Genome integrity sensing by the broad-spectrum Hachiman antiphage defense complex

Abstract

Hachiman is a broad-spectrum antiphage defense system of unknown function. We show here that Hachiman is a heterodimeric nuclease-helicase complex, HamAB. HamA, previously a protein of unknown function, is the effector nuclease. HamB is the sensor helicase. HamB constrains HamA activity during surveillance of intact double-stranded DNA (dsDNA). When the HamAB complex detects DNA damage, HamB helicase activity activates HamA, unleashing nuclease activity. Hachiman activation degrades all DNA in the cell, creating "phantom" cells devoid of both phage and host DNA. We demonstrate Hachiman activation in the absence of phage by treatment with DNA-damaging agents, suggesting that Hachiman responds to aberrant DNA states. Phylogenetic similarities between the Hachiman helicase and enzymes from eukaryotes and archaea suggest deep functional symmetries with other important helicases across domains of life.

Keywords: cryo-EM; genome integrity; helicase; immunity; phage defense.

Figure 1.. Hachiman is a two-component defense system that protects against diverse bacteriophages

(A) Overview of SF1/SF2 helicase-containing phage defense systems found in RefSeq genomes in the DefenseFinder database. (B) Phylogenetic tree of core helicase domains of 329 helicases from defense systems from (A) and representative SF1/SF2 helicases. Helicase superfamily is provided in the outer track (SF1 in black, SF2 in tan) and representative families demarcated in gray clades with labels. Defense-system-associated helicases are colored as shown in (A). Details on tree construction and sequence alignment provided in STAR Methods. (C) Hachiman loci from E. coli strains ECOR04, ECOR28, and ECOR31 tested in this study. HamA genes are shown in purple and HamB genes shown in green. Additional defense systems identified in PADLOC are shown in blue, integrases in yellow, and tRNA genes in red. All other genes are shown in gray. (D) Overview of phage-defense assays. Native Hachiman loci are cloned under an anhydrotetracycline (aTc)-inducible promoter, pTet, and monitored for protection against diverse phages. (E) Representative plaque assays for ECOR31 HamAB against sensitive phages EdH4 and T4, as well as resistant phage T5. Data are presented as the mean of three biological replicates. (F) Comparison of different Hachiman loci against 12 diverse phages representing 12 unique phage genera. Data shown represent the mean of three biological replicates. Plaque assays without EOP reductions, but a measurable difference in plaque size are denoted with an asterisk. (G) Protection against phage EdH4 is complete at low MOI, but insufficient at high MOI. For (E)–(G), Hachiman is induced at 20 nM aTc and for (E) and (F) dCas13d targeting RFP is provided as a negative control. For (G), a negative control is shown in Figure S2D. Data are presented as the mean of three biological replicates ± standard deviation. See also Figures S1 and S2.

Figure 2.. Structural basis of Hachiman complexation and identification of the HamA active site

(A) Cryo-EM density of the E. coli ECOR31 apo HamAB complex. The sharpened map is colored, whereas the unsharpened map is overlaid and transparent. (B) Orthogonal views of the HamAB structure, with domains colored according to the key above. Walker motifs are annotated in the HamB RecA1 and RecA2 domains. (C) Overview of the HamA-HamB NAH interface, with surfaces involved in the interaction shown. (D–F) Detail of three subregions, HamA^– (D), HamA^102–117 (E), and HamA^159–199 (F), contributing to the AB interface. Residues contributing to hydrogen bonding interactions are shown as sticks and are labeled with colors corresponding to the key above each view and in (B). (G) Sequence logo resulting from alignment of HamA DUF1837 ORFs. The ECOR31 HamA sequence and corresponding positions are shown below each residue logo. (H) Structural superimposition of the nuclease domain from the P. aquatile type IIS restriction modification system with HamA. (I) Plaque assays demonstrating the ability of HamAB and various mutants to confer defense against phage EdH4. Individual data points of three independent biological replications are shown along with the mean and standard deviation. The (−) symbol indicates a reduction in plaque size. See also Figure S3.

Figure 3.. HamAB is a DNA nuclease/helicase that degrades plasmids in vitro

(A) Malachite green ATPase assays of HamB against a panel of nucleic acid substrates. Individual data points of three independent biological replicates and the mean and standard deviation are shown. (B–E) HamB DNA unwinding assays on substrates with a 15-bp duplex and a 15-nt 3′ OH (B), forked 15-nt OH (C), 15-nt 5′ OH (D), and no overhang (E). DNA substrates are labeled with 5′ FAM. Gels are representative of three independent biological replicates. (F) Normalized percent unwinding of DNA substrates with 15 bp (circles), 25 bp (squares), and 50 bp (triangles) duplex lengths, all labeled with 5′ FAM and with a 15-nt 3′ OH. Individual data points shown are quantifications of replications of unwinding assays in the format of (B)–(E) normalized against basal unwinding (see STAR Methods). (G) In vitro plasmid clearance assay after 90 min at 37°C with ATP using MBP-HamA, HamB, HamAB, and HamA*B visualized on a 0.75% agarose gel. (H) Time course of HamAB plasmid clearance with addition of ATP or E. coli SSB, visualized on a native agarose gel. (I) Time course assay as in (H) with mutant HamA*B. (J) ATPase activity of HamB, HamAB, and HamA*B, with or without supercoiled plasmid substrates. Individual data points of three independent biological replicates and the mean and standard deviation are shown. (K) Cartoon depicting a model for HamAB-mediated plasmid degradation. See also Figure S4.

Figure 4.. Structural basis of HamB-DNA binding and helicase ratcheting

(A) Cryo-EM density of the 2.8-Å HamB-DNA density. The sharpened map is colored according to domain, whereas the unsharpened map is overlaid and transparent. (B) Orthogonal views of the 2.8-Å HamB-DNA structure. (C) Detail of the 3′ end of the DNA buried within the DNA entry site of HamB. Hydrogen bonds and contributing residues are shown with a dashed line. (D) Detail of the DNA duplex-interacting RecA2 loop. (E) Left, superimposed conformers of HamB-DNA viewed from the DNA side, with conformation 1 (2.8 Å) colored teal and conformation 2 (2.9 Å) colored burgundy. Right, conformations 1 and 2 viewed from the NAH side and transparent, with vectors colored according to domain representing motion between the two conformations. Vectors are scaled 2× and are calculated using modevectors. (F) Representative disruption of the predicted AB interface between the two HamB conformations. AB interactions disrupted by HamB motion are shown and labeled. (G) Native PAGE of reactions of the HamAB complex with the DNA where ratcheting was observed in cryo-EM. ATP and DNA appear to dissociate the AB complex. (H) Model for HamB signal transduction to the NAH and concomitant release of HamA. See also Figure S5.

Figure 5.. Hachiman defends against bacteriophage by nonspecific DNA clearance

(A) EdH4 infection time course in E. coli expressing wild-type HamAB, HamAB* (HamAB^D431A), or HamA*B (HamA^E138A,K140AB) or lacking the Hachiman system (control). Cell membranes were stained with FM4–64 (red) and DNA was stained with DAPI (cyan). Scale bar, 3 μm. MOI ≈ 2. (B) Quantification of intracellular DAPI-stained DNA cross-sectional area over the course of EdH4 infection. Dots represent individual medians from three biological replicates. ** p < 0.01 by Dunnett’s test. n >225 in total across all replicates for each condition (see STAR Methods). (C) Time-to-lysis of EdH4 infecting the control strain based on time-lapse bright-field microscopy under the same growth and infection conditions as the time course in (A) and (B). Black points represent the mean cumulative percentage of total lysed cells that have burst at 10 min intervals over the course of EdH4 infection, measured in triplicate. Shaded region represents the standard deviation.

Figure 6.. DNA damage activates Hachiman

(A–E) Cell growth of E. coli MDS42 expressing wild-type HamAB, HamA*B, and HamAB* at 20 nM aTc in the absence or presence of minimum inhibitory concentrations of nalidixic acid (A), novobiocin (B), bleomycin (C), mitomycin C (D), and gentamycin (E). Growth curves are colored according to condition. See Figure S6 for complete minimum inhibitory concentration determinations. Data are presented as the mean of three biological replicates ± standard deviation. See also Figure S6.

Figure 7.. Hachiman scans intact dsDNA

(A) HamA*B-plasmid +ATP specimen preparation. (B) Representative motion-corrected, dose-weighted cryo-EM micrograph from the HamA*B-plasmid DNA dataset. Plasmid DNA and bound particles are indicated with white arrows. (C) Representative 2D classes of particles bound to plasmid DNA. (D) Cartoon depicting the scanning state resolved here and comparison with the loading state resolved in the HamB-DNA dataset. (E) Composite cryo-EM density colored according to domain. Protein regions are from the 3.2-Å deepEMhancer-sharpened map, whereas DNA is from the 3.2-Å sharp map masked and B-factor refined to display helical features. (F) Orthogonal views of the HamA*B-plasmid DNA structure. The DNA sequence is unknown. (G) Detail of ATP in HamB, with residues and hydrogen bonds shown. The density is masked to ATP. (H) Cartoon showing separation of intact dsDNA from the HamA active site. (I) Model of threshold activation of Hachiman. (J) Proposed mechanism of Hachiman activation. See also Figure S7.