DdmDE eliminates plasmid invasion by DNA-guided DNA targeting

Abstract

Horizontal gene transfer is a key driver of bacterial evolution, but it also presents severe risks to bacteria by introducing invasive mobile genetic elements. To counter these threats, bacteria have developed various defense systems, including prokaryotic Argonautes (pAgos) and the DNA defense module DdmDE system. Through biochemical analysis, structural determination, and in vivo plasmid clearance assays, we elucidate the assembly and activation mechanisms of DdmDE, which eliminates small, multicopy plasmids. We demonstrate that DdmE, a pAgo-like protein, acts as a catalytically inactive, DNA-guided, DNA-targeting defense module. In the presence of guide DNA, DdmE targets plasmids and recruits a dimeric DdmD, which contains nuclease and helicase domains. Upon binding to DNA substrates, DdmD transitions from an autoinhibited dimer to an active monomer, which then translocates along and cleaves the plasmids. Together, our findings reveal the intricate mechanisms underlying DdmDE-mediated plasmid clearance, offering fundamental insights into bacterial defense systems against plasmid invasions.

Keywords: Ago; Argonaute; DNA-guided DNA targeting; DdmD; DdmE; cryo-EM.

Figure 1.. DdmD forms a dimer with helicase activity.

(A) Cryo-EM density map of apo DdmD with the two protomers colored in blue and green, respectively. (B) Cylinder representation of apo DdmD. (C) Cylinder representation of a DdmD protomer, with each domain distinctly colored. (D) Schematic domain organization of DdmD, with residue numbers indicated above. (E) Cylinder representation of DdmD dimerization interfaces I and II, with secondary structures labeled. (F) The helicase activity of DdmD against various substrates with or without ATP. Green dots indicate 5′ Cy3 label. Substates are detailed in Table S2. See also Figure S1.

Figure 2.. Structure of DdmD-DdmE-DNA ternary complex

(A) Gel filtration profiles and SDS-PAGE analyses of DdmD and DdmE after co-incubation without (top panel) or with (bottom panel) a dual-forked DNA substrate, EHJ12. (B) Electrophoretic Mobility Shift Assay (EMSA) analysis of the DdmD dimer and/or DdmE in complex with DNA substrates (EHJ12), with or without ATP. 1E, DdmE monomer; 1D, DdmD monomer; 2D, DdmD dimer. (C) Cryo-EM density map of DdmD-DdmE-DNA (2D-1E-DNA) ternary complex. (D) Schematic sequence of the visible DNA within the DdmD-DdmE-DNA ternary complex. (E) Ribbon representation of DdmD-DdmE-DNA ternary complex. (F) Ribbon representation of DNA substrate within the DdmD-DdmE-DNA ternary complex. The three DNA strands are designated as guide DNA (gDNA), complementary target DNA (ctDNA), and non-complementary target DNA (nctDNA). See also Figure S2 and S3.

Figure 3.. DdmE rectuits DdmD for eliminating plasmids.

(A) Schematic domain organization of DdmE with residue numbers indicated above. (B) Cylinder representation of DdmE, with each domain distinctly colored as shown in (A). (C) Overlaid structures of DdmE and CbAgo reveal the unique DdmDE-interacting domain (DID) in DdmE. (D) Ribbon diagram of DdmE DID domain with secondary structures labeled. (E) Overview of DdmE and its engagement with one DdmD protomer, depicted in cylinder representation with interacted domain distinctly colored and labeled. (F) Interaction interfaces of DdmE and DdmD mediated by L1-PAZ-L2-DID and HD2 domains, with each domain distinctly colored and labeled in cylinder representation. (G) Detailed interactions between DdmE DID domain (pale green) and DdmD HD2 domain (sky blue). (H) Bacterial growth assay showing interactions between DdmD and DdmE are critical for plasmids elimination. See also Figure S3.

Figure 4.. DdmE is a DNA-guide DNA-targeting module.

(A) Cryo-EM density map and cylinder representation of DdmE and DNA complex. gDNA, guide DNA; ctDNA, complementary target DNA; and nctDNA, non-complementary target DNA. Nt-Duplex and gt-Duplex are higlighed in red and blue circles, respectively. (B) Schematic sequence and structure of gDNA, ctDNA, and nctDNA in DdmE. (C) DNA coordination by DdmE. DdmE is shown as electrostatic surface potential model. nt-Duplex and gt-Duplex interact with DdmE differently. (D) Expanded view of the 5’ nucleotide of guide DNA coordinated by residues in DdmE MID domain. (E) DdmE uses 5’-phosphorylated gDNA for DNA targeting, revealed by EMSA. Target DNA and RNA are labeled by Cy-3 fluorophore. (F) DdmE prefers gDNA shorter than 14 nt, revealed by EMSA. See also Figure S4.

Figure 5.. DdmD recognizes ssDNA.

(A) Cylinder representation of DdmD with single strand DNA (ssDNA). (B) A close-up view of the disconnected backbone, as indicated in (A). (C) Ribbon diagram of DdmD with ssDNA. ssDNA, HD1 domain and HD2 domain of DdmD were distinctly colored as indicated. (D) Detailed view of ssDNA coordinated by key residues in DdmD HD1 domain. (E) Detailed view of ssDNA coordinated by key residues in DdmD HD2 domain. (F) Schematic view of residues of DdmD coordinating ssDNA. (G) Representation of the positively charged channel within DdmD HD1 and HD2 domains, crucial for ssDNA binding. See also Figure S5.

Figure 6.. Forked DNA triggers disassembly of DdmD dimer.

(A) Overlaid structures of apo DdmD (cyan) and dimeric ssDNA-bound DdmD (pink). (B) Comparison of key residues of interface II in apo and DNA-bound DdmD dimers. (C) Cryo-EM density maps of fully dissociated and partially dissociated DdmD-DdmE-DNA (1D-1E-DNA) ternary complexes. (D) Forked DNA with a long 3’ overhang triggers the DdmD dimer dissociation, revealed by EMSA. The lengths of the overhang are indicated. All the DNA substrates are detailed in Table S2. (E) Forked DNA with a long 3’overhang triggers the dissociation of the DdmD dimer, revealed by gel filtration and native-PAGE. (F) Low salt conditions promote DdmD disassembly, revealed by EMSA. (G) Cryo-EM density map of ssDNA-bound DdmD monomer. (H) Cylinder representation of ssDNA-bound DdmD monomer. See also Figure S6.

Figure 7.. DdmDE eliminates plasmids by DNA-guided DNA targeting.

(A) DdmD nuclease activity with various divalent metal ions. DdmD_D1059A_H1106A is a catalytic inactive mutant. (B) DdmD nuclease activity against various DNA substrates in the presence of Mn²⁺. DNA substrates are detailed in Table S2. (C) DdmDE nuclease activity against pUC19 in vitro. Nb.BsrDI was used to generate nicking plasmid as control. EcoRI was used to generate linear plasmid. (D) gDNA enhance nuclease activities of DdmDE, but not DdmD alone. (E) Mechanism for the DdmDE-mediated plasmid elimination. See also Figure S7.