and PtuB in the retron Ec83-Septu system assemble into a four-component Retron-Septu complex that remains in an autoinhibited state. Upon phage infection, a phage-derived trigger induces the disassembly of the Retron-Septu complex, leading to the release of the sequestered PtuAB. The liberated PtuAB then indiscriminately cleaves single-stranded DNA (ssDNA), thereby blocking phage replication and preventing the release of phage progeny.