Periplasmic gatekeeping of phage DNA entry by an rSAM enzyme matured effector with HxS repeats

Domain: biology
Published: 2026-03-13
Editorial outcome: major_revision

Abstract
In the relentless arms race between bacteria and phages, bacteria have evolved a variety of defense strategies to combat phage infection. However, no system has been previously demonstrated to specifically inhibit phage genomic DNA ejection. Here, we present a bacterial antiphage system, termed HXS, which provides broad-spectrum and robust antiphage activity by interfering with phage DNA entry. The HXS system consists of two radical S-adenosylmethionine (rSAM) enzymes (HxsB and HxsC), a small protein (HxsD), and the effector HxsA with a peptidoglycan-binding domain and five His-Xaa-Ser (HXS) repeats. HxsB/HxsC catalyze rSAM enzyme-dependent maturation of HxsA, including N-terminal processing and a site-specific +8 Da modification, thereby producing a periplas mic effector required for HXS defense. Biochemical evidence supports a model in which the matured effector likely engages incoming DNA electrostatically to arrest entry, establishing an rSAM enzyme-modified protein effector in antiphage defense.

Introduction
Bacteria and phages are among the most abundant and dynamic biological entities on Earth, engaged in a continuous evolutionary arms race that profoundly shapes microbial diversity and ecosystem function 1,2. Phages initiate their infection by attaching to the bacterial surface receptors and injecting their genomic DNA into host cells. After DNA replication, transcription, and then translation, the phage progenies are assembled and finally released, usually resulting in the lysis of host cells 3. Recent studies have shown that bacteria have evolved a variety of antiphage systems to thwart different stages of the phage replication cycle to counteract phage predation and ensure survival 4-6.

As a first line of defense, bacteria can alter the phage receptors on their cell surfaces, such as surface proteins or lipopolysaccharides (LPS), by mutation or chemical modification to prevent phage recognition and binding 7-9. After phage attachment and DNA entry, bacteria use systems such as restriction-modification (RM) and CRISPR-Cas to identify and degrade phage DNA to protect themselves 10,11. During phage DNA replication, transcription, translation, and virion assembly, a variety of antiphage systems such as CBASS 12,13, Thoeris 14,15, TIR 16, and toxin-antitoxin 17,18 can be activated, usually resulting in premature host cell death or abortion of the infection process, thereby preventing assembly and release of mature phage progeny.

Although many well-characterized bacterial defenses act after phage genomes have entered the cytoplasm or sense DNA entry to trigger downstream effectors 19, surprisingly, no bacterial system has yet been identified that specifically inhibits phage genomic DNA ejection. While the superinfection exclusion (Sie) systems of T4 phage can prevent subsequent infection by other T-even-like phages by blocking the entry of phage DNA, these systems are phage-encoded self-protection with a narrow spectrum, acting mainly against closely related phages 20.

Here, we present a broad and robust bacterial antiphage system, which we call the HXS system, composed of four proteins that work together to uniquely inhibit the phage DNA entry. HxsB and HxsC have a radical S-adenosylmethionine (rSAM) domain, which is part of a diverse family of enzymes known for their roles in various biochemical processes, particularly the modification of peptides or proteins 21. HxsA contains five His-Xaa-Ser (HXS) repeats, a motif that has been identified in bacterial proteins for over a decade but whose function has remained largely mysterious22. We showed that the HxsA is cleaved by the coordinated action of HxsB, HxsC, and HxsD into a functional shorter and covalently modified protein that was translocated into periplasm to inhibit phage DNA entry. The HXS system combines the rSAM domains and HXS repeats and reveals a functional role for these elements in the context of antiphage activity. Our findings reveal a previously unrecognized bacterial defense strategy, expanding our understanding of microbial interactions and providing a potential target for biotechnological applications in phage therapy and bacterial resistance management.

Results
The HXS system is a defense system with a broad antiphage activity
The HXS system was serendipitously discovered while we were trying to identify homologs of viperin proteins in Escherichia coli by sequence and structural domain comparisons. Mammalian viperin is an interferon-induced antiviral protein belonging to the rSAM superfamily 23. It produces the ribonucleotide analog, 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), which acts as a chain terminator during viral RNA synthesis, thereby inhibiting viral replication 23. A recent study has shown that a prokaryotic viperin also has antiphage activity that protects against T7 phage infection by inhibiting viral polymerase-dependent transcription, like mammalian viperin 24.

During the identification of viperin homologs in the E. coli genomes, we identified 114 proteins belonging to the COG0535 rSAM superfamily. These proteins were clustered into 2 clusters, with 80-90% amino acid sequence identity within each cluster. Interestingly, we found that nearly half of these proteins are located within a unique operon structure. This operon contains twin-like rSAM-containing proteins (belong to two 2 clusters respectively) in proximity, accompanied by two additional proteins (Fig. 1a). We cloned one representative operon into the pSEC1 vector and transformed it into E. coli MG1655 cells to assess its antiphage activity against a taxonomically diverse panel of coliphages, which we taxonomically classified into multiple ICTV-recognized genera based on their genome sequences (Supplementary Table 1). HXS inhibited 110 of 113 phages (97.3%), with most exhibiting ≥10^3-fold reductions in efficiency of plating (EOP) (Fig. 1a). The only recurrent exceptions were three phages: two Tequatrovirus (including canonical T4) and one Punavirus (the classical generalized transducing phage P1). These results indicate broad and potent antiphage activity across diverse coliphage lineages.

Single gene deletion experiments showed that all the four genes are necessary for the antiphage activity of the HXS system (Fig. 1b). By surveying 4,541 E. coli genomes available as of November 2025, we found that 77% of detected hxsA-D operons did not overlap with prophage regions predicted by geNomad v1.8.0 25, indicating that HXS is typically chromosomally encoded rather than part of prophage cargo (Supplementary Fig. 1a-b). Bacterial growth curves showed that a high dose of phage infection (MIOI=5) did not induce the rapid death of cells containing the HXS system (Fig. 1c), which is usually a feature of the abortive infection mechanism of many defense systems 26-28. The phage adsorption assay showed that the proportion of phages adsorbed onto E. coli in the presence of the HXS system did not change significantly at different time points after infection (Fig. 1d). These results indicated that the HXS system did not inhibit phage adsorption.

The HXS system inhibits phage genomic DNA entry into E. coli
To obtain escape variants for probing how HXS blocks infection, we serially passaged phage T7 on HXS-expressing E. coli. T7 is a strictly lytic, genetically tractable, well-characterized coliphage that is robustly restricted by HXS. These features make it a practical and informative starting point for dissecting how HXS affects phage infection. After 25 passages, we recovered escape mutants capable of overcoming HXS (Fig. 1e; Supplementary Fig. 2). Genome sequencing of a single-plaque-purified escape clone identified seven mutations within the same phage genome (Supplementary Table 2), including five in the internal core proteins gp15 and gp16 (lower-case gp denoting gene product) (Fig. 1f), one synonymous mutation in gene 2, and one intergenic mutation between gene 0.3 and gene 19.5. We then introduced the single or multiple of these mutations into the wild-type T7 to generate ten T7 phage mutants as shown in Fig. 1g-i. Plaque assays showed that single mutations in the gp15 and the single or double mutation in the gp16 had a limited effect on T7 phage escape from the HXS system, whereas double or triple mutations in gp15 significantly enhanced the ability of T7 phage to escape from the HXS system. The gp15 and gp16 are key components of the phage T7 DNA-ejectosome that initiates genome translocation into the host cell (Fig. 1j) 29-31 32. Gp15 assembles the hexameric translocation tunnel, and gp16 serves as a ‘molecular tape’ that stabilizes gp15-gp15 interfaces. Together, the mutation results suggest that the HXS may interfere with phage DNA entry.

T7 phage translocate its genomic DNA from the left end (highlighted in red in Fig. 2a and the fragment I in Fig. 2b) into E. coli cell 33. Based on the polarity of T7 phage DNA entry and the character of the endonuclease DpnI that can specifically cleave fully methylated but not the unmethylated T7 genomic DNA, previous studies had developed the DpnI digestion assay to directly measure the DNA translocation rate 34. We then used this assay to determine whether the HXS system interfered with T7 DNA translocation. Plaque assays showed that overexpression of Dam methylase did not affect the antiphage activity of HXS (Supplementary Fig. 3). The genomic DNA of T7 (containing 6 DpnI sites) propagated in Dam methylase-overexpressing cells became methylated and was cleaved into 5 large fragments by DpnI (Fig. 2b, right agarose gel). However, the T7 DNA prepared with standard E. coli is incompletely methylated and is DpnI-resistant (Fig. 2b, left agarose gel). During infection of Dam-overexpressing cells by unmethylated T7, the left end that firstly enters the cytoplasm becomes methylated by Dam, whereas the capsid-retained portion remains unmethylated (Fig. 2a). The partially methylated phage DNA was isolated and digested by DpnI. The appearance of DpnI-digested DNA bands, determined by Southern blot with biotin-labeled probes specific for fragments I and V, over the infection course reflects the rate of T7 DNA translocation 34.

We found that in the absence of HXS, fragment I was detected at 8 min post infection (mpi) and fragment V at 12 mpi, consistent with ~10-12 min for complete T7 entry (Fig. 2c) 34. A larger head-to-tail (I+V) band appeared by 12 mpi, indicating the onset of concatemeric replication 35. Strikingly, in the presence of the HXS system, fragment I and fragment V were delayed to ~30 mpi and ~50 mpi, respectively (Fig. 2d), demonstrating a substantial retardation of genome entry. Consistently, the HXS-escape T7 mutant can inject its genomic DNA into E. coli BL21 cells expressing HXS at the similar efficiency (Supplementary Fig. 4).

To visualize genome entry directly, we performed time-resolved transmission electron microscopy (TEM), where DNA-emptied capsids stain dark, whereas DNA-filled capsids appear light 36. Without HXS, most T7 capsids were dark by 10 min post infection (mpi), consistent with completed genome ejection, whereas in HXS-expressing cells, capsids remained largely light even at 25 mpi (Fig. 2e), in agreement with the delay in DNA entry observed in the DpnI assay. Because T7 completes genome delivery via a transcription-dependent mechanism, in which host σ⁷⁰ RNA polymerase initiates from early promoters A1, A2, and A3 before takeover by the viral polymerase gp1 37, we asked whether HXS might act primarily by blocking these early transcriptional events. Deletion of the σ⁷⁰-dependent AI2 and A3 promoters from the T7 genome did not relieve HXS-mediated inhibition in plaque assays (Supplementary Fig. 5a-b). Moreover, when cells carrying an A1 promoter driven GFP reporter plasmid were infected with either wild-type T7 or the ΔA2A3 mutant, HXS neither reduced its restriction efficiency nor detectably altered A1-driven GFP expression (Supplementary Fig. 5c-d). Together with the entry assays above, these data indicate that HXS does not simply inhibit the transcriptional program required for T7 DNA translocation, but instead targets the genome-entry step itself.

To test whether entry inhibition generalizes beyond T7, we randomly picked up a second coliphage from the family Christensenvirus and examined the DNA entry. TEM at matched time points showed the same pattern: without HXS, capsids appeared dark by 10 mpi, whereas with HXS, capsids remained light at 25 mpi (Fig. 2e). Thus, HXS-mediated delay of DNA entry is observed across distinct coliphage lineages, reinforcing the conclusion that HXS targets the genome-delivery step of infection. Together, these results indicate that the HXS interferes with phage DNA entry.

The periplasmic localization of cleaved HxsA protein is indispensable for the antiphage activity of the HXS system
Since the HXS system interferes with phage DNA entry, we hypothesized that some of the four HXS proteins might localize to the bacterial wall. Sequence analysis showed that only the HxsA protein possesses a signal peptide (residues 1-22) and a peptidoglycan-binding domain (Fig. 3a and Supplementary Fig. 6a). Replacement of the signal peptide with a hexa-histidine tag (HxsA ) or mutations on peptidoglycan-binding domain abolished the antiphage activity (Fig. 3b and Supplementary Fig. 6b-c), indicating that the HxsA protein may locate in periplasm and act as an effector of the HXS system.

Periplasmic proteins of E. coli were prepared as described previously 38,39 and analyzed by western blot using home-made HxsA-specific polyclonal antibodies (Supplementary Fig. 7). Although both wild-type HxsA and HxsA were detected in whole-cell lysate, only wild-type HxsA was recovered from the periplasm (Fig. 3c-d). Notably, HxsA was still cleaved, albeit with reduced efficiency, indicating that the observed proteolysis is not simply canonical signal-peptide removal. Indeed, single-gene deletions showed that each of the other Hxs proteins is necessary for HxsA cleavage (Fig. 3e). Taken together, these results suggest that HxsA translocation and N-terminal cleavage follow a specialized, non-canonical pathway. Rather than relying solely on a simple canonical signal peptide, it appears likely that the other Hxs proteins act as essential, dedicated chaperones or accessory translocators to actively mediate the export of HxsA. The precise spatiotemporal dynamics of HxsA processing, whether it occurs co-translocationally or requires the other Hxs proteins as specific chaperones, remains an exciting avenue for future biochemical studies.

Since all the HxsB, HxsC, and HxsD lack a signal peptide, they might participate the cleavage of HxsA in the cytoplasm. To experimentally assess their localization, we generated polyclonal antisera against overexpressed HxsB, HxsC and HxsD (Supplementary Fig. 8), but endogenous levels in cells expressing the native HXS system were below the detection limit of Western blotting, so we cannot yet definitively assign their precise subcellular localization. However, single gene deletion showed that all these proteins are necessary for the HxsA modification (Fig. 3e). HxsB and HxsC proteins are characterized by the radical SAM enzyme with two 4Fe-4S cluster domains (Fig. 3f). Double mutation of the first 4Fe-4S cluster domain of HxsB (C98AC101A) or HxsC (C105AC108A) completely inhibited the HxsA cleavage (Fig. 3f-g), while double mutation of the second 4Fe-4S cluster domain of HxsB (C399AC402A) or HxsC (C342AC345A) decreased the cleavage efficiency. We did not examine single-residue substitutions, which may retain partial cofactor binding and residual activity. HxsD is a small protein belongs to TIGR03976 superfamily, many members of which contain conserved motifs LYW and LLNDYxLRE. Targeted substitutions in HxsD revealed that the LYW motif (Y23A) and the LLNDYxLRE motif (D61A/R65A) also reduce the cleavage efficiency (Fig. 3f-g). In the D61A/R65A mutant, the cleaved HxsA fragment is indeed still abundant, but the fraction of cleaved versus uncleaved HxsA is clearly reduced compared with the wild-type. This pattern indicates that LLNDYxLRE motif partially impairs HxsA processing, with a milder effect than the other mutations. In addition, mutating either of these key motifs of HxsB, HxsC, and HxsD abolished the antiphage activity (Fig. 3h). Together, these results indicated that the HxsB, HxsC, and HxsD work together to cleave the HxsA, which is necessary for its antiphage activity.

The rSAM enzyme-mediated maturation of HxsA establishes a periplasmic barrier to phage DNA entry
To further characterize the cleaved HxsA, the band corresponding to the cleaved HxsA was excised from the SDS-PAGE gel (Supplementary Fig. 9), and the proteins were extracted and digested with trypsin and chymotrypsin, respectively, for mass spectrometric analysis. LC-MS/MS analysis of the cleaved HxsA protein identified eight tryptic and nine chymotryptic peptides, collectively covering nearly all residues 48-214 of HxsA (Fig. 4a). Two fragments, residues 23-47 and 114-139 (highlighted in cyan in Fig. 4a), were not detected. Mutation analysis showed that residues 23-47, but not the residues114-139, are required for antiphage activity (Fig. 4b). Because residues 23-47 harbor four chymotryptic sites, this region was likely fragmented into peptides below the MS detection limit. To determine whether the periplasmic localization of cleaved HxsA alone is sufficient for defense, we expressed four N-terminal truncations starting at each chymotryptic site residues 23-47 (Fig. 4a), each fused to either a Tat or Sec signal peptide (eight constructs; Supplementary Fig. 10a). Western blots confirmed that all eight variants were transported to the periplasm, yet none conferred antiphage activity (Supplementary Fig. 10b-c). Thus, simply placing cleaved HxsA in the periplasm is insufficient; HxsB, HxsC, and HxsD are required beyond cleavage.

Notably, liquid chromatography-high-resolution mass spectrometry (LC-HRMS) detected a peptide matching HxsA (48-79) with a +8.0 Da m/z shift relative to the unmodified species (hereafter denoted HxsA (48-79) [+8]) in the precursor spectrum. High-resolution MS/MS of HxsA (48-79) [+8] yielded diagnostic fragment ladders that unambiguously localized the +8 Da modification to the SHRSS motif within this segment (Fig. 4c-d). The observed isotopic pattern and low-ppm mass error were consistent with a discrete +C/–4H–type alteration rather than adduct formation or oxidation. Site-directed alanine scanning of the SHRSS motif revealed that substituting either the N-terminal Ser (S to A), His (H to A), or C-terminal Ser (S to A) abolished HXS-mediated protection (Fig. 4e), indicating that the +8 Da modification mapped to SHRSS is essential for antiphage function (Fig. 4d-e). However, because mutation of SHRSS or deletion of hxsB, hxsC, or hxsD severely impaired HxsA processing and periplasmic accumulation, we could not recover HxsA from these backgrounds for LC-MS/MS; accordingly, we were unable to directly assess the +8 Da modification state of HxsA in the ΔhxsB/ΔhxsC/ΔhxsD or active-site mutant.

Because HxsB and HxsC each contain an rSAM domain, a hallmark of enzymes involved in peptide and protein modification 21, we hypothesized that they catalyze a covalent maturation of HxsA, consistent with our genetic data showing that active-site substitutions in either protein abolish HxsA cleavage and defense (Fig. 3g-h). To probe how this maturation machinery is organized, we mapped physical interactions among the HXS components. Pull-down assays revealed a HxsC-HxsD complex, whereas no stable association between HxsB and HxsC was detected (Fig. 4f and Supplementary Fig. 11a-c), indicating that HxsC engages HxsD to form the core processing module. Consistently, AlphaFold 3 modelling predicted an HxsC-HxsD heterocomplex with lower free energy than alternative assemblies (Supplementary Fig. 12a-c). Docking the HxsA segment encompassing the SHRSS motif into this model positioned the histidine and terminal serine side chains near the unique-iron/SAM pocket in HxsC, a geometry compatible with the observed +8 Da mass shift on SHRSS and with a site-specific covalent modification at these residues (Fig. 4g). These structural predictions support a model in which the HxsC-HxsD core complex installs a covalent modification on SHRSS motif, although further studies are required to define the precise mechanism.

The breadth of HXS activity suggests that it targets a phage-agnostic step rather than specific structural proteins. Consistent with this, no detectable interaction was observed between periplasmic HxsA and the T7 ejectosome proteins gp15/gp16 in pull-down assays (Supplementary Fig. 13a-d). HxsA carries five tandem HxS repeats enriched in basic residues, including histidine that provides pH-dependent positive charge capable of electrostatic interactions with DNA. We therefore hypothesized that electrostatic engagement of incoming DNA underlies inhibition of genome entry. Functional scanning supported this model: triple-alanine replacement of any single HxS repeat (HxS to AAA) abolished HXS-mediated protection (Fig. 4h), indicating that the repeats act as a multivalent functional array. Moreover, fluorescent DNA-binding assays with periplasmic HxsA showed robust DNA binding (Fig. 4i). Together, these data argue that HxsA does not rely on phage-specific protein recognition; instead, its positively charged, repeated HxS motifs likely sequester the translocating genomic DNA within the periplasm, providing a mechanistic basis for the system’s broad antiphage spectrum.

Taken together, these results suggest that HxsB, HxsC, and HxsD act in the cytoplasm to maturate HxsA, including installing a site-specific +8 Da modification within the last HxS repeat and promoting N-terminal processing. The mature HxsA is exported to the periplasm, where its peptidoglycan-binding domain anchors it to the peptidoglycan layer. Its five basic-residue-enriched HxS repeats, including the modified motif, most likely engage the incoming phage genomic DNA by electrostatic interactions, thereby impeding DNA entry and conferring broad antiphage defense (Fig. 5a).

The distribution and gene structure of the HXS system
Since the HxsA is the effector protein of the HXS system, we determined the distribution of the HXS system by evolutionary analysis of HxsA proteins across all bacteria. Our results showed that 99.7% of the HXS system was found in the phylum Pseudomonadota, whereas there was only a sporadic distribution in other bacterial phyla. Gammaproteobacteria accounted for 91.3% of the HXS system-containing bacteria, which was much more than the other bacterial phyla (Fig. 5b). A species breakdown of HXS-containing bacteria revealed that Klebsiella is the most prevalent species of HXS-containing bacteria, and 27.2% of the 16,000 Klebsiella genomes in the NCBI database (through January 2022) contain the HXS system (Fig. 5c). This suggests that the HXS system may be an important defense system within Klebsiella. In addition, this system is also abundantly distributed in Escherichia and Pseudomonas (Fig. 5c). Evolutionary analysis showed that the HXS systems of Pseudomonas, Acinetobacter, Rhizobium, Vibrio, Proteus, and Enterobacter were well separated from other bacteria. However, the HXS systems of Escherichia, Salmonella and Klebsiella are distributed in different branches and cross each other (Fig. 5c), indicating that the HXS system may be transmitted in these bacterial species.

Gene structure analysis showed that the composition of proteins within the HXS system is relatively conservative, of which more than 99.8% is composed of four proteins, HxsA, HxsB, HxsC, and HxsD (Fig. 5d). HxsB and HxsC are always close together, while HxsA and HxsD are sometimes separated by other proteins. In a few cases the HxsD protein is missing. In addition, 99.6% of HXS systems are organized in the order HxsD-HxsB-HxsC-HxsA, and only a small number of systems have different gene organizations (Fig. 5d).

Methods
Ethics statement
Animal experiments were performed according to the protocols (HZAUMO-2023-0094) approved by the Animal Protection and Ethics Committee of Huazhong Agricultural University, Hubei, China. Mice were housed in specific pathogen-free (SPF) conditions under a 12-hour light/dark cycle at an ambient temperature of 22 ± 2°C and a relative humidity of 50 ± 10%.

Bacterial strains, phages, and plasmids
E. coli DH5α was used for plasmid construction, and E. coli B834 for engineering T7 phage mutants. E. coli MG1655 served as the host for initial antiphage activity screens, and E. coli BL21 was used for mechanistic assays of the HXS defense system. The Zorya type II and RT-TIR defense systems were cloned in previous studies 16,45. The HXS, RT-Abi-P2, and AVAST type 2 systems were PCR-amplified from genomic DNA of E. coli strains MEM (GenBank accession CPO12378), RHB26-C20 (GenBank accession CP057447), and EC6030 (GenBank accession CP183338), respectively, and assembled into pSEC1 by standard methods. The resulting plasmids were individually transformed into E. coli MG1655 and used for antiphage assays. The HXS systems with different mutations were constructed by one-step cloning (Supplementary Table 3). T7 phage (GenBank Accession V01146.1) was maintained in our laboratory stocks. The panel of 109 phages isolated on E. coli MG1655 has been described previously 45 and used for antiphage assays. Genome sequences for these 55 isolated phages were sequenced and deposited in NCBI (GenBank Accession numbers were listed in Supplementary Table 1).

Bioinformatic analysis
A total of 2,289 complete E. coli genome sequences available by October 2020 were downloaded from NCBI GenBank. The genes were annotated using Prodigal version 2.6.3 software, and the resulting protein sequences were functionally annotated and assigned to cluster of orthologous groups (COCG) categories using Eggnog-mapper v2.1.6. The homologs of the eukaryotic viperin protein (rSAM (COG 0535)) were searched within the annotated COG category. All COG0535 proteins were then analyzed for nearby proteins to determine the operon structure. The conserved domains of the HXS system were identified using Motif Search 46. The signal peptides of the HxsA protein were predicted using SignalP 6.0 47.

Phage challenge assay
Phage challenge assays were performed as described previously 45. Briefly, empty vector or pSEC1 constructs expressing individual defense systems were transformed into E. coli MG1655 cells. Transformants were grown with the appropriate antibiotics to mid-log phase (typically OD₆₀₀ ≈ 0.3-0.4). For each assay, 300 μL of culture was mixed with 8 mL of top agar containing the same antibiotics and poured evenly onto LB agar plates. Each of 113 phages (111 phages mentioned above plus T4 and P1 phages) was tenfold serially diluted individually, and 1 μL of each dilution was spotted onto the bacterial lawn. Plates were incubated overnight at 37 °C, and plaque formation was recorded. Antiphage efficacy was expressed as the endpoint-dilution ratio: the highest dilution that produced ≥1 plaque on the empty-vector lawn divided by the highest dilution that produced ≥1 plaque on the defense-system lawn. Unless stated otherwise, data represent ≥3 biological replicates.

Phage-infection dynamics in liquid medium
Overnight culture of E. coli BL21 (DE3) expressing HXS defense system (or empty vector control) was inoculated into fresh LB medium supplemented with 50 µg/mL kanamycin and incubated at 37 ℃ with a shaking speed of 200 rpm. When cultures reached 4×107 cells/mL, 180 µL aliquots were dispensed into microwells of a 100-well plate, and 20 µL of phage suspension was added to achieve multiplicities of infection (MOI) of 5 and 0.05 (two parallel conditions). Plates were instantly placed in an automated growth curve analysis system (Oy Growth Curves Ab Ltd Bioscreen C), and OD600 of the culture was recorded every 10 min for 8h at 37 °C with continuous shaking. Bacterial growth curve was plotted using GraphPad Prism software.

Phage adsorption assay
Overnight cultures of E. coli BL21 (DE3) containing the HXS system or the empty control were transferred to fresh LB medium at a ratio of 1:100 and incubated at 37 ℃ with a shaking speed of 200 rpm. When cultures reached 1×108 cells/mL, T7 phage were added to a final concentration of 2×103 PFU/mL, and the infection mixture was incubated at 37 ℃ with a shaking speed of 200 rpm. At 0, 1, 5, 10, and 15 minutes after infection, 1 mL of the infection mixture was collected and centrifuged at 12,000 g for 90 seconds. 100 uL of the supernatant was collected and titrated for the unadsorbed free phage by plaque assay.

Selection of T7 phage mutants escaping the HXS system
300 μL of an overnight culture of E. coli BL21 (DE3) cells expressing the HXS system was mixed with 8 mL of top agar and poured onto LB agar plates. Tenfold serial dilutions of T7 were prepared, and 2.5 µL of each dilution was spotted onto the HXS lawn. Plates were incubated overnight at 37°C. The resulting plaque was collected into an Eppendorf tube containing 1 mL of Pi-Mg buffer. After 1 h incubation at room temperature with gentle vertexing, phage was titrated and used for the next round of selection (serial passage) using the same procedure. Rounds of selection were repeated until T7 formed plaques on the HXS-expressing BL21(DE3) lawn with an efficiency comparable to the empty-vector control lawn. After the final round, escape phage was purified by single-plaque isolation prior to amplification and downstream analyses.

Genomic sequencing of escaped T7 phage mutants
Escaped phages were propagated as described previously 45. Briefly, 1 L of E. coli BL21(DE3) cells were infected with the escape phage at a MOI of 0.05. After 5 hours incubation at 37°C, E. coli cells were removed by centrifugation at 5,600 g for 20 min, and the supernatant was filtered through a 0.22 μm filter to further remove bacterial debris. The phages in the supernatant were pelleted at 30,000 g for 30 min at 4 °C, then resuspended in Pi-Mg buffer. For genomic DNA extraction, phage resuspensions were treated with DNase I (10 μg/mL) and RNaseA (10 μg/mL) to remove contaminating bacterial DNA. EDTA (5 mM) and SDS (35 mM) were then added to inactivate nuclease and lyse phages for 20 minutes at 65°C. Lysates were then treated with phenol/chloroform/isoamyl alcohol (25:24:1), followed by chloroform/isoamyl alcohol (24:1) to remove proteins. T7 genomic DNAs in the aqueous phase was precipitated with isopropanol, pelleted by centrifugation at 11,000 g, and washed twice with 70% ethanol. The T7 genomic DNA precipitate was dissolved in distilled water and was sent for sequencing. Mutation sites were identified by sequence alignment of wild-type and mutant T7 genome. Primers were designed to amplify the mutation sites, and PCR products were sequenced by sanger sequencing to further confirm the mutations.

Genome editing of T7 phage
Recombinant T7 phages carrying defined mutations in gene 15 or gene 16 were generated individually using CRISPR-Cas technology as described previously 48. Briefly, five CRISPR-Cas12a plasmids, each expressing a single spacer targeting one of the five mutation sites, were constructed. The sequence of spacers is shown in Supplementary Table 4. Donor plasmids containing the desired mutation and flanking homology arms were constructed using standard methods. For each edit, donor plasmid and the corresponding CRISPR-Cas12a plasmid were co-transformed into E. coli B834 cells. 100 μL of Pi-Mg buffer containing different number of wild-type T7 phages were mixed with 300 μL of the freshly cultured E. coli B834 cells, poured onto LB agar plates, and incubated overnight at 37°C to generate the T7 phage mutants. E. coli B834 cells containing only the CRISPR-Cas12a plasmid were used as controls. Individual plaques obtained on the co-transformed strain were picked and plaque-purified, and the targeted loci were PCR-amplified and verified by Sanger sequencing to confirm the intended mutations.

Preparation of T7 phage with methylated genomic DNA
The dam gene was PCR-amplified from the E. coli strain MG1655 and cloned into pET-22b expression vector to generate pET-Dam plasmid, which was then transformed into E. coli BL21. Overnight cultured E. coli BL21 cells were inoculated into fresh LB medium at a 1:100 dilution and grown at 37 °C with shaking. When cultures reached OD =0.6, expression of Dam was induced with IPTG (1 mM) for 2 hours. E. coli BL21 cells were then infected with T7 phage at a MOI of 0.4. After 3 hours incubation at 37 °C, the infection mixture was collected and phages were prepared as described above. Methylation of T7 genomic DNA was confirmed by DpnI digestion, which specifically cleaves the Dam-methylated DNA. T7 phage prepared by infecting E. coli BL21 cells with pET-22b empty vector was used as a control.

Southern blot
E. coli BL21 cells containing pET22b-Dam were transformed with the HXS system-expressing plasmid or empty vector. Transformants were grown in LB medium at 37 °C until the OD600 reached 0.6, then infected with T7 phage at a MOI of 0.4. 100 μL of the infection mixture were collected every 2 min for the first 20 min, and every 10 min thereafter up to 90 min. Each sample was immediately mixed with an equal amount of phenol/alcohol solution. After centrifugation at 10,625 g for 2 min, the pellet was resuspended with Tris-HCl buffer containing proteinase K (100 μg/uL), followed by incubation at 37 °C for 1 h. T7 phage genomic DNAs were extracted using the phenol-chloroform-isoamyl alcohol as described above and treated with DpnI (1 μL/μg) at 37 °C for 1 h to digest methylated DNA. The DNA samples were then analyzed by electrophoresis on 0.8% agarose gels and transferred to nylon membranes using the capillary transfer method with an alkaline transfer solution (400 mM NaOH, 1 M NaCl). Membrane was equilibrated in 6×SSC buffer (0.9 M NaCl, 0.09 M sodium citrate tribasic dehydrate) for 2 min and pre-hybridized in hybridization buffer (6×SSC, 5×Denhardt solution, 0.5% SDS and 1 mg/ml salmon sperm DNA) for 2-4 h at 42 ℃. Head and tail fragments of the T7 phage genome were individually amplified by PCR (Supplementary Table 5), biotin-labelled using a Biotin Random Prime DNA Labeling Kit (Beyotime, China), and used as probes for Southern blot. Membranes were then hybridized overnight with biotin-labelled DNA probes at 42 ℃ and developed with the chemiluminescence detection kit (Beyotime, China) according to the manufacturer’s instructions. Uncropped and unprocessed scans of the blots are provided in the Source Data file.

Expression and purification of HxsA, HxsB, HxsC, and HxsD protein
Recombinant HxsA, HxsB, HxsC, and HxsD proteins each with a hexa-histidine tag were purified by nickel-based affinity chromatography. Briefly, the HxsA, HxsB, HxsC, and HxsD genes were cloned into the pET32a vector. HxsA proteins were induced with 1 mM IPTG at 28°C for 5 hours upon reaching an OD of 0.6-0.8, whereas HxsB, HxsC and HxsD were induced at 16 °C overnight. A Strep-tagged HxsC protein was generated for pull-down assays and expressed in E. coli under the same induction conditions. For His-tagged proteins, E. coli cells were harvested, resuspended in binding buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM imidazole, and 5 mg/mL DNase I), and disrupted by high-pressure cell disruption. Cell debris was removed by high-speed centrifugation, and proteins were then purified using a HisTrap column and analyzed by SDS-PAGE. For Strep-tagged HxsC, cells expressing Strep-HxsC were resuspended in equilibration buffer (100 mM Tris pH 8.0, 150 mM NaCl), lysed and purified using Strep-Tactin affinity resin. Eluted proteins were further purified by size-exclusion chromatography. All purified proteins were analyzed by SDS-PAGE, and concentrations were estimated using a BSA standard.

Preparation of the HxsA-, HxsB-, HxsC-, and HxsD-specific polyclonal antibodies
Six-week-old female BALB/c mice (n=15) were purchased from Laboratory Animal Center of Huazhong Agricultural University (Wuhan, China). Mice were randomly assigned to five groups (n=3 per group). The purified HxsA, HxsB, HxsC, and HxsD proteins were emulsified with complete Freund's adjuvant for the primary immunization and with incomplete Freund's adjuvant for booster immunizations, and administered subcutaneously to mice (100 μg of each protein per dose) at weeks 0 and 2. Mice in the control group (n=3) were immunized with PBS. Serum samples were collected 14 days after the last immunization, and antigen-specific antibody titers were determined by ELISA. Sera were aliquoted and stored at -80°C.

Antibody detection in sera
Serum samples (n = 3) were collected 2 weeks after the second immunization. ELISA plates were coated overnight at 4 °C with 200 ng/well of purified His-tag HxsB, HxsC, or HxsD protein. Plates were washed five times with PBS containing 0.05% Tween 20 (PBST) and blocked with 3% BSA in PBST for 1 h at 37 °C. Serum samples were serially diluted in 1% BSA in PBST and incubated on the coated plates at 37 °C for 1 h. Following five washes with PBST, peroxidase-conjugated goat anti-mouse IgG was added and incubated for 1 h at 37 °C. Plates were washed five times with PBS-T, and bound antibodies were detected using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate. The reaction was stopped with 2 M H2SO4, and absorbance was measured at 450 nm using a microplate reader.

Western blot
E. coli BL21 cells containing the wild-type or mutant HXS systems were cultured overnight, collected by centrifugation, and resuspended in SDS-PAGE loading buffer. Proteins were separated by SDS-PAGE and transferred to 0.22-μm polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk in TBST at 37°C for 2 h, the membranes were incubated with primary antibodies (anti-HxsA, anti-HxsB, anti-HxsC, or anti-HxsD; 1:8,000 dilution in TBST) overnight at 4°C. Membranes were then washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibodies (ABclonal, catalog #AS003; 1:8,000 dilution in TBST) for 1 h at room temperature. Protein bands were visualized using ECL chemiluminescence (Bio-Rad). GFP-specific antibody (Abconal, catalog #AE012; 1:8,000 dilution in TBST) and GAPDH-specific antibody (Abconal, catalog #AC002; 1:8,000 dilution in TBST) were used for western blot analysis of GFP expression under the control of σ70 promoter A1 in the presence of the HXS system. Uncropped and unprocessed scans of the blots are provided in the Source Data file.

Preparation of the bacterial cytoplasmic and periplasmic proteins
Cytoplasmic and periplasmic proteins of E. coli cells were separated as previously described 38,39. Briefly, E. coli expressing the HXS system or its mutants were diluted 1:100 in fresh LB medium supplemented with kanamycin and grown overnight at 37 °C with shaking. Cells were collected by centrifugation at 4,816 g for 15 min and resuspended in PBS (pH 7.5). After centrifugation at 20,817g for 3 min, the supernatant was discarded. The cell pellet was gently resuspended in Buffer I (100 mM Tris-HCl pH 8.0, 500 mM sucrose, 0.5 mM EDTA) and incubated on ice for 5 min, followed by centrifugation at 20,817g for 3 min at 4 ℃. The supernatant was discarded, and the pellet was gently resuspended in 1 mM Mg buffer and incubated on ice for 2 min. After centrifugation at 20,817g for 3 min at 4 ℃, the supernatant (periplasmic fraction) was collected. The cell pellet was washed twice with Buffer II (50 mM Tris-HCl pH 8.0, 250 mM sucrose, 10 mM MgSO4), and then resuspended in Buffer III (50 mM Tris-HCl pH 8.0, 2.5 mM EDTA). Cells were sonicated on ice and centrifuged at 6,708 g for 10 min at 4 °C to remove cell debris. This process was repeated until no visible bacterial cells or debris remained. The supernatant was subjected to ultracentrifugation at 108,726g for 20 min to separate the cytoplasmic proteins (supernatant) from the membrane fractions (pellet).

Expression and purification of gp15 and gp16 proteins
The gp15 and gp16 proteins were produced and purified as previously described 29,49. T7 phage 15 and 16 genes were PCR-amplified from T7 genomic DNA and cloned into the pET-16b expression vector, retaining the native N-terminal His-tag. The resulting plasmids (pET16b-gp15 and pET16b-gp16) was individually transformed into E. coli BL21 (DE3) competent cells. Single colonies were cultured overnight in LB medium containing 100 μg/mL ampicillin. Ten mL starter cultures were inoculated into 1 L LB broth supplemented with ampicillin and grown at 37 ℃ with shaking. Production of gp15 and gp16 proteins was induced with 0.5 mM IPTG at 37 ℃ for 2 hours when cultures reached an OD₆₀₀ of 0.6-0.8. Cells were harvested by centrifugation at 4,300g for 15 min at 4 ℃ and resuspended in lysis buffer (50 mM HEPES pH 8.0, 300 mM NaCl, 100 mM NDSB-201, 10% glycerol, and 1 mM PMSF) . Cell pellets were lysed at 4 ℃ using a high-pressure cell disruptor, and cell debris was removed by centrifugation at 35,000 g for 20 min at 4°C. The supernatant containing the recombinant proteins was incubated with Ni-NTA resin, washed with 50 mL wash buffer (50 mM HEPES pH 8.0, 300 mM NaCl, 20 mM NDSB-201, 10 mM imidazole, 5% glycerol, and 1 mM PMSF), and eluted with elution buffer (20 mM HEPES pH 8.0, 200 mM NaCl, and 400 mM imidazole). Proteins were further purified by size-exclusion chromatography (Superdex200 10/300 column, GE Healthcare Life Sciences) using gel filtration buffer (50 mM HEPES pH 8.0, 100 mM sodium citrate, and 10 mM MgCl2). Peak fractions were collected, and the purity of gp15 and gp16 proteins was analyzed by SDS-PAGE.

Interaction assay of gp15 or gp16 proteins with cleaved HxsA
To determine whether cleaved HxsA associates with gp15 or gp16, periplasmic protein fractions enriched for cleaved HxsA were prepared as described above. Pull-down assays were carried out using His-tag purification resin (Biyuntian Biotech). Briefly, 20 μg of His-tagged gp15 or gp16 proteins were incubated on ice overnight with 90 μL of periplasmic fraction containing cleaved HxsA protein. A total of 150 μL His-NTA resin was packed into a gravity-flow column, washed with pre-chilled water, and equilibrated with gel filtration buffer (50 mM HEPES pH 8.0, 100 mM sodium citrate, and 10 mM MgCl2). The protein mixture was then incubated with the equilibrated resin at 4 ℃ for 7 hours. As a negative control, 90 μL of periplasmic fraction (no hig-tagged gp15 or gp16) were incubated with resin under the same conditions. After incubation, the resin was washed with 10 mL wash buffer (50 mM Tris pH 7.5, 300 mM NaCl, and 20 mM imidazole), then resuspended in 100 μL of buffer (50 mM Tris pH 7.5, and 300 mM NaCl). Resin-bound proteins were analyzed by SDS-PAGE followed by immunoblotting using anti-HxsA antibodies (prepared as described above).

Mass spectrometry analysis of the cleaved HxsA protein
The cleaved HxsA protein was analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). Briefly, the E. coli BL21(DE3) cells containing the HXS system was cultured overnight at 37°C and collected by centrifugation at 6,708 g for 10 min. The pellets were resuspended in SDS-PAGE loading buffer, and the cleaved HxsA protein was separated on an SDS-PAGE gel and confirmed by western blotting using HxsA-specific antibodies. The band corresponding to cleaved HxsA on the second SDS-PAGE gel was carefully excised, and the HxsA proteins were recovered using electrophoresis. The sample was sent to the Sangon Biotech (Shanghai, China) for LC-MS/MS analysis. The recovered HxsA protein was divided into two aliquots and subjected to parallel in-solution digestion using either trypsin or chymotrypsin. Both digests were analyzed independently by LC-MS/MS.

MS data were acquired on a Q Exactive Plus mass spectrometer (Thermo scientific) coupled to an EASY nLC 1200 system (Thermo scientific). Briefly, samples were first loaded onto a nanoViper C18 trap column (3 μm, Thermo Scientific) for on-line desalting. After washing the trap column with loading solvent to remove salts, peptides were eluted from the trap and separated on an analytical C18 column (Acclaim PepMap RSLC, 75 μm × 25 cm, C18, 2 μm, 100 Å; Thermo Scientific) using solvent A (0.1% formic acid in water) and solvent B (80% acetonitrile, 0.1% formic acid). Peptide separation was performed at a flow rate of 0.300 μL/min with the following gradient: 5% B at 5.0 min, increased to 40% B at 12.0 min, ramped to 95% B at 14.0 min, and held at 95% B until 20.0 min for column washing. The system was then returned to 5% B at 20.1 min and re-equilibrated prior to the next injection. The total method time was 25 min. MS full scans were performed in the Orbitrap mass analyzer in the m/z range of 300-1500 with a resolution of 70,000, an automatic gain control (AGC) set at 3e6 and with a maximum injection time of 100 ms. The 20 most intense ions were isolated and further fragmented via high-energy collision dissociation (HCD). MS/MS (MS2) spectra were acquired in the Orbitrap over an m/z range of 200-2000 at a resolution of 17,500, with an AGC set at 1e5 and with a maximum injection time of 50 ms. We selected ions with charge state from 2+ to 5+ for screening. Normalized collision energy (NCE) was set at 28 and 30 and a dynamic exclusion of 25 s.

Raw MS data were processed using Proteome Discoverer (version 2.5, Thermo Fisher Scientific) and searched against the HxsA protein sequence. Carbamidomethylation of cysteine residues (+57.021 Da) was set as a static modification, whereas oxidation of methionine and tyrosine (+15.995 Da), as well as HxsB/C-catalyzed modifications (+12.000 Da or -2.016 Da) on histidine, serine and arginine residues, were set as dynamic modifications. The precursor ion mass tolerance was set to 10 ppm and the fragment ion mass tolerance to 0.02 Da. Peptide-spectrum matches were filtered using a false discovery rate of 1% at the peptide level. Modification site assignment was confirmed by manual inspection of fragment ion spectra.

Transmission electron microscopy
Overnight-grown E. coli Bl21 (DE3) harboring the HXS defense system or an empty vector were subcultured into fresh LB medium to 1×108 cells/mL and infected with purified phages at a multiplicity of infection (MOI) of 50. Samples were collected at 10-, 15-, and 25-min post-infection (empty vector control at 10 min only), chilled on ice, and centrifuged at 12,000g for 3 min at 4 °C to pellet cells with adsorbed phages. Pellets were washed twice and resuspended in pre-chilled, filter-sterilized Pi-Mg buffer. For transmission electron microscopy, 20 μL of the suspension was applied to copper grids for 1 min, blotted, reapplied once, negatively stained twice with 2% phosphotungstic acid, and imaged using a Talos L120C transmission electron microscope at 120 kV.

DNA-binding assay
The DNA-binding activity of cleaved HxsA was determined using the periplasmic fraction containing cleaved HxsA prepared as described above. 15 μL of the periplasmic fraction was mixed with 3′- and 5′-Cy3-labeled DNA in 20 μL of DNA-binding buffer (200 mM HEPES, 500 mM NaCl, pH 8.0) and incubated at 37 °C for 1 h. The reaction mixtures were run on a 6.5% native polyacrylamide gel in 0.5× TBE on ice for 1.5 h and visualized by chemiluminescence (Bio-Rad).

Identification of the HxsA cleavage site and signal peptide characterization
The Sec signal peptide MalE or the Tat signal peptide CueO 50,51 were inserted upstream of the HxsA cleavage site identified by mass spectrometry, as well as three upstream chymotrypsin cleavage sites. All constructs were cloned into the arabinose-inducible pBAD vector, and the resulting plasmids were transformed into E. coli BL21, respectively. Overnight-grown bacteria were inoculated into fresh medium, cultured to an OD600 of 0.6-0.8, and induced overnight with 0.2% arabinose. Whole-cell lysates and periplasmic fractions were prepared as described above and analyzed by western blotting with an anti-HxsA polyclonal antibodies.

Interaction assays of HxsB, HxsC, and HxsD
To examine interactions among HxsB, HxsC, and HxsD, proteins were prepared as described above and subjected to pull-down assays using Strep-tag affinity resin (Biyuntian Biotech). Briefly, Strep-tagged HxsC (300 pmol) was incubated with HxsB and HxsD at a molar ratio of 1:3 (HxsC:HxsB or HxsC:HxsD) or 1:3:3 (HxsC:HxsB:HxsD) on ice for 2 h. Strep-Tactin Sepharose resin was washed with water and equilibrated with equilibration buffer (100 mM Tris pH 8.0, 150 mM NaCl). The protein mixture was then loaded onto the equilibrated resin, washed with 8 mL equilibration buffer, and eluted with elution buffer (100 mM Tris pH 8.0, 150 mM NaCl, and 2.5 mM desthiobiotin). Resin-bound proteins were analyzed by SDS-PAGE. The structures of HxsB, HxsC, and HxsD proteins were predicted using AlphaFold352, and interface solvation free energies were analyzed with PDBePISA 53.

Assay of HXS effect on T7 phage transcription and translation
The T7 phage σ70 promoter A1, ribosome binding site, and intergenic region were cloned into a T vector by Gibson Assembly, and the GFP gene was placed under the control of σ70 promoter A1 to generate the plasmid PA1-GFP. The plasmid was transformed into E. coli MG1655 harboring the HXS system or an empty vector (EV). Colonies were inoculated into LB medium containing ampicillin and kanamycin and grown overnight. Plaque assays were performed to assess defense against wild-type T7 phage and a T7 mutant lacking the σ70 promoters A2 and A3 (previously constructed in the laboratory). GFP expression was quantified by western blotting using anti-GFP antibodies, with GAPDH as a loading control.

Statistics and reproducibility
Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software Inc., USA). Sample sizes were chosen according to similar experiments published in previous literatures16,54. No data were excluded from the analyses. Multi-group comparisons were assessed using one-way analysis of variance (ANOVA). Data are presented as the mean ± SD of three replicates, and p value < 0.05 was considered statistically significant. All plaque assays were performed in triplicate and the representative images were shown. The experiments were not randomized. The investigators were not blinded to outcome assessment.

Data Availability
The data generated in this study are presented in the Source Data file (see the Source Data file for the details). The GenBank accession numbers for all the 59 coliphages were included in the Supplementary Table 1. The protein mass spectrometry raw data have been deposited in the PRIDE repository under accession PXD063886.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Sun et al discover and characterize a new anti-phage defense system called HXS, which is conserved in Enterobacteria, and they find it defends against numerous phages in E. coli. This is a four-gene system with 3 proteins that are predicted to use Fe-S clusters to participate in transactions with SAM and radical derivatives. The authors demonstrate that all four genes and conserved residues within them are required for phage interference. They take a clever approach to obtain evidence supporting the idea that the HXS system specifically prevents DNA injection by T7 phage. Finally, they demonstrate that HxsA is a periplasmic component of the defense that is subject to proteolytic cleavage, which is dependent on the other Hxs proteins. The findings that DNA injection is affected, the periplasmic HxsA component of the defense, and T7 phage escape mutations in the DNA injection channel all support the idea that HXS-mediated interference occurs at the earliest stages of infection after phage adsorption.\nThis is a notable finding and relatively novel mode of anti-phage defense. The breadth of phages tested with HXS and other defenses is impressive, and I appreciate the unique approach to measure T7 phage DNA injection. However, some conclusions are significantly overstated and must be toned down. Furthermore, the mechanism of this system, beyond affecting DNA injection, remains largely unclear. My concerns and additional suggestions are listed below.\nMajor concerns:\n1. In the abstract, introduction, and discussion, there is extensive mention of the radical SAM nature of the HXS system. However, there is not a single experimental demonstration that any of these factors interact with SAM or generate radical SAM. The claim in the abstract “To our knowledge, this is the first report of a SAM-dependent system involved in antiphage defense” is unsupported by the data and regardless, untrue. The White group reported in Nature a SAM-dependent type III CRISPR effector CorA that forms membrane pores in response to infection (PMID: 37853119). Without additional data the authors must remove or at least qualify all conclusions of SAM-dependent enzymatic activity. Furthermore, the authors should at least speculate in the Discussion as to what the SAM-dependent reaction is. How does SAM fit into the model of anti-phage defense by HXS?\n2. Is the periplasmic localization of cleaved HxsA sufficient to mediate phage defense? Can the authors deliver the HxsA fragment to the periplasm by appending a signal peptide, then measure T7 interference? Or are the other components still required in this case?\n3. While the experiments in Fig. 3c-d provide good evidence that DNA injection is affected by HXS, additional support for this conclusion is warranted. Phage DNA injection is often associated with measurable potassium ion efflux. Is this affected by HXS? It would also perhaps be expected that the phage DNA remains in the capsid during HXS defense, this is a prediction that can be tested via electron microscopy or other methods.\nMinor concerns:\n1. The abstract and introduction are quite repetitive with the claims that “no anti-phage defense that affects DNA injection has been discovered”. The distinction between superinfection exclusion systems on phages and bacterial defenses is noted, but is somewhat semantic.\n2. Line 92 HxsAis\n3. Line 96 the phage DNA injection > remove “the”\n4. Line 128 unknow\n5. Line 128 HxsA…has been identified for more than a decade… citation?", "reviewer_2": "The manuscript describes a novel bacterial antiphage defense system, HXS, that reportedly blocks phage DNA injection through a mechanism involving radical SAM enzymes modifying a peptidoglycan-binding protein, HxsA. While the discovery of a SAM-dependent antiphage system is intriguing and potentially significant, I find the current study too preliminary to support the strong conclusions drawn.\nFirst, the functional role of the individual proteins (HxsB, HxsC, HxsD, and HxsA) is not clearly demonstrated beyond the proposed modification of HxsA. The mechanistic details of how these modifications disrupt phage DNA injection remain speculative and require further biochemical and structural validation.\nSecond, the authors claim broad-spectrum activity against most phages tested, but it is unclear if their phage collection includes phages capable of plasmid mobilization via transduction. Testing whether the HXS system affects the transduction of plasmids would provide strong evidence that DNA injection is indeed the inhibited step. This functional assay would greatly strengthen the conclusions.\nBy testing transduction or the mobility of other mobile genetic elements, such as phage satellites that rely on conserved mechanisms of DNA injection, the authors could further expand the novelty and relevance of their findings.\nOverall, the concept is promising, but more rigorous experiments clarifying the molecular mechanism and directly demonstrating inhibition of DNA injection, preferably with transducing phages that can inject different DNAs/MGEs, are necessary before this system can be firmly established.", "reviewer_3": "The manuscript by Sun et al. describes a novel anti-phage defense system they have named HXS, which is made up of a four-gene cassette HxsABCD. Expression of this system in an E. coli background resulted in reduced sensitivity to a large panel of E. coli phages. A fragment of HxsA is shown to be exported into the periplasm. Using phage T7 as an experimental system, the authors show greatly reduced DNA translocation in the presence of the HXS system. However, the rationale for the choice of phage T7 as the test system is not explained, and because this phage uses an unusual transcription-dependent DNA translocation mechanism, the major conclusion of the paper — that the HXS system broadly inhibits DNA translocation — is not firmly supported by the evidence provided. The provided results could also be consistent with a system that prevents early phage transcription or translation, and these issues need to be resolved. In general, inhibition of DNA translocation would need to be demonstrated for multiple phages with different DNA ejection strategies for paper’s claims on the HXS system mechanism of action to be supported.\nThe paper is generally well written, however the Materials and Methods section has a larger number of grammatical and language usage issues.\nGENERAL COMMENTS\nWhat types of phages are included among the 111 phages in the panel? Are they all of the same type, or does the collection comprise phages from different families or groups? This information would help to support the claim of broad anti-phage activity as claimed in Line 123.\nIt is not clear from the manuscript why phage T7 was selected specifically for follow-on experiments on HXS system activity. Phage T7 translocates its DNA via an unusual transcription-dependent mechanism (see below) that is not typical of most phages.\nPhage T7 translocates its DNA by an unusual mechanism that is dependent on initial transcription from sigma70 promoters followed by transcription driven by the phage RNA polymerase (gp1). Based on the evidence provided, it is not clear if the HXS system is interfering with DNA translocation per se or if it is simply inhibiting the transcription/translation functions that are required for DNA translocation in this phage. Most other phages do not rely on transcription/translation for DNA ejection, and it would be important to observe inhibition of DNA translocation in other phages to generalize this as the mode of action across all phages.\nRelated to the above comment, mutations in gp16 that allow for transcription-dependent translocation of T7 DNA have been reported (https://doi.org/10.1128/jb.178.23.6921-6929.1996), and these mutations would be consistent with the paper’s observations and would support the idea that the HXS system inhibits transcription/translation rather than translocation per se.\nHxsBCD are implicated in the cytoplasmic processing of HxsA, deletions of these genes result in loss of the 15 kD version of the protein. How do the authors reconcile N-terminal processing of the protein in the cytoplasm with its secretion via the sec system? If the N terminus of HxsA is removed it should no longer be able to be exported via sec. The cellular localization of HxsBCD were not confirmed in this study.\nSPECIFIC COMMENTS\nLine 41-42: SAM-dependent systems involved in anti-phage defense have previously been characterized. Examples include the SAM-dependent type 1 restriction-modification BREX mentioned in the work of Andriianov A et al., 2023 (Phage T3 overcome the BREX defense through SAM cleavage and inhibition of SAM synthesis by SAM lyase. 1016/j.celrep.2023.112972), or the SAM-AMP second messenger for antiviral signaling from Chi H et al., 2023 (Antiviral type III CRISPR signalling via conjugation of ATP and SAM. 10.1038/s41586-023006620-5)\nL 78: repetitive with lines 63-64 above.\nLine 90: The term \"radical S-adenosylmethionine domain\" should be abbreviated as \"rSAM domain,\" since \"SAM domain\" typically refers to the sterile alpha motif domain. This correction should be applied throughout the paper.\nLine 94: In the work of Haft and Basu, 2011 cited here, they say that genes neighboring the four-gene cassette are enriched in mobility markers which include some phage proteins homologs. Do you check the presence of this cassette in prophage genomes?\nLine 95: A space is missing between HxsAis\nLine 119 and 123: Why is Fig. 1c named before Fig. 1a? This causes confusion in the reading.\nLine 119: From which specific bacteria was the HXS system selected and cloned for the analysis in the paper? The methods section claims that the defense systems were cloned from a collection of E. coli, but it could be better to give more information on the specific strains that were selected for cloning each defense system.\nLine 130: Does a single mutation inside the conserved domains of HxsB and HxsC affect the antiphage activity?\nLine 145: Since phages are neither alive nor dead, the term \"generations\" could be replaced with \"cycles of dilution,\" as stated in Meyer J et al., 2016 (Ecological speciation of bacteriophage lambda in allopatry and sympatry, 10.1126/science.aai8446)\nLine 146-147: Are the mutants obtained isogenic, or did the mutations in Gp16 co-occur with mutations in Gp15?\nL148: silent\nLine 148: gp15 and gp16 as proteins should be written with the first letter as capital letter. This needs to be corrected all along the paper.\nLine 148: Word “gene” should not be in italics. Just the name of the gene should be written in italics. Correct all the manuscript as needed.\nLine 149: The non-coding region between which genes?\nFig 2a: The PFU should be removed as no PFU calculation was shown here.\nLines 152-153: Enhanced ability of T7 to escape could be observed in single mutation III on Gp15. Does this mutation occur in a sequence involved in the interaction zone between Gp15 and Gp16?\nLine 164: The statement that HXS lacks nuclease properties is based on experimental data?\nFig 2f: Is Gp16 involved in pore formation in the plasma membrane as represented in green? Please clarify.\nLine 171: DpnI instead of Dpn I all along the paper. Also, the 3 first letters of restriction enzymes names should be written in italics. Correct all the manuscript as needed.\nLine 195: The figures that show southern blot analysis are just 3c and 3d.\nLine 201-202: Why is the fragment I + V detected at the 10 mpi while the V band is not detected in Fig. 3c?\nLine 218: gene should not be in italics.\nLine 219: Since the unbound phages were not washed away, a negative control for the PCR assay could be performed. Does the PCR assay add support to the idea of HXS system blocking the translocation? As a control, can an assay of T7 genome electroporation be performed coupled with the PCR?\nLine 231: The correct form to name amino-terminus should be N-terminus or NH2-terminus\nLine 238: recombinant HxsA purified protein\nLine 263: The amount of protein in the double mutation D61A R65A could not be enough to support this idea. The results show that most of the protein is obtained as a cleaved peptide at the bottom of the image.\nLine 268-269: There is no visible signal in the cleavage peptide region so “completely inhibited the cleavage” could be a better observation.\nLines 304-305: As you are talking about genus, it should be only Escherichia and Pseudomonas.\nLine 307: The same as the previous point. It should be Escherichia and not E. coli.\nLine 336: NH2-terminal domain since NH2 is not a designated domain.\nLine 455: MOI\nLine 484: As a gene dam should be written without capital letter.\nLine 499: every instead of very.\nLine 505: Add space between number and units (1 µg/µL). Check the whole manuscript for similar mistakes. Check the same for temperatures as 4°C in line 545.\nLine 568: If you are using gp15 and gp16 as proteins, you should write them in capital letters. Also, genes are expressed not proteins. You can change the term “expressed” to “produced”.\nLine 569: gene without italics.\nLine 574: As genes, gp15 and gp16 should be in italics.\nLine 570-571: For interaction assays the native His-tag from pET16b was conserved?\nLine 763: Rounds of selection or cycles of selection instead of generations of selection (Line 30 of Supplementary materials should be corrected accordingly)\nLine 807: Western blot instead of West blot\nFigures\nFig. 1e: A color different from light gray could be picked to not get lost in the picture.\nFig. 2c: Correct the II in the Figure.\nFig. 2 and Fig. 4: The dilution factors in the image could be fixed. It is usually marked as dilution 10-1, 10-2, 10-3, etc.\nFig. 4a caption: this is not an efficiency of plating as you are not providing the actual number. This could be called a plaque assay or an infection assay.\nFig. 5b: Remove PFU\nSupplementary Table 1: The typography of deletion symbols (Δ) must be changed in columns 5 and 6 of the table.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank the reviewer for the very positive and thoughtful assessment of our work.\nWe are grateful for the recognition that HXS represents a relatively novel mode of anti-\nphage defense, that the breadth of the phage panel is a strength of the study, and that\nour approach to monitoring DNA entry and mapping HxsA as a periplasmic effector is\ninformative for dissecting the earliest stages of infection. At the same time, we fully\nagree with the reviewer that some of our original conclusions were overstated and that\nimportant aspects of the mechanism remain to be clarified. In the revised manuscript,\nwe have therefore (i) toned down and refined our mechanistic claims, (ii) added new\nbiochemical and genetic data on HxsA maturation and the roles of HxsB-D, and (iii)\nclarified that we present an rSAM enzyme-mediated periplasmic interception\nmechanism as a working model supported by our data rather than as a fully resolved\npathway.\nMajor concerns:\n1. In the abstract, introduction, and discussion, there is extensive mention of the radical\nSAM nature of the HXS system. However, there is not a single experimental\ndemonstration that any of these factors interact with SAM or generate radical SAM.\nThe claim in the abstract “To our knowledge, this is the first report of a SAM-dependent\nsystem involved in antiphage defense” is unsupported by the data and regardless, untrue.\nThe White group reported in Nature a SAM-dependent type III CRISPR effector CorA\nthat forms membrane pores in response to infection (PMID: 37853119). Without\nadditional data the authors must remove or at least qualify all conclusions of SAM-\ndependent enzymatic activity. Furthermore, the authors should at least speculate in the\nDiscussion as to what the SAM-dependent reaction is. How does SAM fit into the\nmodel of anti-phage defense by HXS?\nResponse:\nWe thank the reviewer for pointing this out. In our original submission, our\nwording was indeed imprecise: our system relies specifically on radical SAM (rSAM)\nenzymes HxsB and HxsC, rather than being a generic SAM-dependent defense system,\nexamples of which have already been reported. In the revised manuscript we have both\n(i) substantially toned down and refined our claims regarding SAM enzyme dependence\nand (ii) added new biochemical data on rSAM enzyme-dependent maturation of HxsA.\n(i) Toning down and clarifying rSAM enzyme-related claims\nFirst, throughout the manuscript we have replaced the term “SAM-dependent\ndefense system” with “rSAM enzyme-dependent defense system” when referring to\nHXS, to more accurately reflect that our data concern the radical SAM enzymes HxsB\nand HxsC. Second, in the revised Discussion (Page 13, lines 365-368 of the “Marked\nUp Manuscript”) we now explicitly cite SAM-utilizing defense pathways such as SAM-\ndependent restriction in BREX and SAM-AMP signaling in type III CRISPR-Cas\nsystems, and we clearly distinguish these systems from HXS. The former employ SAM\nin canonical methyltransferase reactions or as a small-molecule second messenger and\ndo not involve rSAM enzymes modifying a protein effector in the manner we observe\nfor HXS. We therefore now emphasize that our work instead establishes an rSAM\nenzyme-modified protein effector in antiphage defense, rather than claiming that we\nhave discovered the first SAM-dependent defense system.\nWe propose that HxsB/HxsC act as rSAM enzymes that install a covalent\nmodification within the SHRSS motif, thereby enabling mature HxsA to engage\nincoming phage DNA electrostatically in the periplasm (see below) (Pages 10-11, lines\n265-295 of the “Marked Up Manuscript”). We explicitly note that we do not yet\npinpoint the exact chemical nature of the +8 Da modification or directly detect SAM\nradicals, and we frame this as an important avenue for future biochemical work.\n(ii) New biochemical evidence for rSAM enzymes-mediated maturation of HxsA\nIn the revised Results (Pages 9-11, lines 252-310 of the “Marked Up Manuscript”),\nwe now show that HxsA is proteolytically processed into a ~15 kDa form whose peptide\ncoverage spans residues 48-214 of HxsA. Liquid chromatography-high-resolution mass\nspectrometry reveals a peptide corresponding to HxsA (48-79) carrying a discrete +8.0\nDa mass shift. High-resolution MS/MS localizes this +8 Da modification to the SHRSS\nmotif within this segment. Site-directed mutagenesis of the SHRSS motif (S to A, H to\nA, or terminal S to A) abolishes HXS-mediated phage resistance. Active-site mutations\nin the two rSAM enzymes HxsB and HxsC (4Fe-4S cluster motifs) abolish HxsA\ncleavage and defense activity. These data collectively support a model in which\nHxsB/HxsC (together with HxsD) catalyze an rSAM enzyme-dependent covalent\nmaturation of HxsA, involving both N-terminal processing and a site-specific\nmodification at the SHRSS motif, which is essential for antiphage function.\n2. Is the periplasmic localization of cleaved HxsA sufficient to mediate phage defense?\nCan the authors deliver the HxsA fragment to the periplasm by appending a signal\npeptide, then measure T7 interference? Or are the other components still required in\nthis case?\nResponse:\nWe thank the reviewer for this constructive suggestion regarding the sufficiency\nof periplasmic HxsA, and we have performed additional experiments to address it\ndirectly in the revised manuscript (Page 10, lines 256-264 of the “Marked Up\nManuscript”).\nBased on LC-MS/MS mapping, the processed HxsA comprises residues 48-214.\nWithin the upstream region (residues 23-47), we identified four chymotryptic cleavage\nsites and showed by mutagenesis that residues 23-47 are required for antiphage activity\n(Fig. 4a-b).\nWe then constructed four N-terminal truncations of HxsA starting at each of these\nsites within residues 23-47 and fused each truncation to either a Tat or Sec signal peptide\n(eight constructs in total) (Supplementary Fig. 11). All eight constructs were expressed\nin cells lacking the native HXS system. Subcellular fractionation and Western blotting\nconfirmed that all eight HxsA variants are efficiently exported to the periplasm.\nHowever, none of these periplasm-targeted HxsA constructs conferred detectable\nantiphage protection in plaque assays.\nThese data are now described in the Results (Page 10, lines 257-263 of the\n“Marked Up Manuscript”) and shown in Fig. 4a-b and Supplementary Fig. 11. They\ndemonstrate that simply localizing cleaved HxsA to the periplasm is not sufficient for\ndefense. The activity of HxsB, HxsC, and HxsD is required beyond cleavage and\nperiplasmic targeting, consistent with their role in rSAM enzyme-guided maturation\nand possibly in setting up the correct modification/processing state of HxsA. We discuss\nthis explicitly in the revised Results and Discussion.\n3. While the experiments in Fig. 3c-d provide good evidence that DNA injection is\naffected by HXS, additional support for this conclusion is warranted. Phage DNA\ninjection is often associated with measurable potassium ion efflux. Is this affected by\nHXS? It would also perhaps be expected that the phage DNA remains in the capsid\nduring HXS defense, this is a prediction that can be tested via electron microscopy or\nother methods.\nResponse:\nWe appreciate the suggestion and have strengthened our evidence that HXS\ninhibits phage DNA entry in several ways.\n(i) Direct visualization of DNA entry by transmission electron microscopy\nFollowing the reviewer’s suggestion, we performed time-resolved transmission\nelectron microscopy of T7 infection (Fig. 2e) (Page 7, lines 178-183 of the “Marked\nUp Manuscript”). In this assay, DNA-emptied capsids stain dark, whereas DNA filled\ncapsids appear light (Nguyen H T et al, Sensors and Actuators Reports, 2024). We\nfound that in control cells lacking the HXS system, most T7 capsids are dark by 10 min\npost-infection, indicating completed genome entry. By contrast, in cells expressing\nHXS, T7 capsids remain largely light even at 25 min, in agreement with the delayed\nentry detected in the DpnI assay. These images provide direct evidence that HXS\nimpede genome ejection.\n(ii) A second phage as an independent test\nTo address generality across phages, we extended DNA-entry assays to a\nChristensenvirus phage, which is taxonomically distinct from T7 and is likely to use a\ndifferent DNA entry mechanism. We performed time-resolved transmission electron\nmicroscopy analysis (Fig. 2e). These experiments show that HXS also impairs genome\nentry of this second phage, supporting DNA entry as the primary step affected rather\nthan a T7-specific phenomenon (Pages 8-9, lines 195-198 of the “Marked Up\nManuscript”).\nAs the reviewer notes, global potassium efflux assays can report on membrane\npermeabilization associated with DNA ejection. We considered this approach but there\nare no such instruments in our university can do efflux assay. Given the clear,\northogonal evidence from DpnI digestion and direct visualization by TEM for two\ndifferent phages, we believe the mechanistic conclusion that HXS impairs phage\ngenome entry is now well supported.\nMinor concerns:\n1. The abstract and introduction are quite repetitive with the claims that “no anti-phage\ndefense that affects DNA injection has been discovered”. The distinction between\nsuperinfection exclusion systems on phages and bacterial defenses is noted, but is\nsomewhat semantic.\nResponse:\nWe have revised the Abstract and streamlined the Introduction to soften the\nnovelty claims and reduce repetition. We now state that inhibition of genome entry has\nhistorically been attributed to phage-encoded superinfection exclusion (Sie) systems\n(e.g., T4 Imm/Sp) and that bacterial defenses acting at this step have remained\n“underexplored.” rather than completely unknown. We emphasize that HXS is an\nintrinsic bacterial defense that interferes with DNA entry, but we no longer claim that\nit is the first system ever to do so, and we now compare it explicitly with Sie systems.\n2. Line 92 HxsAis\n3. Line 96 the phage DNA injection > remove “the”\n4. Line 128 unknow\n5. Line 128 HxsA…has been identified for more than a decade… citation?\nHave done."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have undertaken a great deal of new experiments to address reviewer concerns. The additions of TEM images are helpful, and MS identification of the +8 Da modification is a significant advance. It is somewhat surprising that no single HXS-sensitive transducing phage could be found to test the idea posed by Reviewer 3. While I concur with the other reviewers that the exact modification and mechanism of action remains incomplete for the HXS defense, it seems a tall order to have it addressed in this single paper. I think the paper is suitable for publication and only have one minor point.\nMinor point:\nAbstract: “no bacterial system has been identified that specifically target the phage DNA entry phase” This is still not quite accurate. I would argue the periplasmic Zorya system targets phage DNA entry. See doi.org/10.1038/s41586-024-08493-8. Perhaps a more accurate phrasing would be “no system has been demonstrated to inhibit phage DNA ejection”", "reviewer_2": "The authors have substantially strengthened the manuscript and addressed the major concerns raised in my initial review. The revised study now provides convincing, orthogonal evidence that the HXS system interferes with phage genome entry, including DNA-entry kinetics, escape mutations in ejectosome components, and time-resolved electron microscopy. The identification of HxsA as a post-translationally matured, periplasmic effector, supported by genetic and mass spectrometric analyses, significantly improves the mechanistic clarity.\nThe authors have appropriately tempered claims regarding SAM dependence and now present rSAM enzyme-mediated maturation of HxsA as a well-supported model rather than a fully resolved biochemical mechanism. While important biochemical details remain to be elucidated, these limitations are clearly acknowledged and do not undermine the central conclusions. Overall, the manuscript is no longer preliminary and I support publication.", "reviewer_3": "I would like to thank the authors for their consideration of my comments and revisions to the manuscript. I have two remaining major comments on the revised work.\n1) The issue with protein secretion despite N-terminal processing remains unresolved. While the additional experiments conducted by the authors show the N-terminal signal is required for secretion and activity, the fusion of the HxsA signal with GFP does not prove that it is not sufficient. GFP is known to be a poor substrate for secretion by the sec system (cf Linton et al 2012 PMID: 21834133), and a positive control for GFP secretion (e.g., GFP fused to a known good SPI signal) was not included in the experiment shown in Figure S8. Thus the evidence that the HxsA signal is abnormal or insufficient is lacking. If anything this finding makes the proposed model more inconsistent, as it requires N-terminal processing of HxsA in the cytoplasm while also showing that the N-terminal SPI signal is required for its activity. Is processing of HxsA after initiation of secretion possible, or could the other Hxs proteins also play a role in secretion?\n2) The model for HxsA activity in the periplasm shown in Figure 5 and described on lines 377-384 is inconsistent with what is known about the phage DNA ejection process. There is no evidence that phage DNA is ever exposed in the periplasm; thorough structural studies of the T7 infection process in particular show the presence of a complex protein channel that spans the periplasmic space and protects phage DNA. Likewise, the myophage tail tube would provide a barrier for HxsA accessing the phage DNA in the periplasm. It’s possible HxsA could be associated with the inner or outer membrane and detect the formation of a channel by the phage tail, or perhaps respond to a signal such as PMF loss transduced from the cytoplasm.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We sincerely thank Reviewer #1 for taking the time to re-evaluate our manuscript and for the positive assessment. We are also deeply grateful for your highly constructive comments during the initial review, which were instrumental in improving the rigor and overall quality of our work. We deeply appreciate your recognition of our revision efforts and your understanding that fully resolving the mechanistic details of the HXS system is a complex undertaking best suited for future studies.\nMinor point:\nAbstract: “no bacterial system has been identified that specifically target the phage DNA entry phase” This is still not quite accurate. I would argue the periplasmic Zorya system targets phage DNA entry. See doi.org/10.1038/s41586-024-08493-8. Perhaps a more accurate phrasing would be “no system has been demonstrated to inhibit phage DNA ejection”\nWe sincerely thank the reviewer for pointing this out. We agree that the previous statement was overly broad. Following your suggestion, we have revised the Abstract and Introduction to be more precise. Modifications made in the revised manuscript: The sentence in the Abstract (Lines 30-31) has been changed to: \"However, no system has been previously demonstrated to specifically inhibit phage genomic DNA translocation.\" We have also ensured that the Zorya system's role in targeting phage DNA entry is appropriately contextualized in the Introduction (Page 3, Lines 73-74).\nReviewer #2 (Remarks to the Author):\nThe authors have substantially strengthened the manuscript and addressed the major concerns raised in my initial review. The revised study now provides convincing, orthogonal evidence that the HXS system interferes with phage genome entry, including DNA-entry kinetics, escape mutations in ejectosome components, and time-resolved electron microscopy. The identification of HxsA as a post-translationally matured, periplasmic effector, supported by genetic and mass spectrometric analyses, significantly improves the mechanistic clarity.\nThe authors have appropriately tempered claims regarding SAM dependence and now present rSAM enzyme-mediated maturation of HxsA as a well-supported model rather than a fully resolved biochemical mechanism. While important biochemical details remain to be elucidated, these limitations are clearly acknowledged and do not undermine the central conclusions. Overall, the manuscript is no longer preliminary and I support publication.\nWe are grateful to Reviewer #2 for the highly encouraging comments and for recognizing our efforts during the revision process. We also wish to thank you for your insightful suggestions in the first round, which significantly helped us strengthen the manuscript. We sincerely appreciate your positive evaluation of our revised mechanistic model and your understanding that further biochemical details will be the focus of our ongoing research.\nReviewer #3 (Remarks to the Author):\nI would like to thank the authors for their consideration of my comments and revisions to the manuscript. I have two remaining major comments on the revised work.\nWe would like to express our sincere appreciation to Reviewer #3 for the rigorous, highly professional, and constructive evaluation of our revised manuscript. We are also deeply grateful for your profound insights and invaluable suggestions during the first round of review. By incorporating your suggestions, we have been able to correct our previous over-interpretations and significantly refine our working model. Detailed responses to your two remaining major comments are provided below.\n1) The issue with protein secretion despite N-terminal processing remains unresolved. While the additional experiments conducted by the authors show the N-terminal signal is required for secretion and activity, the fusion of the HxsA signal with GFP does not prove that it is not sufficient. GFP is known to be a poor substrate for secretion by the sec system (cf Linton et al 2012 PMID: 21834133), and a positive control for GFP secretion (e.g., GFP fused to a known good SPI signal) was not included in the experiment shown in Figure S8. Thus the evidence that the HxsA signal is abnormal or insufficient is lacking. If anything this finding makes the proposed model more inconsistent, as it requires N-terminal processing of HxsA in the cytoplasm while also showing that the N-terminal SPI signal is required for its activity. Is processing of HxsA after initiation of secretion possible, or could the other Hxs proteins also play a role in secretion?\nWe sincerely thank the reviewer for highlighting the insightful work by Linton et al. (2012) regarding the incompatibility of GFP with canonical Sec-dependent secretion. Initially, we intended to use the GFP fusion assay to complement our mutational analyses of the native HXS system (Fig. 3c-e), aiming to collectively demonstrate that HxsA translocation and N-terminal cleavage follow a highly specialized, non-canonical pathway, rather than relying solely on a simple canonical signal peptide (such as the classical Sec- or Tat-dependent pathways). However, since the exact nature of the predicted HxsA signal peptide remains undetermined, we fully realize now that using GFP, which is a notoriously poor Sec-substrate, might mislead readers into concluding that the native HxsA signal peptide alone is \"abnormal\" or \"insufficient.\" To eliminate this confusion and maintain the rigorous logic of the manuscript, we have removed this supplementary experiment (formerly Supplementary Fig. 8) from the revised manuscript.\nConsequently, our conclusion that HxsA translocation and N-terminal cleavage follow a highly specialized, non-canonical pathway remains supported by our mutational analyses of the native system (Fig. 3c-e). As we detailed in the manuscript, when the predicted signal peptide was deleted (HxsA ), the protein was still cleaved (Fig. 3c-d). Crucially, this atypical cleavage is strictly dependent on the presence of HxsB, HxsC, and HxsD (Fig. 3e). These results align with your insightful question regarding the role of other Hxs proteins. It is highly likely that the other Hxs proteins act as essential, dedicated chaperones or accessory translocators to actively mediate the export of HxsA, rather than it relying solely on a simple canonical signal peptide.\nAs appropriately noted by other reviewers during this revision process, fully resolving the precise biochemical choreography and spatial dynamics of this novel rSAM-mediated maturation and secretion pathway is a complex undertaking that exceeds the scope of this single, foundational study. Accordingly, we have revised the relevant text in the Results section (Pages 8-9, Lines 219-225) to explicitly embrace your proposed model. We now clearly state that the other Hxs proteins likely mediate this specialized secretion and acknowledge that \"the precise spatiotemporal dynamics of HxsA processing, whether it occurs co-translocationally or requires the other Hxs proteins as specific chaperones, remains an exciting avenue for future biochemical studies.\"\n2) The model for HxsA activity in the periplasm shown in Figure 5 and described on lines 377-384 is inconsistent with what is known about the phage DNA ejection process. There is no evidence that phage DNA is ever exposed in the periplasm; thorough structural studies of the T7 infection process in particular show the presence of a complex protein channel that spans the periplasmic space and protects phage DNA. Likewise, the myophage tail tube would provide a barrier for HxsA accessing the phage DNA in the periplasm. It’s possible HxsA could be associated with the inner or outer membrane and detect the formation of a channel by the phage tail, or perhaps respond to a signal such as PMF loss transduced from the cytoplasm.\nWe sincerely appreciate the reviewer’s rigorous correction regarding the structural mechanics of the phage DNA ejection process. We completely agree that for phages such as T7 and T4, the genomic DNA transits through a complex proteinaceous channel (e.g., the ejectisome or tail tube) that spans the periplasmic space to enter the host cell.\nFirst, we would like to clarify that the omission of the trans-envelope channel in the Fig. 5a was an inadvertent drawing oversight rather than an intentional depiction of \"naked\" DNA in the periplasm. We apologize for this confusion. Accordingly, we have corrected Fig. 5a to accurately depict the trans-envelope channel (Please see the revised Fig. 5a).\nFurthermore, while our in vitro data demonstrate that HxsA has the capacity to bind DNA due to its five tandem HxS repeats enriched in basic amino acids, we did not intend to claim that HxsA directly binds \"naked\" DNA in vivo. Instead, the in vivo interference with phage DNA injection is complex. We completely agree with your excellent alternative hypotheses and propose that HxsA might impede DNA entry through one or a combination of the following highly plausible mechanisms:\n1. Local electrostatic perturbations and transient binding: The highly positively charged HxS repeats may exert local electrostatic perturbations on the translocating genome precisely as it passes through the channel. Furthermore, if the phage DNA becomes momentarily exposed as it exits the inner membrane pore, HxsA could directly engage it.\n2. Sensing channel formation or secondary signals: As you insightfully suggested, HxsA might detect the formation of the tail channel, interact at the inner membrane interface, or respond to infection-induced secondary signals (such as the loss of proton motive force). Such sensing events could trigger a conformational activation of HxsA, prompting it to occlude the inner membrane pore or create a steric blockade that effectively seals off the DNA entry path.\n3. Dynamic interactions with the channel complex: Although our initial in vitro interaction assays did not detect strong binding between HxsA and the T7 ejectisome components (gp15 and gp16), we certainly cannot rule out weak, transient associations. It is possible that the infection process induces a conformational change in HxsA, subsequently allowing it to interact with gp15/gp16 to disrupt or jam the channel assembly, thereby mechanically obstructing DNA passage.\nAs appropriately noted by other reviewers during this revision process, fully resolving the precise biochemical choreography and spatial dynamics of this in vivo periplasmic interference is a complex undertaking that exceeds the scope of this single, foundational study. To ensure scientific rigor, we have corrected our working model in Fig. 5a to accurately depict the phage DNA enclosed within a spanning protein channel. We have also thoroughly revised the Discussion (Page 14, Line 379-395) to explicitly present these alternative mechanistic hypotheses, establishing them as the primary focus for our future investigations.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe would like to express our sincere appreciation to Reviewer #4 for participating in the evaluation of our manuscript. We are grateful for the time and effort dedicated to the review process, and we highly value the constructive feedback provided."}]

Decision letter