Cross-genus phage design through branching domain and conserved peptide interactions

Cross-genus phage design through branching domain and conserved peptide interactions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cross-genus phage design through branching domain and conserved peptide interactions Agnieszka Latka, Dorien Dams, Lennert Scholiers, Britt Van Mieghem, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8669920/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background Branched receptor-binding protein (RBP) systems enable bacteriophages to broaden their host range through the incorporation of two or more RBPs. Using the Klebsiella podophages KP32, K11, and KP34 as model systems, we experimentally validated the interaction between the branching domain of the primary RBP (RBP1) and the conserved docking peptide of the secondary RBP (RBP2) as an essential architectural pair enabling dual-RBP incorporation into the virion. Results Systematic engineering revealed that loss of either of these domains, the branching domain or the conserved peptide, abolishes RBP2 assembly, underscoring their structural role in organizing the branched configuration and demonstrating that the anchor domain is the sole element directly attaching the RBP complex to the virion. Exploiting this interaction, we engineered a chimeric phage based on the Klebsiella KP32 scaffold that was capable of cross-genus infection and productive propagation on both Klebsiella and Escherichia hosts. In contrast to previous approaches that required replacement of entire tail modules, this strategy achieved host-range reprogramming through modular domain swapping and positional relocation of RBPs (i.e., exchanging RBP1 and RBP2 positions). Conversely, an Escherichia phage K1F scaffold was also successfully engineered to infect Klebsiella . Conclusions Our study confirms that the RBP branching domain and the conserved peptide function as specific interacting partners. Our findings establish the conserved peptide as a universal docking element and highlight the structural flexibility of podoviruses to accommodate RBPs from different positional and taxonomic contexts. Collectively, this work provides a mechanistic framework for rational phage engineering and defines a general design principle for generating customized therapeutic phages with an expanded host spectrum, including cross-genus infectivity. Klebsiella phage host range receptor-binding protein depolymerase tailspike branching domain conserved peptide Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Bacteriophages evolved elaborate strategies to recognize and infect their host with high specificity. Initial contact with receptors present on the bacterial cell wall is made via phage receptor-binding proteins (RBPs), which can adopt the morphology of a tail fiber (TF, long fibrous) or tailspike (TSP, shorter and thicker protein, equipped with enzymatic activity) ( 1 ). While bacterial defense systems are crucial in blocking phage infection at various stages of its replication cycle, the receptor recognition by RBPs is widely regarded as the primary and most pivotal factor determining phage specificity ( 2 ). This was also highlighted by the application of newly developed AI tools for phage host prediction, pinpointing RBPs as key to predict a phage-host interaction at the strain level ( 3 , 4 ). The high RBP specificity is strongly exemplified by capsule-targeting Klebsiella phages, which are equipped with capsule-specific depolymerases. There are many monospecific Klebsiella phages, recognizing and infecting strains limited to one particular capsular type, with their RBP (TSP) responsible for capsule polysaccharide degradation ( 5 , 6 ). Klebsiella phages with two TSP types also exist (e.g., KP32, K11, K5-2, K5-4, KP192) ( 5 , 7 – 9 ). Multi-specific Klebsiella jumbo phages were also described with at least ten (RaK2), eleven (phage K64-1), or even fourteen (phage ϕKp24) different RBPs (TSPs), able to infect hosts with a broad range of capsular serotypes ( 10 – 12 ). Cryo-EM analysis revealed a hyperbranched complex organization of their RBP systems ( 10 , 12 ). Genome synteny typically observed for closely related phages is often broken in the RBP region ( 5 ). While the N-terminal part of the RBP remains conserved amongst close relatives, enabling RBP attachment to the virion (‘anchor’), the remaining central and C-terminal domains are highly variable ( 5 , 13 ). The latter are responsible for host specificity, substrate binding, folding, and trimerization and are together with the central domain frequently exchanged between phages through horizontal gene transfer events, reprogramming phage specificity ( 14 – 16 ). In some cases, as visualized using cryo-EM, the anchor is not directly fused with an enzymatic domain, but rather functions as a separate, short ‘adaptor’ protein to which other RBPs attach non-covalently (e.g., E. coli phage K1-5 and Salmonella phage SP6) ( 13 ). The concept of anchor as a minimal module sufficient for RBP attachment to the virion was experimentally confirmed via Klebsiella phage K11 engineering ( 16 ). When more RBPs are present in the branching system, only one type of RBP (primary RBP) bears an anchor responsible for the anchorage of all RBPs to the virion ( 5 , 13 ). In the case of a dual RBP system, the primary RBP is equipped with a branching domain (called a T4gp10-like domain or XD2/XD3 domain), immediately following the anchor domain, and providing an attachment site for the secondary RBP ( 5 , 10 , 17 – 22 ). Such a branched structure becomes clear when comparing mono-RBP Escherichia phage K1F (capsular type K1) and phage K1-5, possessing two enzymatically active RBPs (capsular types K1 and K5) ( 13 ). In the latter, a short conserved, N-terminal peptide of the secondary RBP was predicted as responsible for the attachment to the T4gp10-like domain of the primary RBP. Another model is Escherichia phage G7C equipped with two RBPs. Domains responsible for RBP complex formation were identified on the protein level: residues 138–375 of gp66 (primary RBP) were predicted to be homologous to T4gp10 domains XD2 and XD3, while the N-terminus of gp63.1 (secondary RBP) serves as an interaction partner ( 17 ). In the case of phages with multiple RBPs in a single system, the secondary RBP can also bear T4gp10-like domains, enabling attachment of the next RBP layer ( 10 , 12 , 18 , 20 ). The RBP assembly was in-depth examined for Escherichia phage CBA120 ( 18 ), where equivalent branching domains were detected for primary and secondary RBP, organizing its four RBPs into a three-layer system. The importance of the structural domains in complex assembly was also examined for Enterobacteria phage S117, a close relative of CBA120. Phage S117 was used as a scaffold for host range engineering, utilizing analogous RBPs from one phage family, equipped with the same structural domains (anchor, branching domain) but different specificity domains ( 22 ). The exchange of RBPs occupying an analogous position resulted in maintaining the full branched RBP complex but modified specificity. The structural domains (anchor, branching domain, and conserved peptide) were also detected in Klebsiella phages, and their RBPs systems were thoroughly modelled across different taxonomic clusters ( 5 ). In the past years, we have intensively studied the RBPs of Klebsiella phage KP32 as a model system. Klebsiella phage KP32 is a dual-RBP Przondovirus with podovirus morphology. Its system is composed of KP32gp37 as the primary RBP (RBP1), and KP32gp38 as the secondary RBP (RBP2). RBP1 starts with the conserved anchor domain at its N-terminus, followed by the branching domain, the enzymatic domain specific towards K3 capsular type, and finally an autocleavable chaperone in its C-terminus ( 5 , 7 ). The branching domain interacts at the protein level with the N-terminal conserved peptide of RBP2 ( 23 ). The conserved peptide of RBP2 is followed by an enzymatic domain with specificity towards K21/KL163 capsular types, a linker, carbohydrate-binding module, and lectin-like domain at its C-terminus. Previously, we described the function of the C-terminal domains ( 23 , 24 ) and the function of the anchor domain of RBP1 in attachment to the virion ( 16 ). In this work, we confirm the interaction between the branching domain of RBP1 and the conserved peptide of RBP2 on the level of the phage particle and exploit this interaction pair to create a cross-genus phage infecting K. pneumoniae and E. coli . We also investigated whether an Escherichia coli- specific phage could be turned into a Klebsiella- specific phage. Materials and methods Bacterial growth conditions Tryptone Soy Broth (TSB, Oxoid, Thermo Scientific) or Tryptone Soy Agar (TSA; TSB supplemented with 1.5 w/v % bacteriological agar, VWR) was used to culture K. pneumoniae strains. E. coli TOP10 (Invitrogen, Thermo Fisher Scientific) for plasmid propagation, and E. coli BL21(DE3) (Invitrogen, Thermo Fisher Scientific) for protein expression, were cultured in standard lysogeny broth (LB; 10 g Tryptone (VWR), 5 g yeast extract (VWR), 10 g sodium chloride (Fisher Scientific) or LB agar (LB supplemented with 1.5 w/v % bacteriological agar). For culturing of E. coli transformed with an entry (pVTE) or destination (pVTD3 or pVTD23) vectors, LB was supplemented with 5 w/v % sucrose (Fisher Scientific) and 100 µg/mL ampicillin (Carl Roth), or 50 µg /mL kanamycin (Carl Roth), respectively. Phage engineering Construction of chimeric RBP clusters using the VersaTile method Chimeric RBP clusters were assembled using a modified VersaTile protocol, as previously described by ( 24 , 25 ). First, a repository of tiles was created in the pVTE vector with VersaTile cloning. The selected tiles were assembled in a two-step tile assembly reaction. In the initial step, only the individual tiles were combined. The second step introduced the preliminary assembly into the destination vector. The first reaction was prepared with 46 nM of each tile per position, 15 U of BsaI, 3.5 U of T4 DNA ligase, 10× ligation buffer, and ultrapure water to a final volume of 20 µl. This mixture underwent 30 cycles of restriction-ligation (5 minutes at 37°C followed by 5 minutes at 22°C per cycle). Subsequently, 23 nM of the pVTD23 destination vector, 5 U of BsaI, and 1.875 U of T4 DNA ligase were added to the preassembled mixture. An additional 30 cycles of restriction-ligation were performed under the same conditions, followed by enzyme inactivation (5 minutes at 50°C and 5 minutes at 80°C). The final product was used for the transformation of chemically competent E. coli TOP10 cells. Plasmid DNA was extracted from overnight cultures using the GeneJET plasmid miniprep kit. Correct assembly of the RBP clusters was confirmed via colony PCR and sequencing. Primer sequences used for tile generation and the composition of RBP clusters are provided in Supplementary Tables S1, S2 (successfully rebooted phages) and S3 (phages for which rebooting was not successful), respectively. Construction of engineered phage genomes The phage genome was segmented into five overlapping fragments (F1-F5), each amplified using either PrimeStar polymerase (TaKaRa) or KAPA HiFi DNA polymerase (Roche) with primers listed in Supplementary Table S4. Each fragment included approximately 200 bp of overlap with adjacent fragments to facilitate seamless assembly via Gibson reaction. Fragment F4 encompassed the RBP cluster. To substitute the wild-type F4, PCR amplification was performed on a plasmid harboring the engineered RBP cluster (eF4, constructed as described above), yielding a linearized product. Gibson assembly was carried out using 0.05 pM of each purified linear fragment (DNA Clean & Concentrator kit, ZYMO RESEARCH) and Gibson master mix (( 26 ); Supplementary Table S5), incubated at 37°C for 30 minutes, followed by 60°C for 1 hour, and enzyme inactivation at 80°C for 20 minutes. The assembled genome was dialyzed against ultrapure water using MF-Millipore 0.025 µm MCE membranes (Fisher Scientific) and subsequently electroporated into electrocompetent E. coli 10G ELITE cells (LGC Genomics). Post-electroporation, cells were recovered in SOC medium (LGC Biosearch Technologies) at 37°C with shaking for 3 hours. Engineered virions were released by adding chloroform (1:10 v/v), followed by vortexing and centrifugation (5 min, 1700 × g). The supernatant was plated on a lawn of the respective Klebsiella pneumoniae host using the double agar overlay method ( 27 ). After overnight incubation, individual plaques were isolated, propagated in liquid culture (described below), and the phage DNA was extracted using the Quick-DNA Viral kit (ZYMO RESEARCH). Genome integrity was confirmed via Nanopore sequencing (Plasmidsaurus). Phage assays Phage propagation K. pneumoniae and E. coli host strains were cultured at 37°C with shaking in TSB or LB, respectively. Phage propagation was performed in three sequential rounds, each involving a 10-fold dilution of the phage suspension (single plaque resuspended in 200 µL PBS) into fresh bacterial cultures recognized by either RBP1 or RBP2. In each round, bacterial cultures at OD₆₀₀ ≈ 0.1–0.2 were mixed with phage suspensions at a 9:1 ratio and incubated for 1 hour at 37°C with shaking. Following incubation, 100 µL of chloroform was added, the mixture was vortexed, and centrifuged (1700 × g, 5 min, 4°C). The resulting supernatant was used for the subsequent propagation round. Phage titers were assessed before and after the three propagation rounds using a spot test. Bacterial lawns were prepared in triplicate by mixing 200 µL of K. pneumoniae or E. coli cultures (OD₆₀₀ ≥ 1) with 4 mL of soft agar (0.5 w/v % agar in TSB) and overlaying onto TSA plates. After solidification and drying, lawns were spotted with 10 µL of serial 10-fold dilutions of each phage suspension prepared in PBS. Plates were incubated overnight at 37°C, and plaques were counted to calculate phage titers (log₁₀ PFU/mL). To determine effective propagation, the initial titer, adjusted for the cumulative 1000-fold dilution across three rounds, was subtracted from the final titer, yielding Δlog₁₀ (PFU/mL). Statistical analysis Data distributions were assessed for normality and lognormality using the Shapiro-Wilk test, followed by visual inspection via quantile–quantile (QQ) plots. Statistical significance among groups was evaluated using one-way ANOVA, followed by Tukey’s post hoc multiple comparisons test (GraphPad Prism 9, San Diego, CA, USA). Significance levels were indicated as follows: * for p < 0.05; ** for 0.01 < p < 0.05; *** for 0.0001 < p < 0.01; **** for p < 0.0001. Results General approach To investigate and exploit the interaction between the branching domain of RBP1 and the conserved peptide of RBP2 at the phage level, different engineered phages were designed and constructed. The respective engineered phages were rebooted to infective phage particles, which were subsequently tested for their ability to propagate. As a source of RBP domains, other viruses with podovirus morphology were used. They differ in Klebsiella capsular serotype specificity: K11 (RBP1 of phage K11), K63 (RBP2 of phage KP34), compared to K3 and K21/KL163 (phage KP32 RBP1 and RBP2, respectively), as well as host genus specificity: Escherichia K1 (RBP1 of K1F). The RBP architecture or position in the RBP cluster also varied (Fig. 1 ). The RBP-donating phages also had a variable RBP architecture. Whereas Klebsiella phage K11 has a similar dual-RBP system similar to Klebsiella phage KP32, the first RBP of Klebsiella phage KP34 is truncated, resulting in a short adaptor protein to which a full-length RBP2 is attached via a similar branching domain. In contrast, E. coli phage K1F has a simple, mono-RBP system. Presence of non-cognate branching domain ensures proper RBP2 assembly Klebsiella phages KP32 and K11 belong to the same genus, Przondovirus , exhibiting 89.94% nt sequence identity across 88% of their genomes. Both phages have a typical branched organization in their dual-RBP system. The RBP1s share a 88% amino acid identity in their anchor domain and 67% identity in their corresponding branching domain. While the identity in the branching domain is somewhat lower at the sequence level, it shows a high structural similarity (TM score 0.95223). The C-terminal specificity domains are dissimilar, corresponding to different capsular type specificities (Fig. 2 A). In a previous study, we proved that the anchors of phages K11 and KP32 can be used interchangeably, without disturbing virion infectivity (Latka et al. 2021). In this work, we transplanted the entire RBP1 of phage K11 (K11gp17) to the phage KP32 scaffold, replacing its original full-length RBP1 (KP32gp37). The resulting engineered phage (KP32_gp37_A_B_E::K11gp17) and both the donating and accepting wild-type phages underwent three rounds of propagation on their respective hosts for RBP1 and RBP2. Afterwards, the phage titer was determined on both hosts (for RBP1 and RBP2), and the increase in phage titer was expressed relative to the titer of the starting, non-propagated phage suspension (Dlog10 PFU/mL) (Fig. 2 B). The host range of the engineered KP32 phage shifted to the host of the newly acquired RBP1 (capsular type K11) instead of the original host, while retaining infectivity on the original host of RBP2 (capsular type KL163) without apparent loss in infectivity. This indicates that the non-cognate branching domain, originating from phage K11, can still serve as a docking site for the wild-type RBP2. In other words, the conserved peptide of RBP2 of phage KP32 functionally interacts with the non-cognate branching domain of phage K11. We conclude that both the anchor domain and branching domain of phage KP32 can be exchanged with its functional equivalents of phage K11. Absence of the branching domain prevents incorporation of RBP2 into the virion In the previous experiment, we demonstrated that the branching domains of phages KP32 and K11 are functional equivalents, allowing proper assembly of RBP2 even when the primary RBP originates from a different phage. However, this does not prove that docking of RBP2 can be confined exclusively to the branching domain. To address this, we applied a domain deletion strategy to verify whether removal of the branching domain blocks RBP2 incorporation into the virion. First, we deleted the native branching domain from RBP1 (construct KP32_gp37ΔB), while retaining all other RBP1 domains, including its enzymatic region. This construct could not be rebooted into an infective phage (Supplementary Table S3). As an alternative approach with potentially higher chances of proper folding, we designed a second construct (KP32_gp37ΔE) in which the enzymatic domain was deleted, but the structural domains, including the anchor and branching domains, were preserved. This phage also failed to be rebooted (Supplementary Table S3). These outcomes indicate that the domain deletion strategy was ineffective for generating infective phages, possibly because truncations destabilize RBP1 or the overall architecture required for virion particle formation. Therefore, we adopted a third approach: replacing the branching and enzymatic domains of phage KP32 RBP1 with the full RBP2 of phage KP34 (construct KP32_gp37_B_E::KP34gp57), resulting in an RBP1 variant lacking the branching domain entirely (Fig. 3 ). This design was based on the rationale that phage KP34 RBP2 can fold independently from preceding structural domains, increasing the likelihood of successful assembly. Whereas wild-type phage KP32 propagated efficiently on hosts recognized by both RBPs (K3 and KL163), the engineered phage gained the ability to infect the KP34-specific host (K63) and accordingly lost infectivity on the K3 and KL163 host. This demonstrates that, although RBP2 remained encoded in the genome of the engineered phage, it could not be incorporated into the virion in the absence of the branching domain on RBP1. These findings strongly support the hypothesis that the branching domain is essential for RBP2 assembly. Conserved peptide is responsible for the attachment of RBP2 to the virion Having established that the branching domain of RBP1 is indispensable for RBP2 assembly, we next examined whether the conserved peptide of RBP2 fulfils its predicted role ( 5 ) as the interaction partner for the branching domain on the phage level. To test this, we engineered phage KP32_gp38ΔCP, in which the first 29 amino acids of RBP2 corresponding to the conserved peptide were deleted, while all other domains of RBP2 and the complete RBP1 remained intact. Propagation assays revealed that phage KP32_gp38ΔCP behaved similarly to wild-type phage KP32 on the RBP1-specific host (K3), confirming that deletion of conserved peptide does not impair RBP1 function (Fig. 4 ). In contrast, the engineered phage failed to propagate on the RBP2-specific host (KL163), indicating that RBP2 was not incorporated into the virion. This demonstrates that conserved peptide is essential for RBP2 assembly, even though the remaining domains of RBP2 were present in the genome. These findings align with previous protein-level observations: truncated RBP2 variants lacking conserved peptide were shown to fold correctly and retain enzymatic activity but failed to interact with RBP1 in vitro on the protein level ( 23 , 24 ). Together, the phage-level and protein-level data confirm that conserved peptide is not only critical for protein-protein interaction but also acts as a structural module required for the correct assembly of a dual-RBP system onto the virion. Conserved peptide enables accommodation of chimeric RBP2 and supports cross-genus host range expansion Having demonstrated that the branching domain of RBP1 and the conserved peptide of RBP2 are indispensable for proper assembly of the dual-RBP system, we next asked whether the role of the conserved peptide as a docking module is sufficiently generic to accommodate non-native RBPs. If the conserved peptide can mediate the incorporation of an RBP from a different phage scaffold, even one originating from a phage infecting another genus, it would reveal a remarkable level of structural flexibility and establish the conserved peptide as a universal connector for rational phage engineering. To test this, we prepared a phage (KP32_gp38_E_LIN_CBM_LEC::K1Fgp17E) wherein we fused the conserved peptide of phage KP32 RBP2 to the enzymatic domain of the Escherichia phage K1F endosialidase (K1Fgp17). The latter can digest polysialic acid of the Escherichia K1 capsule, a receptor absent in Klebsiella . There is no significant amino acid sequence identity between KP32gp38 and K1Fgp17. Additionally, they are located in different positions in the original phage scaffolds, with RBP2 in phage KP32 and RBP1 in phage K1F, respectively. This design probes thus the limits of conserved peptide functionality. Propagation assays confirmed that the engineered phage retained infectivity on its original Klebsiella host (K3, recognized by RBP1) and, importantly, gained the ability to propagate on E. coli K1 (Fig. 5 ). This shows that conserved peptide can accommodate a chimeric RBP2 carrying an enzymatic domain from a phage infecting a different genus, allowing the KP32 scaffold to cross the genus barrier without compromising its original specificity. These findings underscore the architectural modularity of the conserved peptide: it acts as a universal docking element that mediates virion assembly independently of the origin or structural context of the attached enzymatic domain. This property provides a powerful design principle for engineering phages with tailored or broadened host ranges, including cross-genus targeting. Importantly, achieving such cross-genus host range expansion is far from trivial. It requires overcoming differences between host genera and phage genera, as well as adapting RBPs to new positional contexts within the virion. Klebsiella phage KP32 and Escherichia phage K1F share only 69.15% identity across 36% of their genomes, and although both belong to the order Autographivirales , they represent different genera ( Przondovirus and Kayfunavirus ). In our design, the Escherichia phage-derived RBP, naturally functioning as a primary RBP, had to be repositioned to occupy the secondary RBP position in a Klebsiella phage scaffold, illustrating the structural flexibility and complexity required for successful cross-genus engineering. In addition, the engineered phage KP32 variant demonstrated that it can complete its replication cycle in an E. coli environment despite local metabolic dependencies, highlighting the versatility of the used phage scaffold. Adaptation of E. coli phage scaffold to infect Klebsiella Building on the successful incorporation of a chimeric RBP2 into the phage KP32 scaffold, which enabled propagation on both Klebsiella and Escherichia hosts, we next explored whether this versatility could be extended in the reverse direction, i.e., reprogramming an Escherichia phage to infect Klebsiella host. This approach evaluates other limits of structural flexibility within RBP systems by testing whether a scaffold that naturally supports a single RBP can accommodate an RBP originating from a dual-RBP system and whether an E. coli phage scaffold can be adapted to infect Klebsiella . To achieve this, we engineered phage K1F by replacing its native RBP with KP32gp38, the Klebsiella -specific RBP2. In its original context, KP32gp38 attaches to RBP1 via the conserved peptide and functions as a secondary RBP. In the engineered construct, KP32gp38 was fused to the anchor domain of K1Fgp17, enabling its integration at the primary RBP position within the K1F scaffold. Propagation assays revealed that the engineered phage K1F_gp17_E::KP32gp38 gained the ability to propagate on K. pneumoniae KL163 (Fig. 6 ). This outcome demonstrates that the phage K1F scaffold can accommodate a Klebsiella -specific RBP, that positional relocation also here does not prevent functional assembly, and that an Escherichia phage scaffold is functional in a K. pneumoniae cell. The ability to reprogram an Escherichia phage to infect Klebsiella (and vice versa) suggests that the modularity within RBP systems is not restricted by genus (at least for E. coli and K. pneumoniae , both belonging to Enterobacteriaceae ) or positional context. Further work will be needed to determine the limits of this adaptability and its applicability across more divergent scaffolds. Discussion Branched RBP systems represent a sophisticated adaptation in phage architecture, enabling modular assembly and host-range flexibility. Initially visualized in Escherichia phages K1-5 and K1E through cryo-electron microscopy ( 13 ), these systems were later characterized in Escherichia phages such as CBA120 and G7C, where protein-level interactions within branched complexes were elucidated ( 17 , 18 ). The presence of similar branched RBP structures in phages infecting Klebsiella spp. was predicted by our group previously ( 5 ). Since then, multiple aspects of RBP modularity have been explored. Notably, we demonstrated that the anchor domain, originally identified in Escherichia phages ( 13 , 28 ), can function as a modular acceptor for RBP specificity domains in Klebsiella phages ( 16 ). This finding enabled targeted reprogramming of host specificity with Klebsiella phage K11 as a scaffold, underscoring the potential of anchor domains in phage engineering. However, despite its conceptual appeal, experimental validation of the branching domain function in Klebsiella phages remained limited until now. This study addresses this gap by systematically probing the role of the branching domain and its interaction partner (the conserved peptide) in the assembly of dual RBP systems. Understanding these interactions provides a foundation for exploiting RBP modularity for designing therapeutic phages. Branching domain from RBP1 and the conserved peptide from RBP2 are interacting partners Our initial targeted deletion strategy aimed to systematically assess the contribution of individual domains (Supplementary Tables S2 and S3). However, the rebooting efficiency of engineered constructs was limited, and not all designed engineered phages yielded infective virions. While failure to reboot constructs lacking essential domains indirectly suggested their functional importance, the low efficiency prevented definitive confirmation. This challenge reflects a broader issue in phage engineering: rebooting remains non-trivial, as also reported by others ( 29 ). The rebooting efficiency varies between phage species and can be restricted by host antiviral defense systems ( 30 ). Another study noted that the rebooting efficiency differs depending on the assembly method and phage type, with Golden Gate assembly combined with cell-free transcription-translation (TXTL) designated as the most efficient, but for large or complex genomes, it is still challenging ( 31 ). Optimization of Gibson-based phage genome assembly, combined with TXTL rebooting, was proposed by Levrier and co-workers to avoid transformation biases ( 32 ). Despite these constraints, successful constructs allowed us to confirm the central hypothesis: the branching domain of RBP1 and the conserved peptide of RBP2 are indispensable for assembly of the dual-RBP system. We successfully replaced the RBP1 of phage KP32 with the homologous RBP1 from phage K11. Although the branching domains of phages KP32 and K11 exhibit high structural similarity, their amino acid sequence identity is relatively low. This replacement was also performed in the reverse direction, introducing phage KP32 RBP1 into the RBP1 position of the phage K11 scaffold ( 16 ). However, the assembly of RBP2 in the phage K11 scaffold could not be confirmed due to the absence of a permissive host recognized by phage K11 RBP2. The engineered phage KP32 carrying phage K11 RBP1 (KP32_gp37_A_B_E::K11gp17) was successfully propagated not only on the host recognized by the newly introduced RBP1, but also on the host specific to RBP2, confirming correct assembly of both RBPs into the virion. In contrast, when phage KP32 RBP1 was replaced with the branching domain-lacking RBP2 from phage KP34, the resulting engineered phage (KP32_gp37_B_E::KP34gp57) propagated only on the host recognized by the new RBP1, but not on the RBP2-specific host. The interaction between the conserved peptide and the branching domain was further supported by earlier in silico modelling of the KP32gp37–KP32gp38 complex ( 33 ). Four residues of KP32gp37 (Arg224, Val227, His261, and Arg275) were predicted to form hydrogen bonds with the N-terminal amino acids of KP32gp38 (Leu2, Asp3, Phe5, Asn6). Notably, Arg224 and Val227 are located within the branching domain and are conserved between the RBP1 proteins of phages KP32 and K11 (Fig. 2 A, brown arrows). The remaining two residues are absent at the corresponding positions in K11gp17. At the protein level, isothermal titration calorimetry confirmed that a truncated variant of RBP2, lacking the conserved peptide, failed to interact with RBP1, whereas the full-length protein retained its binding capability ( 23 ). In the presented study, we engineered a corresponding truncation directly into the phage genome, generating a phage variant lacking the conserved peptide. The engineered phage KP32_gp38ΔCP was unable to propagate on a host for RBP2, indicating that deletion of the conserved peptide disrupts RBP2 incorporation into the virion. Although direct visualization of RBP2 absence via cryo-electron microscopy (cryo-EM) would provide definitive evidence, the lack of propagation on an RBP2-dependent host serves as strong indirect evidence for unsuccessful RBP2 assembly. Techniques such as RNA sequencing could further support this by confirming the production of the truncated KP32gp38ΔCP protein during phage propagation. Notably, the truncated RBP2 variant (KP32gp38_ΔCP) was previously shown to be properly folded, as demonstrated by circular dichroism spectroscopy ( 23 ), and to retain enzymatic activity ( 24 ). Thus, while conserved peptide deletion prevents RBP2 assembly into the virion, it does not impair protein folding or function, supporting its role as a distinct architectural module required specifically for RBP2 incorporation. Chimeric fusion of the conserved peptide with the enzymatic domain of the Escherichia K1-specific endosialidase enabled its incorporation into the phage virion, further supporting the role of conserved peptide in RBP2 assembly. This result highlights the conserved peptide as a distinct and modular architectural element, capable of mediating virion integration independently of the native RBP2 context. Baykov et al. ( 9 ) investigated the exchange of TSP genes between two genomic scaffolds of Przondoviruses , KP192 and KP195. KP192 encodes two TSPs designated in our study as a dual-RBP system, with tspA192 specific to KL111 and tspB192 specific to K2 capsule types. In contrast, phage KP195 carries a mono-RBP system with tspA195 specific to the K64 capsule type. Despite a ~ 20% difference in amino acid sequence between the anchor domains of the RBPs, the proteins were successfully exchanged, resulting in infective virions with altered host specificity. This finding aligns with our previous study ( 16 ) and further confirms the results of our work. Additionally, the authors observed that phages with identical RBP but different genomic scaffolds can exhibit substantial variation in replication efficiency when infecting the same Klebsiella strain. This phenomenon was hypothesized to relate to the activity of bacterial phage defense systems, underscoring the importance of scaffold context in phage engineering and therapeutic applications. Recent studies demonstrated that the host range of Kuttervirus phages S117 and STDP.1 can be modified by replacing their RBP genes or receptor-binding domains with those from related phages ( 34 ). Specifically, substitution of TSP3 and TSP4 in phage S117 with homologs from phage CBA120, sharing conserved N-termini but differing in receptor specificity, resulted in altered host range. Moreover, the TSP2 gene of phage S117 was successfully replaced by that of Agtrevirus AV101, highlighting that the conserved N-terminal architecture enables RBP engineering within the Ackermannviridae family ( 22 ). Up to now other studies mostly explored switches between RBPs occupying the same position within the virion. In this study as well as in our previous work ( 16 ) we deviated from this approach and applied also positional relocation of RBPs. The RBP1 of engineered phage KP32_gp37_B_E::KP34gp57 originates from a RBP2 position. Engineered phage KP32_gp38_E_LIN_CBM_LEC::K1Fgp17_E has K1F RBP1 engineered to the position of RBP2. In the engineered phage K1F_gp17_E::KP32gp38, KP32gp38, normally positioned as RBP2, became RBP1. An alternative strategy described earlier to expand the host range of Kuttervirus S117 involved the introduction of a fifth TSP into the phage genome. However, this modification did not broaden the host range. Instead, it led to rearrangements within the TSP complex, including recombination events and deletions, ultimately altering the phage’s host spectrum ( 22 ). These findings suggest that while phage virions exhibit a notable capacity for structural adaptation, this flexibility is not without limits. Cross-genus phages infecting strains belonging to different genera Ando et al. ( 28 ) demonstrated successful host range reprogramming of Escherichia and Klebsiella phages, enabling reciprocal targeting of these genera, but utilizing a different approach. Initial attempts to swap only the tail fiber gene (gp17) between Escherichia phage T7 and Klebsiella phage K11 did not yield viable phage particles. Hybrid tail fibers constructed from fragments of gp17 from both phages also failed to produce functional virions. Ultimately, host range conversion was achieved by exchanging a complete set of tail-associated genes: gp11 (adaptor), gp12 (nozzle), and gp17 (tail fiber/tailspike), allowing the phage T7 scaffold to infect Klebsiella sp. 390 and the phage K11 scaffold to infect E. coli. In the present study, we successfully crossed the genus barrier by swapping only a fragment of the RBP gene, achieving bi-directional host range conversion (KP32_gp38_E_LIN_CBM_LEC::K1Fgp17_E and K1F_gp17_E::KP32gp38 phages). Remarkably, the engineered phages were able to propagate on hosts from different genera, despite potential differences in cellular metabolism and defense mechanisms. This finding is particularly surprising given that even strains within the same genus and capsular type can exhibit variable susceptibility to phage infection ( 9 ). Consistent with previous observations ( 9 , 28 ) this study showed that Klebsiella phage promoters are recognized by E. coli RNA polymerase, enabling the transcription of early phage genes, synthesis of phage proteins, and assembly of infectious particles during phage rebooting. That, together with a proper set of specific RBPs for the primary receptor, contributes to a successful infection cycle. Limitations and future perspectives The narrow host range of phages remains a major limitation in the development of effective phage therapy, and phage engineering can be part of the solution. To fully harness the therapeutic potential of phages, it is essential to refine phage engineering strategies and promote rational, structure-informed design of engineered phages. The limited rebooting efficiency encountered in this study might be attributed to technical limitations inherent to the rebooting protocol or structural incompatibilities introduced during the design process, indicating that a conceptually robust method requires further optimization. As previously demonstrated ( 24 , 35 ), suboptimal domain delineations and the presence of two-amino acid assembly scars in some RBP cluster constructs may have contributed to reduced rebooting performance. The integration of advanced structural prediction tools, such as AlphaFold3, which enables rapid modelling of RBP trimers, alongside the growing suite of machine learning-based design platforms, holds promise for enhancing the efficiency of chimeric protein and phage engineering. Moreover, the incorporation of new synthetic biology tools being developed may substantially increase the robustness and scalability of the rebooting protocol. An additional validation step at the protein level of chimeric RBPs could be implemented to ensure proper protein production and maintenance of enzymatic activity, thereby helping to mitigate potential structural constraints. Such advancements will enable the creation of customized phages tailored to specific bacterial pathogens, improving treatment outcomes in clinical settings. Future work should focus on integrating high-resolution structural data, predictive modelling, and synthetic biology tools to enhance the precision and reliability of phage engineering. Additionally, expanding our understanding of phage biology and phage-host interactions across diverse bacterial genera will be critical for designing broadly applicable therapeutic platforms of engineered phages. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Funding A.L. was supported by Research Foundation–Flanders, Belgium (FWO: 1240021N, 1251224N). Z.D.-K. was supported by Narodowe Centrum Nauki, Poland in the frame of UMO-2017/26/M/NZ1/00233 and UMO-2022/47/I/NZ1/01450 projects. Author Contribution Agnieszka Latka: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft, Writing – review & editing. Dorien Dams: Methodology, Writing – review & editing. Lennert Scholiers: Investigation, Writing – review & editing. Britt Van Mieghem: Investigation, Writing – review & editing. Zuzanna Drulis-Kawa: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. Yves Briers: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. Data Availability All data generated or analysed during this study are included in this published article [and its supplementary information files]. References Dunne M, Prokhorov NS, Loessner MJ, Leiman PG. Reprogramming bacteriophage host range: design principles and strategies for engineering receptor binding proteins. Curr Opin Biotechnol. 2021;68:272–81. Lood C, Boeckaerts D, Stock M, De Baets B, Lavigne R, Van Noort V, et al. Digital phagograms: predicting phage infectivity through a multilayer machine learning approach. Curr Opin Virol. 2022;52:174–81. Boeckaerts D, Stock M, Ferriol-González C, Oteo-Iglesias J, Sanjuán R, Domingo-Calap P et al. Prediction of Klebsiella phage-host specificity at the strain level. Nat Commun. 2024;15(1). Gaborieau B, Vaysset H, Tesson F, Charachon I, Dib N, Bernier J, et al. Prediction of strain level phage–host interactions across the Escherichia genus using only genomic information. Nat Microbiol. 2024;9(11):2847–61. Latka A, Leiman PG, Drulis-Kawa Z, Briers Y. Modeling the Architecture of Depolymerase-Containing Receptor Binding Proteins in Klebsiella Phages. Front Microbiol. 2019;10. Cheetham MJ, Huo Y, Stroyakovski M, Cheng L, Wan D, Dell A, et al. Specificity and diversity of Klebsiella pneumoniae phage-encoded capsule depolymerases. Essays Biochem. 2024;68(5):661–77. Majkowska-Skrobek G, Latka A, Berisio R, Squeglia F, Maciejewska B, Briers Y et al. Phage-Borne Depolymerases Decrease Klebsiella pneumoniae Resistance to Innate Defense Mechanisms. Front Microbiol. 2018;9. Hsieh P-F, Lin H-H, Lin T-L, Chen Y-Y, Wang J-T. Two T7-like Bacteriophages, K5-2 and K5-4, Each Encodes Two Capsule Depolymerases: Isolation and Functional Characterization. Sci Rep. 2017;7(1). Baykov IK, Kurchenko OM, Mikhaylova EE, Miroshnikova AV, Morozova VV, Khlebnikova MI et al. Replacement of the Genomic Scaffold Improves the Replication Efficiency of Synthetic Klebsiella Phages. Int J Mol Sci. 2025;26(14). Noreika A, Rutkiene R, Dumalakiene I, Viliene R, Laurynenas A, Poviloniene S et al. Insights into the Alcyoneusvirus Adsorption Complex. Int J Mol Sci. 2023;24(11). Pan Y-J, Lin T-L, Chen C-C, Tsai Y-T, Cheng Y-H, Chen Y-Y, et al. Klebsiella Phage ΦK64-1 Encodes Multiple Depolymerases for Multiple Host Capsular Types. J Virol. 2017;91(6):JVI02457–16. Ouyang R, Costa AR, Cassidy CK, Otwinowska A, Williams VCJ, Latka A et al. High-resolution reconstruction of a Jumbo-bacteriophage infecting capsulated bacteria using hyperbranched tail fibers. Nat Commun. 2022;13(1). Leiman PG, Battisti AJ, Bowman VD, Stummeyer K, Mühlenhoff M, Gerardy-Schahn R, et al. The Structures of Bacteriophages K1E and K1-5 Explain Processive Degradation of Polysaccharide Capsules and Evolution of New Host Specificities. J Mol Biol. 2007;371(3):836–49. Scholl D, Adhya S, Merril CR. 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Sorensen AN, Woudstra C, Sorensen MCH, Brondsted L. Subtypes of tail spike proteins predicts the host range of Ackermannviridae phages. Comput Struct Biotechnol J. 2021;19:4854–67. Sorensen AN, Kalmar D, Lutz VT, Klein-Sousa V, Taylor NMI, Sorensen MC, et al. Agtrevirus phage AV101 recognizes four different O-antigens infecting diverse E. coli . Microlife. 2024;5:uqad047. Sørensen AN, Brøndsted L. Renewed insights into Ackermannviridae phage biology and applications. npj Viruses. 2024;2(1). Sørensen AN, Woudstra C, Kalmar D, Poppeliers J, Lavigne R, Sørensen MCH, et al. The branched receptor-binding complex of Ackermannviridae phages promotes adaptive host recognition. iScience. 2024;27(9):110813. Squeglia F, Maciejewska B, Łątka A, Ruggiero A, Briers Y, Drulis-Kawa Z, et al. Structural and Functional Studies of a Klebsiella Phage Capsule Depolymerase Tailspike: Mechanistic Insights into Capsular Degradation. Structure. 2020;28(6):613–e244. Latka A, Dams D, Scholiers L, Otwinowska A, Olejniczak S, Drulis-Kawa Z et al. Role of the C-terminal Modules of Klebsiella Phage KP32 Receptor-Binding Protein gp38 in Protein and Phage Functionality. 2025. Gerstmans H, Grimon D, Gutiérrez D, Lood C, Rodríguez A, Van Noort V, et al. A VersaTile-driven platform for rapid hit-to-lead development of engineered lysins. Sci Adv. 2020;6(23):eaaz1136. Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, Smith HO. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009;6(5):343–5. Adams M, Bacteriophages. Geneva: Interscience; 1959. Ando H, Lemire S, Diana. Timothy. Engineering Modular Viral Scaffolds for Targeted Bacterial Population Editing. Cell Syst. 2015;1(3):187–96. Sun Q, Shen L, Zhang B-L, Yu J, Wei F, Sun Y et al. Advance on Engineering of Bacteriophages by Synthetic Biology. Infection and Drug Resistance. 2023;Volume 16:1941-53. Ipoutcha T, Racharaks R, Huttelmaier S, Wilson CJ, Ozer EA, Hartmann EM. A synthetic biology approach to assemble and reboot clinically relevant Pseudomonas aeruginosa tailed phages. Microbiol Spectr. 2024;12(3). Kristensen CS, Petersen AO, Kilstrup M, van der Helm E, Takos A. Cell-free synthesis of infective phages from in vitro assembled phage genomes for efficient phage engineering and production of large phage libraries. Synth Biol (Oxf). 2024;9(1):ysae012. Levrier A, Karpathakis I, Nash B, Bowden SD, Lindner AB, Noireaux V. PHEIGES: all-cell-free phage synthesis and selection from engineered genomes. Nat Commun. 2024;15(1). Napolitano V, Privitera M, Drulis-Kawa Z, Marasco D, Fallarini S, Berisio R, et al. Structural and functional features of Klebsiella pneumoniae capsular degradation by the phage depolymerase KP32gp38: implications for vaccination. Int J Antimicrob Agents. 2025;66(6):107596. Gil J, Paulson J, Brown M, Zahn H, Nguyen MM, Eisenberg M et al. Tailoring the Host Range of Ackermannviridae Bacteriophages through Chimeric Tailspike Proteins. Viruses. 2023;15(2). Maciejewska B, Squeglia F, Latka A, Privitera M, Olejniczak S, Switala P et al. Klebsiella phage KP34gp57 capsular depolymerase structure and function: from a serendipitous finding to the design of active mini-enzymes against K. pneumoniae . mBio. 2023;14(5). Additional Declarations No competing interests reported. Supplementary Files LatkaAdditionalfile1260122.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 23 Feb, 2026 Reviews received at journal 20 Feb, 2026 Reviews received at journal 16 Feb, 2026 Reviewers agreed at journal 02 Feb, 2026 Reviewers agreed at journal 02 Feb, 2026 Reviewers invited by journal 02 Feb, 2026 Editor assigned by journal 23 Jan, 2026 Submission checks completed at journal 23 Jan, 2026 First submitted to journal 22 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8669920","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":584727168,"identity":"8cad73fc-cd80-419d-9b59-77b61ec8715a","order_by":0,"name":"Agnieszka Latka","email":"","orcid":"","institution":"Ghent University","correspondingAuthor":false,"prefix":"","firstName":"Agnieszka","middleName":"","lastName":"Latka","suffix":""},{"id":584727169,"identity":"a3170b9a-638e-4f32-a9b0-c589c70a5c2c","order_by":1,"name":"Dorien Dams","email":"","orcid":"","institution":"Ghent University","correspondingAuthor":false,"prefix":"","firstName":"Dorien","middleName":"","lastName":"Dams","suffix":""},{"id":584727170,"identity":"653f47a8-2703-48a0-90cb-05b2f9877591","order_by":2,"name":"Lennert Scholiers","email":"","orcid":"","institution":"Ghent University","correspondingAuthor":false,"prefix":"","firstName":"Lennert","middleName":"","lastName":"Scholiers","suffix":""},{"id":584727171,"identity":"425eb6e9-e06e-4a9f-a990-7291cd2fe429","order_by":3,"name":"Britt Van Mieghem","email":"","orcid":"","institution":"Ghent University","correspondingAuthor":false,"prefix":"","firstName":"Britt","middleName":"Van","lastName":"Mieghem","suffix":""},{"id":584727172,"identity":"6e3cdcfc-3706-4b1f-8796-bb3a0491fdcc","order_by":4,"name":"Zuzanna Drulis-Kawa","email":"","orcid":"","institution":"University of Wrocław","correspondingAuthor":false,"prefix":"","firstName":"Zuzanna","middleName":"","lastName":"Drulis-Kawa","suffix":""},{"id":584727176,"identity":"c91bad4c-38e7-4e24-952e-0a4c234bbf17","order_by":5,"name":"Yves Briers","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIie2PsQrCMBBAUwLtcrFrin5EQYiL2F9pKXQSl446dEqX6OznKIW6FPyAgiiCc8SlmybdXFJHh7zhchfucXcIWSx/SHhQwekQ6OcOG/0Hvyiib8NTqLUwoASFVnifu2NS/KD4NK2kIy4Tf3x6rEkxX0WIHK8mxaVZRp0uh2C3ZC0pshzQKA2NCjRM3RJD2IDbkl2VqLsYNSvnl3R4DFHjPXLC31qZdUbFE4hqJQTEMOGHforJUApnNFGL0Qamwb5OE1GNUvNiGN/ls4sjX3g3KTeLpCy3R2kco4m/KjzYb7FYLJYhPm/eQP4qHn49AAAAAElFTkSuQmCC","orcid":"","institution":"Ghent University","correspondingAuthor":true,"prefix":"","firstName":"Yves","middleName":"","lastName":"Briers","suffix":""}],"badges":[],"createdAt":"2026-01-22 12:54:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8669920/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8669920/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101840891,"identity":"b033c3b2-98b3-4644-a01e-15913d176553","added_by":"auto","created_at":"2026-02-04 08:32:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":146437,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModel of the dual-RBP system of Klebsiella phages KP32, K11, KP34 (Latka et al., 2019) and the mono-RBP system of Escherichia\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ephage K1F (13).\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Schematic models of RBP systems in phage particles used in this study: anchor-branch attachment mode with two complete RBPs (KP32, K11 phages); anchor-branch attachment mode with the first RBP truncated (phage KP34); anchor attachment mode with one RBP (phage K1F). \u003cstrong\u003e(B)\u003c/strong\u003eThe modular composition of RBP genes of the abovementioned phages. The RBP system is attached to the virion through the anchor domain (A) of RBP1. The branching domain (B), present on RBP1, provides an attachment spot for the conserved peptide (CP) of RBP2. Structural domains (A, B, CP) of both RBP1 and RBP2 are followed by a central enzymatic domain (E) with diverse capsular serotype specificities (K3 – cyan, K21/KL163 – dark blue, K11 – dark grey, K31 – light grey, K63 – light green, K1 – brown). The RBP might be equipped with a C-terminal autoproteolytic chaperone (that is not present at the phage level) or carbohydrate-specific domain(s) like a carbohydrate-binding module (CBM), and in some cases a lectin-like domain (LEC).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8669920/v1/0782375ce7cee8682fa3d02a.png"},{"id":101881281,"identity":"451bc9da-bc0f-4242-92b2-83096cebbc0a","added_by":"auto","created_at":"2026-02-04 15:11:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":384062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural similarity between the RBP1 branching domain in phage KP32 and phage K11 allows for functional exchange. (A) \u003c/strong\u003e3D structures of RBP1 of phage KP32 (KP32gp37) and phage K11 (K11gp17), predicted with AlphaFold3, along with superposition of their branching domains (TM-align, Zhang \u0026amp; Skolnick 2005) and sequence identity (Blastp). Brown arrows indicate conserved amino acids mentioned in the discussion section. \u003cstrong\u003e(B) \u003c/strong\u003eEngineered phage KP32_gp37_A_B_E::K11gp17 propagation on hosts for new RBP1 and RBP2. The results are presented as log10 changes in phage titer (Δlog10(PFU/mL)) after propagation on each host compared to the start phage titer. The start phage titer is depicted as black circles; propagation on K3 (Kp271) – phage KP32 RBP1 host as cyan squares; propagation on KL163 (Kp968) – phage KP32 RBP2 host as dark blue diamonds; propagation on K11 (Kp390) – phage K11 RBP1 host as grey circles. “start” – Δlog10(PFU/mL) of the phage suspension before propagation, “prop.” – Δlog10(PFU/mL) of the phage suspension after propagation. The first bacterial strain indicates on which the propagation cycles were done, whereas the second bacterial strain indicates on which the spotting was done. Phages plated on a K3 lawn are presented on a cyan background, KL163 on a blue background, and K11 on a grey background. With **** statistically significant differences with p\u0026lt;0.0001 are indicated (Ordinary one-way ANOVA, GraphPad Prism 9.0.0). Visual representation of the engineered phage KP32_gp37_A_B_E::K11gp17 virion, where RBP1 originates from phage K11 RBP1 and is transplanted into the phage KP32 scaffold at the RBP1 position, and wild-type phages KP32 and K11 are presented below the graphs.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8669920/v1/08ba3e2da1a5dc2dd9c82ad8.png"},{"id":101840893,"identity":"f0afffea-04cf-43b1-ad47-3690abb0568f","added_by":"auto","created_at":"2026-02-04 08:32:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":120337,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe native branching domain is essential for RBP2 assembly. \u003c/strong\u003eThe results are presented as log10 changes in phage titer (Δlog10(PFU/mL) after propagation on each host compared to the start phage titer. The start phage titers are depicted as black circles; propagation on K3 (Kp271) – phage KP32 RBP1 host as cyan squares; propagation on KL163 (Kp968) – phage KP32 RBP2 host as dark blue diamonds; propagation on K63 (Kp77) – phage KP34 RBP2 host as green triangles. “start” – Δlog10(PFU/mL) of the phage suspension before propagation, “prop.” – Δlog10(PFU/mL) of the phage suspension after propagation. The first bacterial strain indicates on which the propagation cycles were done, whereas the second bacterial strain indicates on which strain spotting was done. Phages plated on a K3 lawn are presented on a cyan background, KL163 on a blue background, and K63 on a green background. With **** statistically significant differences with p\u0026lt;0.0001 are indicated (Ordinary one-way ANOVA, GraphPad Prism 9.0.0). Visual representation of the engineered phage KP32_gp37_B_E::KP34gp57 virion, in which the RBP1 enzymatic part originates from phage KP34 RBP2, and it is transplanted into the phage KP32 scaffold at the RBP1 position as a chimeric fusion with wild-type phage KP32 anchor, and wild-type phages KP32 and KP34 are presented below the graphs.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8669920/v1/cbd4178b2d0a93dd7871b718.png"},{"id":101881458,"identity":"67d5f5d7-7264-4011-ad27-3099f0f7c40c","added_by":"auto","created_at":"2026-02-04 15:12:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":84924,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConserved peptide is essential for RBP2 incorporation into the virion. \u003c/strong\u003eThe results are presented as log10 changes in phage titer (Δlog10(PFU/mL) after propagation on each host compared to the start phage titer). The start phage titers are depicted as black circles; propagation on K3 (Kp271) – phage KP32 RBP1 host as cyan squares; propagation on KL163 (Kp968) – phage KP32 RBP2 host as dark blue diamonds. “start” – Δlog10(PFU/mL) of the phage suspension before propagation, “prop.” – Δlog10(PFU/mL) of the phage suspension after propagation. The first bacterial strain indicates on which the propagation cycles were done, whereas the second bacterial strain indicates on which strain spotting was done. Phages plated on a K3 lawn are presented on a cyan background, KL163 on a blue background. With **** statistically significant differences with p\u0026lt;0.0001 are indicated (Ordinary one-way ANOVA, GraphPad Prism 9.0.0). Visual representation of the engineered phage KP32_gp38ΔCP virion, which RBP2 lacks CP, and the wild-type phage KP32 are presented below the graphs.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8669920/v1/2dce22c9c68d44846f975a68.png"},{"id":101881835,"identity":"31aa4eca-ebc4-49d7-8a3c-5f454fdbe897","added_by":"auto","created_at":"2026-02-04 15:16:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":121090,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConserved peptide-mediated assembly of a chimeric RBP2 enables cross-genus host range extension. \u003c/strong\u003eThe results are presented as log10 changes in phage titer (Δlog10(PFU/mL) after propagation on each host compared to the start phage titer. The start phage titers are depicted as black circles; propagation on K3 (Kp271) – phage KP32 RBP1 host as cyan squares; propagation on KL163 (Kp968) – phage KP32 RBP2 host as dark blue diamonds; propagation on Ec K1 (Ec23503) – phage K1F RBP1 host as brown triangles. “start” – Δlog10(PFU/mL) of the phage suspension before propagation, “prop.” – Δlog10(PFU/mL) of the phage suspension after propagation. The first bacterial strain indicates on which the propagation cycles were done, whereas the second bacterial strain indicates on which strain spotting was done. Phages plated on a K3 lawn are presented on a cyan background, KL163 on a blue background, and Ec K1 on a brown background. With **** statistically significant differences with p\u0026lt;0.0001 are indicated (Ordinary one-way ANOVA, GraphPad Prism 9.0.0). Visual representation of the engineered phage KP32_gp38_E_LIN_CBM_LEC::K1Fgp17E, in which RBP2 is formed by chimeric fusion between KP32gp38 CP and the enzymatic part of K1Fgp17, while RBP1 is unchanged compared to phage KP32 WT, and wild-type phages KP32 and K1F are presented below the graphs.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8669920/v1/11a48b489cae5f488481bc42.png"},{"id":101840898,"identity":"35a6df49-d88b-4e44-ab4c-0a5a0f210f96","added_by":"auto","created_at":"2026-02-04 08:32:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":111940,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEngineered Escherichia phage propagation on a new \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eKlebsiella\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ehost. \u003c/strong\u003eThe results are presented as log10 changes in phage titer (Δlog10(PFU/mL) after propagation on each host compared to the start phage titer. The start phage titers are depicted as black circles; propagation on K3 (Kp271) – phage KP32 RBP1 host as cyan squares; propagation on KL163 (Kp968) – phage KP32 RBP2 host as dark blue diamonds; propagation on Ec K1 (Ec23503) – phage K1F RBP1 host as brown triangles. “start” – Δlog10(PFU/mL) of the phage suspension before propagation, “prop.” – Δlog10(PFU/mL) of the phage suspension after propagation. The first bacterial strain indicates on which the propagation cycles were done, whereas the second bacterial strain indicates on which strain spotting was done. Phages plated on a K3 lawn are presented on a cyan background, KL163 on a blue background, and Ec K1 on a brown background. With **** statistically significant differences with p\u0026lt;0.0001 are indicated (Ordinary one-way ANOVA, GraphPad Prism 9.0.0). Visual representation of the engineered phage K1F_gp17_E::KP32gp38, where the Escherichia phage K1F scaffold was used to assemble a chimeric RBP1, being a fusion between the wild-type K1Fgp17 anchor and whole phage KP32 RBP2 (KP32gp38), and wild-type phages KP32 and K1F are presented below the graphs.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8669920/v1/0be25557fc778c04ad8d9241.png"},{"id":101943091,"identity":"62c7ef6e-ecb2-4d1a-b574-a828f37f73d3","added_by":"auto","created_at":"2026-02-05 09:40:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1973263,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8669920/v1/afbe31e0-3b69-4b00-9962-c4238338dbd2.pdf"},{"id":101840895,"identity":"4ecb0c12-fb86-4122-831f-a5e6f3c3c251","added_by":"auto","created_at":"2026-02-04 08:32:35","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":706283,"visible":true,"origin":"","legend":"","description":"","filename":"LatkaAdditionalfile1260122.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8669920/v1/e059347302332542fd061033.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cross-genus phage design through branching domain and conserved peptide interactions","fulltext":[{"header":"Background","content":"\u003cp\u003eBacteriophages evolved elaborate strategies to recognize and infect their host with high specificity. Initial contact with receptors present on the bacterial cell wall is made via phage receptor-binding proteins (RBPs), which can adopt the morphology of a tail fiber (TF, long fibrous) or tailspike (TSP, shorter and thicker protein, equipped with enzymatic activity) (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). While bacterial defense systems are crucial in blocking phage infection at various stages of its replication cycle, the receptor recognition by RBPs is widely regarded as the primary and most pivotal factor determining phage specificity (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). This was also highlighted by the application of newly developed AI tools for phage host prediction, pinpointing RBPs as key to predict a phage-host interaction at the strain level (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The high RBP specificity is strongly exemplified by capsule-targeting Klebsiella phages, which are equipped with capsule-specific depolymerases. There are many monospecific Klebsiella phages, recognizing and infecting strains limited to one particular capsular type, with their RBP (TSP) responsible for capsule polysaccharide degradation (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Klebsiella phages with two TSP types also exist (e.g., KP32, K11, K5-2, K5-4, KP192) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Multi-specific Klebsiella jumbo phages were also described with at least ten (RaK2), eleven (phage K64-1), or even fourteen (phage ϕKp24) different RBPs (TSPs), able to infect hosts with a broad range of capsular serotypes (\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Cryo-EM analysis revealed a hyperbranched complex organization of their RBP systems (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGenome synteny typically observed for closely related phages is often broken in the RBP region (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). While the N-terminal part of the RBP remains conserved amongst close relatives, enabling RBP attachment to the virion (\u0026lsquo;anchor\u0026rsquo;), the remaining central and C-terminal domains are highly variable (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). The latter are responsible for host specificity, substrate binding, folding, and trimerization and are together with the central domain frequently exchanged between phages through horizontal gene transfer events, reprogramming phage specificity (\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). In some cases, as visualized using cryo-EM, the anchor is not directly fused with an enzymatic domain, but rather functions as a separate, short \u0026lsquo;adaptor\u0026rsquo; protein to which other RBPs attach non-covalently (e.g., \u003cem\u003eE. coli\u003c/em\u003e phage K1-5 and Salmonella phage SP6) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). The concept of anchor as a minimal module sufficient for RBP attachment to the virion was experimentally confirmed via Klebsiella phage K11 engineering (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhen more RBPs are present in the branching system, only one type of RBP (primary RBP) bears an anchor responsible for the anchorage of all RBPs to the virion (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In the case of a dual RBP system, the primary RBP is equipped with a branching domain (called a T4gp10-like domain or XD2/XD3 domain), immediately following the anchor domain, and providing an attachment site for the secondary RBP (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Such a branched structure becomes clear when comparing mono-RBP Escherichia phage K1F (capsular type K1) and phage K1-5, possessing two enzymatically active RBPs (capsular types K1 and K5) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In the latter, a short conserved, N-terminal peptide of the secondary RBP was predicted as responsible for the attachment to the T4gp10-like domain of the primary RBP. Another model is Escherichia phage G7C equipped with two RBPs. Domains responsible for RBP complex formation were identified on the protein level: residues 138\u0026ndash;375 of gp66 (primary RBP) were predicted to be homologous to T4gp10 domains XD2 and XD3, while the N-terminus of gp63.1 (secondary RBP) serves as an interaction partner (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). In the case of phages with multiple RBPs in a single system, the secondary RBP can also bear T4gp10-like domains, enabling attachment of the next RBP layer (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The RBP assembly was in-depth examined for Escherichia phage CBA120 (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), where equivalent branching domains were detected for primary and secondary RBP, organizing its four RBPs into a three-layer system. The importance of the structural domains in complex assembly was also examined for Enterobacteria phage S117, a close relative of CBA120. Phage S117 was used as a scaffold for host range engineering, utilizing analogous RBPs from one phage family, equipped with the same structural domains (anchor, branching domain) but different specificity domains (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). The exchange of RBPs occupying an analogous position resulted in maintaining the full branched RBP complex but modified specificity.\u003c/p\u003e \u003cp\u003eThe structural domains (anchor, branching domain, and conserved peptide) were also detected in Klebsiella phages, and their RBPs systems were thoroughly modelled across different taxonomic clusters (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). In the past years, we have intensively studied the RBPs of Klebsiella phage KP32 as a model system. Klebsiella phage KP32 is a dual-RBP \u003cem\u003ePrzondovirus\u003c/em\u003e with podovirus morphology. Its system is composed of KP32gp37 as the primary RBP (RBP1), and KP32gp38 as the secondary RBP (RBP2). RBP1 starts with the conserved anchor domain at its N-terminus, followed by the branching domain, the enzymatic domain specific towards K3 capsular type, and finally an autocleavable chaperone in its C-terminus (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The branching domain interacts at the protein level with the N-terminal conserved peptide of RBP2 (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The conserved peptide of RBP2 is followed by an enzymatic domain with specificity towards K21/KL163 capsular types, a linker, carbohydrate-binding module, and lectin-like domain at its C-terminus. Previously, we described the function of the C-terminal domains (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) and the function of the anchor domain of RBP1 in attachment to the virion (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). In this work, we confirm the interaction between the branching domain of RBP1 and the conserved peptide of RBP2 on the level of the phage particle and exploit this interaction pair to create a cross-genus phage infecting \u003cem\u003eK. pneumoniae\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. We also investigated whether an \u003cem\u003eEscherichia coli-\u003c/em\u003especific phage could be turned into a \u003cem\u003eKlebsiella-\u003c/em\u003especific phage.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial growth conditions\u003c/h2\u003e \u003cp\u003eTryptone Soy Broth (TSB, Oxoid, Thermo Scientific) or Tryptone Soy Agar (TSA; TSB supplemented with 1.5 w/v % bacteriological agar, VWR) was used to culture \u003cem\u003eK. pneumoniae\u003c/em\u003e strains. \u003cem\u003eE. coli\u003c/em\u003e TOP10 (Invitrogen, Thermo Fisher Scientific) for plasmid propagation, and \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) (Invitrogen, Thermo Fisher Scientific) for protein expression, were cultured in standard lysogeny broth (LB; 10 g Tryptone (VWR), 5 g yeast extract (VWR), 10 g sodium chloride (Fisher Scientific) or LB agar (LB supplemented with 1.5 w/v % bacteriological agar). For culturing of \u003cem\u003eE. coli\u003c/em\u003e transformed with an entry (pVTE) or destination (pVTD3 or pVTD23) vectors, LB was supplemented with 5 w/v % sucrose (Fisher Scientific) and 100 \u0026micro;g/mL ampicillin (Carl Roth), or 50 \u0026micro;g /mL kanamycin (Carl Roth), respectively.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhage engineering\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of chimeric RBP clusters using the VersaTile method\u003c/h2\u003e \u003cp\u003eChimeric RBP clusters were assembled using a modified VersaTile protocol, as previously described by (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). First, a repository of tiles was created in the pVTE vector with VersaTile cloning. The selected tiles were assembled in a two-step tile assembly reaction. In the initial step, only the individual tiles were combined. The second step introduced the preliminary assembly into the destination vector.\u003c/p\u003e \u003cp\u003eThe first reaction was prepared with 46 nM of each tile per position, 15 U of BsaI, 3.5 U of T4 DNA ligase, 10\u0026times; ligation buffer, and ultrapure water to a final volume of 20 \u0026micro;l. This mixture underwent 30 cycles of restriction-ligation (5 minutes at 37\u0026deg;C followed by 5 minutes at 22\u0026deg;C per cycle). Subsequently, 23 nM of the pVTD23 destination vector, 5 U of BsaI, and 1.875 U of T4 DNA ligase were added to the preassembled mixture. An additional 30 cycles of restriction-ligation were performed under the same conditions, followed by enzyme inactivation (5 minutes at 50\u0026deg;C and 5 minutes at 80\u0026deg;C). The final product was used for the transformation of chemically competent \u003cem\u003eE. coli\u003c/em\u003e TOP10 cells.\u003c/p\u003e \u003cp\u003ePlasmid DNA was extracted from overnight cultures using the GeneJET plasmid miniprep kit. Correct assembly of the RBP clusters was confirmed via colony PCR and sequencing. Primer sequences used for tile generation and the composition of RBP clusters are provided in Supplementary Tables S1, S2 (successfully rebooted phages) and S3 (phages for which rebooting was not successful), respectively.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eConstruction of engineered phage genomes\u003c/h3\u003e\n\u003cp\u003eThe phage genome was segmented into five overlapping fragments (F1-F5), each amplified using either PrimeStar polymerase (TaKaRa) or KAPA HiFi DNA polymerase (Roche) with primers listed in Supplementary Table S4. Each fragment included approximately 200 bp of overlap with adjacent fragments to facilitate seamless assembly via Gibson reaction. Fragment F4 encompassed the RBP cluster. To substitute the wild-type F4, PCR amplification was performed on a plasmid harboring the engineered RBP cluster (eF4, constructed as described above), yielding a linearized product.\u003c/p\u003e \u003cp\u003eGibson assembly was carried out using 0.05 pM of each purified linear fragment (DNA Clean \u0026amp; Concentrator kit, ZYMO RESEARCH) and Gibson master mix ((\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e); Supplementary Table S5), incubated at 37\u0026deg;C for 30 minutes, followed by 60\u0026deg;C for 1 hour, and enzyme inactivation at 80\u0026deg;C for 20 minutes. The assembled genome was dialyzed against ultrapure water using MF-Millipore 0.025 \u0026micro;m MCE membranes (Fisher Scientific) and subsequently electroporated into electrocompetent \u003cem\u003eE. coli\u003c/em\u003e 10G ELITE cells (LGC Genomics).\u003c/p\u003e \u003cp\u003ePost-electroporation, cells were recovered in SOC medium (LGC Biosearch Technologies) at 37\u0026deg;C with shaking for 3 hours. Engineered virions were released by adding chloroform (1:10 v/v), followed by vortexing and centrifugation (5 min, 1700 \u0026times; g). The supernatant was plated on a lawn of the respective \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e host using the double agar overlay method (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). After overnight incubation, individual plaques were isolated, propagated in liquid culture (described below), and the phage DNA was extracted using the Quick-DNA Viral kit (ZYMO RESEARCH). Genome integrity was confirmed via Nanopore sequencing (Plasmidsaurus).\u003c/p\u003e\n\u003ch3\u003ePhage assays\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhage propagation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eK. pneumoniae\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e host strains were cultured at 37\u0026deg;C with shaking in TSB or LB, respectively. Phage propagation was performed in three sequential rounds, each involving a 10-fold dilution of the phage suspension (single plaque resuspended in 200 \u0026micro;L PBS) into fresh bacterial cultures recognized by either RBP1 or RBP2. In each round, bacterial cultures at OD₆₀₀ \u0026asymp; 0.1\u0026ndash;0.2 were mixed with phage suspensions at a 9:1 ratio and incubated for 1 hour at 37\u0026deg;C with shaking. Following incubation, 100 \u0026micro;L of chloroform was added, the mixture was vortexed, and centrifuged (1700 \u0026times; g, 5 min, 4\u0026deg;C). The resulting supernatant was used for the subsequent propagation round.\u003c/p\u003e \u003cp\u003ePhage titers were assessed before and after the three propagation rounds using a spot test. Bacterial lawns were prepared in triplicate by mixing 200 \u0026micro;L of \u003cem\u003eK. pneumoniae\u003c/em\u003e or \u003cem\u003eE. coli\u003c/em\u003e cultures (OD₆₀₀ \u0026ge; 1) with 4 mL of soft agar (0.5 w/v % agar in TSB) and overlaying onto TSA plates. After solidification and drying, lawns were spotted with 10 \u0026micro;L of serial 10-fold dilutions of each phage suspension prepared in PBS. Plates were incubated overnight at 37\u0026deg;C, and plaques were counted to calculate phage titers (log₁₀ PFU/mL). To determine effective propagation, the initial titer, adjusted for the cumulative 1000-fold dilution across three rounds, was subtracted from the final titer, yielding Δlog₁₀ (PFU/mL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData distributions were assessed for normality and lognormality using the Shapiro-Wilk test, followed by visual inspection via quantile\u0026ndash;quantile (QQ) plots. Statistical significance among groups was evaluated using one-way ANOVA, followed by Tukey\u0026rsquo;s post hoc multiple comparisons test (GraphPad Prism 9, San Diego, CA, USA). Significance levels were indicated as follows: * for p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ** for 0.01\u0026thinsp;\u0026lt;\u0026thinsp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; *** for 0.0001\u0026thinsp;\u0026lt;\u0026thinsp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; **** for p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGeneral approach\u003c/h2\u003e \u003cp\u003eTo investigate and exploit the interaction between the branching domain of RBP1 and the conserved peptide of RBP2 at the phage level, different engineered phages were designed and constructed. The respective engineered phages were rebooted to infective phage particles, which were subsequently tested for their ability to propagate. As a source of RBP domains, other viruses with podovirus morphology were used. They differ in \u003cem\u003eKlebsiella\u003c/em\u003e capsular serotype specificity: K11 (RBP1 of phage K11), K63 (RBP2 of phage KP34), compared to K3 and K21/KL163 (phage KP32 RBP1 and RBP2, respectively), as well as host genus specificity: \u003cem\u003eEscherichia\u003c/em\u003e K1 (RBP1 of K1F). The RBP architecture or position in the RBP cluster also varied (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The RBP-donating phages also had a variable RBP architecture. Whereas Klebsiella phage K11 has a similar dual-RBP system similar to Klebsiella phage KP32, the first RBP of Klebsiella phage KP34 is truncated, resulting in a short adaptor protein to which a full-length RBP2 is attached via a similar branching domain. In contrast, \u003cem\u003eE. coli\u003c/em\u003e phage K1F has a simple, mono-RBP system.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePresence of non-cognate branching domain ensures proper RBP2 assembly\u003c/h2\u003e \u003cp\u003eKlebsiella phages KP32 and K11 belong to the same genus, \u003cem\u003ePrzondovirus\u003c/em\u003e, exhibiting 89.94% nt sequence identity across 88% of their genomes. Both phages have a typical branched organization in their dual-RBP system. The RBP1s share a 88% amino acid identity in their anchor domain and 67% identity in their corresponding branching domain. While the identity in the branching domain is somewhat lower at the sequence level, it shows a high structural similarity (TM score 0.95223). The C-terminal specificity domains are dissimilar, corresponding to different capsular type specificities (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eIn a previous study, we proved that the anchors of phages K11 and KP32 can be used interchangeably, without disturbing virion infectivity (Latka et al. 2021). In this work, we transplanted the entire RBP1 of phage K11 (K11gp17) to the phage KP32 scaffold, replacing its original full-length RBP1 (KP32gp37). The resulting engineered phage (KP32_gp37_A_B_E::K11gp17) and both the donating and accepting wild-type phages underwent three rounds of propagation on their respective hosts for RBP1 and RBP2. Afterwards, the phage titer was determined on both hosts (for RBP1 and RBP2), and the increase in phage titer was expressed relative to the titer of the starting, non-propagated phage suspension (Dlog10 PFU/mL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The host range of the engineered KP32 phage shifted to the host of the newly acquired RBP1 (capsular type K11) instead of the original host, while retaining infectivity on the original host of RBP2 (capsular type KL163) without apparent loss in infectivity. This indicates that the non-cognate branching domain, originating from phage K11, can still serve as a docking site for the wild-type RBP2. In other words, the conserved peptide of RBP2 of phage KP32 functionally interacts with the non-cognate branching domain of phage K11. We conclude that both the anchor domain and branching domain of phage KP32 can be exchanged with its functional equivalents of phage K11.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAbsence of the branching domain prevents incorporation of RBP2 into the virion\u003c/h2\u003e \u003cp\u003eIn the previous experiment, we demonstrated that the branching domains of phages KP32 and K11 are functional equivalents, allowing proper assembly of RBP2 even when the primary RBP originates from a different phage. However, this does not prove that docking of RBP2 can be confined exclusively to the branching domain. To address this, we applied a domain deletion strategy to verify whether removal of the branching domain blocks RBP2 incorporation into the virion.\u003c/p\u003e \u003cp\u003eFirst, we deleted the native branching domain from RBP1 (construct KP32_gp37ΔB), while retaining all other RBP1 domains, including its enzymatic region. This construct could not be rebooted into an infective phage (Supplementary Table S3).\u003c/p\u003e \u003cp\u003eAs an alternative approach with potentially higher chances of proper folding, we designed a second construct (KP32_gp37ΔE) in which the enzymatic domain was deleted, but the structural domains, including the anchor and branching domains, were preserved. This phage also failed to be rebooted (Supplementary Table S3). These outcomes indicate that the domain deletion strategy was ineffective for generating infective phages, possibly because truncations destabilize RBP1 or the overall architecture required for virion particle formation.\u003c/p\u003e \u003cp\u003eTherefore, we adopted a third approach: replacing the branching and enzymatic domains of phage KP32 RBP1 with the full RBP2 of phage KP34 (construct KP32_gp37_B_E::KP34gp57), resulting in an RBP1 variant lacking the branching domain entirely (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This design was based on the rationale that phage KP34 RBP2 can fold independently from preceding structural domains, increasing the likelihood of successful assembly. Whereas wild-type phage KP32 propagated efficiently on hosts recognized by both RBPs (K3 and KL163), the engineered phage gained the ability to infect the KP34-specific host (K63) and accordingly lost infectivity on the K3 and KL163 host. This demonstrates that, although RBP2 remained encoded in the genome of the engineered phage, it could not be incorporated into the virion in the absence of the branching domain on RBP1. These findings strongly support the hypothesis that the branching domain is essential for RBP2 assembly.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eConserved peptide is responsible for the attachment of RBP2 to the virion\u003c/h2\u003e \u003cp\u003eHaving established that the branching domain of RBP1 is indispensable for RBP2 assembly, we next examined whether the conserved peptide of RBP2 fulfils its predicted role (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) as the interaction partner for the branching domain on the phage level. To test this, we engineered phage KP32_gp38ΔCP, in which the first 29 amino acids of RBP2 corresponding to the conserved peptide were deleted, while all other domains of RBP2 and the complete RBP1 remained intact.\u003c/p\u003e \u003cp\u003ePropagation assays revealed that phage KP32_gp38ΔCP behaved similarly to wild-type phage KP32 on the RBP1-specific host (K3), confirming that deletion of conserved peptide does not impair RBP1 function (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In contrast, the engineered phage failed to propagate on the RBP2-specific host (KL163), indicating that RBP2 was not incorporated into the virion. This demonstrates that conserved peptide is essential for RBP2 assembly, even though the remaining domains of RBP2 were present in the genome.\u003c/p\u003e \u003cp\u003eThese findings align with previous protein-level observations: truncated RBP2 variants lacking conserved peptide were shown to fold correctly and retain enzymatic activity but failed to interact with RBP1 \u003cem\u003ein vitro\u003c/em\u003e on the protein level (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Together, the phage-level and protein-level data confirm that conserved peptide is not only critical for protein-protein interaction but also acts as a structural module required for the correct assembly of a dual-RBP system onto the virion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eConserved peptide enables accommodation of chimeric RBP2 and supports cross-genus host range expansion\u003c/h2\u003e \u003cp\u003eHaving demonstrated that the branching domain of RBP1 and the conserved peptide of RBP2 are indispensable for proper assembly of the dual-RBP system, we next asked whether the role of the conserved peptide as a docking module is sufficiently generic to accommodate non-native RBPs. If the conserved peptide can mediate the incorporation of an RBP from a different phage scaffold, even one originating from a phage infecting another genus, it would reveal a remarkable level of structural flexibility and establish the conserved peptide as a universal connector for rational phage engineering.\u003c/p\u003e \u003cp\u003eTo test this, we prepared a phage (KP32_gp38_E_LIN_CBM_LEC::K1Fgp17E) wherein we fused the conserved peptide of phage KP32 RBP2 to the enzymatic domain of the Escherichia phage K1F endosialidase (K1Fgp17). The latter can digest polysialic acid of the \u003cem\u003eEscherichia\u003c/em\u003e K1 capsule, a receptor absent in \u003cem\u003eKlebsiella\u003c/em\u003e. There is no significant amino acid sequence identity between KP32gp38 and K1Fgp17. Additionally, they are located in different positions in the original phage scaffolds, with RBP2 in phage KP32 and RBP1 in phage K1F, respectively. This design probes thus the limits of conserved peptide functionality. Propagation assays confirmed that the engineered phage retained infectivity on its original \u003cem\u003eKlebsiella\u003c/em\u003e host (K3, recognized by RBP1) and, importantly, gained the ability to propagate on \u003cem\u003eE. coli\u003c/em\u003e K1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This shows that conserved peptide can accommodate a chimeric RBP2 carrying an enzymatic domain from a phage infecting a different genus, allowing the KP32 scaffold to cross the genus barrier without compromising its original specificity.\u003c/p\u003e \u003cp\u003eThese findings underscore the architectural modularity of the conserved peptide: it acts as a universal docking element that mediates virion assembly independently of the origin or structural context of the attached enzymatic domain. This property provides a powerful design principle for engineering phages with tailored or broadened host ranges, including cross-genus targeting. Importantly, achieving such cross-genus host range expansion is far from trivial. It requires overcoming differences between host genera and phage genera, as well as adapting RBPs to new positional contexts within the virion. Klebsiella phage KP32 and Escherichia phage K1F share only 69.15% identity across 36% of their genomes, and although both belong to the order \u003cem\u003eAutographivirales\u003c/em\u003e, they represent different genera (\u003cem\u003ePrzondovirus\u003c/em\u003e and \u003cem\u003eKayfunavirus\u003c/em\u003e). In our design, the Escherichia phage-derived RBP, naturally functioning as a primary RBP, had to be repositioned to occupy the secondary RBP position in a Klebsiella phage scaffold, illustrating the structural flexibility and complexity required for successful cross-genus engineering. In addition, the engineered phage KP32 variant demonstrated that it can complete its replication cycle in an \u003cem\u003eE. coli\u003c/em\u003e environment despite local metabolic dependencies, highlighting the versatility of the used phage scaffold.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAdaptation of\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003ephage scaffold to infect\u003c/b\u003e \u003cb\u003eKlebsiella\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBuilding on the successful incorporation of a chimeric RBP2 into the phage KP32 scaffold, which enabled propagation on both \u003cem\u003eKlebsiella\u003c/em\u003e and \u003cem\u003eEscherichia\u003c/em\u003e hosts, we next explored whether this versatility could be extended in the reverse direction, i.e., reprogramming an Escherichia phage to infect \u003cem\u003eKlebsiella\u003c/em\u003e host. This approach evaluates other limits of structural flexibility within RBP systems by testing whether a scaffold that naturally supports a single RBP can accommodate an RBP originating from a dual-RBP system and whether an \u003cem\u003eE. coli\u003c/em\u003e phage scaffold can be adapted to infect \u003cem\u003eKlebsiella\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTo achieve this, we engineered phage K1F by replacing its native RBP with KP32gp38, the \u003cem\u003eKlebsiella\u003c/em\u003e-specific RBP2. In its original context, KP32gp38 attaches to RBP1 via the conserved peptide and functions as a secondary RBP. In the engineered construct, KP32gp38 was fused to the anchor domain of K1Fgp17, enabling its integration at the primary RBP position within the K1F scaffold. Propagation assays revealed that the engineered phage K1F_gp17_E::KP32gp38 gained the ability to propagate on \u003cem\u003eK. pneumoniae\u003c/em\u003e KL163 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This outcome demonstrates that the phage K1F scaffold can accommodate a \u003cem\u003eKlebsiella\u003c/em\u003e-specific RBP, that positional relocation also here does not prevent functional assembly, and that an Escherichia phage scaffold is functional in a \u003cem\u003eK. pneumoniae\u003c/em\u003e cell. The ability to reprogram an Escherichia phage to infect \u003cem\u003eKlebsiella\u003c/em\u003e (and vice versa) suggests that the modularity within RBP systems is not restricted by genus (at least for \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eK. pneumoniae\u003c/em\u003e, both belonging to \u003cem\u003eEnterobacteriaceae\u003c/em\u003e) or positional context. Further work will be needed to determine the limits of this adaptability and its applicability across more divergent scaffolds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBranched RBP systems represent a sophisticated adaptation in phage architecture, enabling modular assembly and host-range flexibility. Initially visualized in Escherichia phages K1-5 and K1E through cryo-electron microscopy (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), these systems were later characterized in Escherichia phages such as CBA120 and G7C, where protein-level interactions within branched complexes were elucidated (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). The presence of similar branched RBP structures in phages infecting \u003cem\u003eKlebsiella spp.\u003c/em\u003e was predicted by our group previously (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Since then, multiple aspects of RBP modularity have been explored. Notably, we demonstrated that the anchor domain, originally identified in Escherichia phages (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), can function as a modular acceptor for RBP specificity domains in Klebsiella phages (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). This finding enabled targeted reprogramming of host specificity with Klebsiella phage K11 as a scaffold, underscoring the potential of anchor domains in phage engineering. However, despite its conceptual appeal, experimental validation of the branching domain function in Klebsiella phages remained limited until now. This study addresses this gap by systematically probing the role of the branching domain and its interaction partner (the conserved peptide) in the assembly of dual RBP systems. Understanding these interactions provides a foundation for exploiting RBP modularity for designing therapeutic phages.\u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBranching domain from RBP1 and the conserved peptide from RBP2 are interacting partners\u003c/h2\u003e \u003cp\u003eOur initial targeted deletion strategy aimed to systematically assess the contribution of individual domains (Supplementary Tables S2 and S3). However, the rebooting efficiency of engineered constructs was limited, and not all designed engineered phages yielded infective virions. While failure to reboot constructs lacking essential domains indirectly suggested their functional importance, the low efficiency prevented definitive confirmation. This challenge reflects a broader issue in phage engineering: rebooting remains non-trivial, as also reported by others (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The rebooting efficiency varies between phage species and can be restricted by host antiviral defense systems (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Another study noted that the rebooting efficiency differs depending on the assembly method and phage type, with Golden Gate assembly combined with cell-free transcription-translation (TXTL) designated as the most efficient, but for large or complex genomes, it is still challenging (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Optimization of Gibson-based phage genome assembly, combined with TXTL rebooting, was proposed by Levrier and co-workers to avoid transformation biases (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite these constraints, successful constructs allowed us to confirm the central hypothesis: the branching domain of RBP1 and the conserved peptide of RBP2 are indispensable for assembly of the dual-RBP system. We successfully replaced the RBP1 of phage KP32 with the homologous RBP1 from phage K11. Although the branching domains of phages KP32 and K11 exhibit high structural similarity, their amino acid sequence identity is relatively low. This replacement was also performed in the reverse direction, introducing phage KP32 RBP1 into the RBP1 position of the phage K11 scaffold (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). However, the assembly of RBP2 in the phage K11 scaffold could not be confirmed due to the absence of a permissive host recognized by phage K11 RBP2.\u003c/p\u003e \u003cp\u003eThe engineered phage KP32 carrying phage K11 RBP1 (KP32_gp37_A_B_E::K11gp17) was successfully propagated not only on the host recognized by the newly introduced RBP1, but also on the host specific to RBP2, confirming correct assembly of both RBPs into the virion. In contrast, when phage KP32 RBP1 was replaced with the branching domain-lacking RBP2 from phage KP34, the resulting engineered phage (KP32_gp37_B_E::KP34gp57) propagated only on the host recognized by the new RBP1, but not on the RBP2-specific host. The interaction between the conserved peptide and the branching domain was further supported by earlier \u003cem\u003ein silico\u003c/em\u003e modelling of the KP32gp37\u0026ndash;KP32gp38 complex (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Four residues of KP32gp37 (Arg224, Val227, His261, and Arg275) were predicted to form hydrogen bonds with the N-terminal amino acids of KP32gp38 (Leu2, Asp3, Phe5, Asn6). Notably, Arg224 and Val227 are located within the branching domain and are conserved between the RBP1 proteins of phages KP32 and K11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, brown arrows). The remaining two residues are absent at the corresponding positions in K11gp17. At the protein level, isothermal titration calorimetry confirmed that a truncated variant of RBP2, lacking the conserved peptide, failed to interact with RBP1, whereas the full-length protein retained its binding capability (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the presented study, we engineered a corresponding truncation directly into the phage genome, generating a phage variant lacking the conserved peptide. The engineered phage KP32_gp38ΔCP was unable to propagate on a host for RBP2, indicating that deletion of the conserved peptide disrupts RBP2 incorporation into the virion. Although direct visualization of RBP2 absence via cryo-electron microscopy (cryo-EM) would provide definitive evidence, the lack of propagation on an RBP2-dependent host serves as strong indirect evidence for unsuccessful RBP2 assembly. Techniques such as RNA sequencing could further support this by confirming the production of the truncated KP32gp38ΔCP protein during phage propagation. Notably, the truncated RBP2 variant (KP32gp38_ΔCP) was previously shown to be properly folded, as demonstrated by circular dichroism spectroscopy (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), and to retain enzymatic activity (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Thus, while conserved peptide deletion prevents RBP2 assembly into the virion, it does not impair protein folding or function, supporting its role as a distinct architectural module required specifically for RBP2 incorporation. Chimeric fusion of the conserved peptide with the enzymatic domain of the Escherichia K1-specific endosialidase enabled its incorporation into the phage virion, further supporting the role of conserved peptide in RBP2 assembly. This result highlights the conserved peptide as a distinct and modular architectural element, capable of mediating virion integration independently of the native RBP2 context.\u003c/p\u003e \u003cp\u003eBaykov et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) investigated the exchange of TSP genes between two genomic scaffolds of \u003cem\u003ePrzondoviruses\u003c/em\u003e, KP192 and KP195. KP192 encodes two TSPs designated in our study as a dual-RBP system, with tspA192 specific to KL111 and tspB192 specific to K2 capsule types. In contrast, phage KP195 carries a mono-RBP system with tspA195 specific to the K64 capsule type. Despite a\u0026thinsp;~\u0026thinsp;20% difference in amino acid sequence between the anchor domains of the RBPs, the proteins were successfully exchanged, resulting in infective virions with altered host specificity. This finding aligns with our previous study (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) and further confirms the results of our work. Additionally, the authors observed that phages with identical RBP but different genomic scaffolds can exhibit substantial variation in replication efficiency when infecting the same \u003cem\u003eKlebsiella\u003c/em\u003e strain. This phenomenon was hypothesized to relate to the activity of bacterial phage defense systems, underscoring the importance of scaffold context in phage engineering and therapeutic applications.\u003c/p\u003e \u003cp\u003eRecent studies demonstrated that the host range of \u003cem\u003eKuttervirus\u003c/em\u003e phages S117 and STDP.1 can be modified by replacing their RBP genes or receptor-binding domains with those from related phages (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Specifically, substitution of TSP3 and TSP4 in phage S117 with homologs from phage CBA120, sharing conserved N-termini but differing in receptor specificity, resulted in altered host range. Moreover, the TSP2 gene of phage S117 was successfully replaced by that of \u003cem\u003eAgtrevirus\u003c/em\u003e AV101, highlighting that the conserved N-terminal architecture enables RBP engineering within the \u003cem\u003eAckermannviridae\u003c/em\u003e family (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Up to now other studies mostly explored switches between RBPs occupying the same position within the virion. In this study as well as in our previous work (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) we deviated from this approach and applied also positional relocation of RBPs. The RBP1 of engineered phage KP32_gp37_B_E::KP34gp57 originates from a RBP2 position. Engineered phage KP32_gp38_E_LIN_CBM_LEC::K1Fgp17_E has K1F RBP1 engineered to the position of RBP2. In the engineered phage K1F_gp17_E::KP32gp38, KP32gp38, normally positioned as RBP2, became RBP1. An alternative strategy described earlier to expand the host range of \u003cem\u003eKuttervirus\u003c/em\u003e S117 involved the introduction of a fifth TSP into the phage genome. However, this modification did not broaden the host range. Instead, it led to rearrangements within the TSP complex, including recombination events and deletions, ultimately altering the phage\u0026rsquo;s host spectrum (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). These findings suggest that while phage virions exhibit a notable capacity for structural adaptation, this flexibility is not without limits.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCross-genus phages infecting strains belonging to different genera\u003c/h2\u003e \u003cp\u003eAndo et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) demonstrated successful host range reprogramming of Escherichia and Klebsiella phages, enabling reciprocal targeting of these genera, but utilizing a different approach. Initial attempts to swap only the tail fiber gene (gp17) between Escherichia phage T7 and Klebsiella phage K11 did not yield viable phage particles. Hybrid tail fibers constructed from fragments of gp17 from both phages also failed to produce functional virions. Ultimately, host range conversion was achieved by exchanging a complete set of tail-associated genes: gp11 (adaptor), gp12 (nozzle), and gp17 (tail fiber/tailspike), allowing the phage T7 scaffold to infect \u003cem\u003eKlebsiella\u003c/em\u003e sp. 390 and the phage K11 scaffold to infect \u003cem\u003eE. coli.\u003c/em\u003e In the present study, we successfully crossed the genus barrier by swapping only a fragment of the RBP gene, achieving bi-directional host range conversion (KP32_gp38_E_LIN_CBM_LEC::K1Fgp17_E and K1F_gp17_E::KP32gp38 phages). Remarkably, the engineered phages were able to propagate on hosts from different genera, despite potential differences in cellular metabolism and defense mechanisms. This finding is particularly surprising given that even strains within the same genus and capsular type can exhibit variable susceptibility to phage infection (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Consistent with previous observations (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) this study showed that Klebsiella phage promoters are recognized by \u003cem\u003eE. coli\u003c/em\u003e RNA polymerase, enabling the transcription of early phage genes, synthesis of phage proteins, and assembly of infectious particles during phage rebooting. That, together with a proper set of specific RBPs for the primary receptor, contributes to a successful infection cycle.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eLimitations and future perspectives\u003c/h2\u003e \u003cp\u003eThe narrow host range of phages remains a major limitation in the development of effective phage therapy, and phage engineering can be part of the solution. To fully harness the therapeutic potential of phages, it is essential to refine phage engineering strategies and promote rational, structure-informed design of engineered phages. The limited rebooting efficiency encountered in this study might be attributed to technical limitations inherent to the rebooting protocol or structural incompatibilities introduced during the design process, indicating that a conceptually robust method requires further optimization. As previously demonstrated (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), suboptimal domain delineations and the presence of two-amino acid assembly scars in some RBP cluster constructs may have contributed to reduced rebooting performance. The integration of advanced structural prediction tools, such as AlphaFold3, which enables rapid modelling of RBP trimers, alongside the growing suite of machine learning-based design platforms, holds promise for enhancing the efficiency of chimeric protein and phage engineering. Moreover, the incorporation of new synthetic biology tools being developed may substantially increase the robustness and scalability of the rebooting protocol. An additional validation step at the protein level of chimeric RBPs could be implemented to ensure proper protein production and maintenance of enzymatic activity, thereby helping to mitigate potential structural constraints. Such advancements will enable the creation of customized phages tailored to specific bacterial pathogens, improving treatment outcomes in clinical settings. Future work should focus on integrating high-resolution structural data, predictive modelling, and synthetic biology tools to enhance the precision and reliability of phage engineering. Additionally, expanding our understanding of phage biology and phage-host interactions across diverse bacterial genera will be critical for designing broadly applicable therapeutic platforms of engineered phages.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eA.L. was supported by Research Foundation\u0026ndash;Flanders, Belgium (FWO: 1240021N, 1251224N). Z.D.-K. was supported by Narodowe Centrum Nauki, Poland in the frame of UMO-2017/26/M/NZ1/00233 and UMO-2022/47/I/NZ1/01450 projects.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAgnieszka Latka: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Validation, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Dorien Dams: Methodology, Writing \u0026ndash; review \u0026amp; editing. Lennert Scholiers: Investigation, Writing \u0026ndash; review \u0026amp; editing. Britt Van Mieghem: Investigation, Writing \u0026ndash; review \u0026amp; editing. Zuzanna Drulis-Kawa: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Yves Briers: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDunne M, Prokhorov NS, Loessner MJ, Leiman PG. Reprogramming bacteriophage host range: design principles and strategies for engineering receptor binding proteins. Curr Opin Biotechnol. 2021;68:272\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLood C, Boeckaerts D, Stock M, De Baets B, Lavigne R, Van Noort V, et al. Digital phagograms: predicting phage infectivity through a multilayer machine learning approach. 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Front Microbiol. 2019;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheetham MJ, Huo Y, Stroyakovski M, Cheng L, Wan D, Dell A, et al. Specificity and diversity of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e phage-encoded capsule depolymerases. Essays Biochem. 2024;68(5):661\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMajkowska-Skrobek G, Latka A, Berisio R, Squeglia F, Maciejewska B, Briers Y et al. Phage-Borne Depolymerases Decrease \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e Resistance to Innate Defense Mechanisms. Front Microbiol. 2018;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHsieh P-F, Lin H-H, Lin T-L, Chen Y-Y, Wang J-T. Two T7-like Bacteriophages, K5-2 and K5-4, Each Encodes Two Capsule Depolymerases: Isolation and Functional Characterization. Sci Rep. 2017;7(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaykov IK, Kurchenko OM, Mikhaylova EE, Miroshnikova AV, Morozova VV, Khlebnikova MI et al. Replacement of the Genomic Scaffold Improves the Replication Efficiency of Synthetic \u003cem\u003eKlebsiella\u003c/em\u003e Phages. Int J Mol Sci. 2025;26(14).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNoreika A, Rutkiene R, Dumalakiene I, Viliene R, Laurynenas A, Poviloniene S et al. Insights into the \u003cem\u003eAlcyoneusvirus\u003c/em\u003e Adsorption Complex. Int J Mol Sci. 2023;24(11).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePan Y-J, Lin T-L, Chen C-C, Tsai Y-T, Cheng Y-H, Chen Y-Y, et al. Klebsiella Phage ΦK64-1 Encodes Multiple Depolymerases for Multiple Host Capsular Types. J Virol. 2017;91(6):JVI02457\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOuyang R, Costa AR, Cassidy CK, Otwinowska A, Williams VCJ, Latka A et al. 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The genome of bacteriophage K1F, a T7-like phage that has acquired the ability to replicate on K1 strains of \u003cem\u003eEscherichia coli\u003c/em\u003e. J Bacteriol. 2005;187(24):8499\u0026ndash;503.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLatka A, Lemire S, Grimon D, Dams D, Maciejewska B, Lu T et al. Engineering the Modular Receptor-Binding Proteins of \u003cem\u003eKlebsiella\u003c/em\u003e Phages Switches Their Capsule Serotype Specificity. mBio. 2021;12(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eProkhorov NS, Riccio C, Zdorovenko EL, Shneider MM, Browning C, Knirel YA, et al. Function of bacteriophage G7C esterase tailspike in host cell adsorption. Mol Microbiol. 2017;105(3):385\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePlattner M, Shneider MM, Arbatsky NP, Shashkov AS, Chizhov AO, Nazarov S, et al. Structure and Function of the Branched Receptor-Binding Complex of Bacteriophage CBA120. J Mol Biol. 2019;431(19):3718\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSorensen AN, Woudstra C, Sorensen MCH, Brondsted L. Subtypes of tail spike proteins predicts the host range of \u003cem\u003eAckermannviridae\u003c/em\u003e phages. Comput Struct Biotechnol J. 2021;19:4854\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSorensen AN, Kalmar D, Lutz VT, Klein-Sousa V, Taylor NMI, Sorensen MC, et al. Agtrevirus phage AV101 recognizes four different O-antigens infecting diverse \u003cem\u003eE. coli\u003c/em\u003e. Microlife. 2024;5:uqad047.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS\u0026oslash;rensen AN, Br\u0026oslash;ndsted L. 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Role of the C-terminal Modules of Klebsiella Phage KP32 Receptor-Binding Protein gp38 in Protein and Phage Functionality. 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGerstmans H, Grimon D, Guti\u0026eacute;rrez D, Lood C, Rodr\u0026iacute;guez A, Van Noort V, et al. A VersaTile-driven platform for rapid hit-to-lead development of engineered lysins. Sci Adv. 2020;6(23):eaaz1136.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, Smith HO. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009;6(5):343\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdams M, Bacteriophages. Geneva: Interscience; 1959.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndo H, Lemire S, Diana. Timothy. Engineering Modular Viral Scaffolds for Targeted Bacterial Population Editing. Cell Syst. 2015;1(3):187\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Q, Shen L, Zhang B-L, Yu J, Wei F, Sun Y et al. Advance on Engineering of Bacteriophages by Synthetic Biology. Infection and Drug Resistance. 2023;Volume 16:1941-53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIpoutcha T, Racharaks R, Huttelmaier S, Wilson CJ, Ozer EA, Hartmann EM. A synthetic biology approach to assemble and reboot clinically relevant \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e tailed phages. Microbiol Spectr. 2024;12(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKristensen CS, Petersen AO, Kilstrup M, van der Helm E, Takos A. Cell-free synthesis of infective phages from in vitro assembled phage genomes for efficient phage engineering and production of large phage libraries. Synth Biol (Oxf). 2024;9(1):ysae012.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLevrier A, Karpathakis I, Nash B, Bowden SD, Lindner AB, Noireaux V. PHEIGES: all-cell-free phage synthesis and selection from engineered genomes. Nat Commun. 2024;15(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNapolitano V, Privitera M, Drulis-Kawa Z, Marasco D, Fallarini S, Berisio R, et al. Structural and functional features of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e capsular degradation by the phage depolymerase KP32gp38: implications for vaccination. Int J Antimicrob Agents. 2025;66(6):107596.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGil J, Paulson J, Brown M, Zahn H, Nguyen MM, Eisenberg M et al. Tailoring the Host Range of \u003cem\u003eAckermannviridae\u003c/em\u003e Bacteriophages through Chimeric Tailspike Proteins. Viruses. 2023;15(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaciejewska B, Squeglia F, Latka A, Privitera M, Olejniczak S, Switala P et al. \u003cem\u003eKlebsiella\u003c/em\u003e phage KP34gp57 capsular depolymerase structure and function: from a serendipitous finding to the design of active mini-enzymes against \u003cem\u003eK. pneumoniae\u003c/em\u003e. mBio. 2023;14(5).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-biological-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jbie","sideBox":"Learn more about [Journal of Biological Engineering](http://jbioleng.biomedcentral.com/)","snPcode":"13036","submissionUrl":"https://submission.nature.com/new-submission/13036/3","title":"Journal of Biological Engineering","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Klebsiella phage, host range, receptor-binding protein, depolymerase, tailspike, branching domain, conserved peptide","lastPublishedDoi":"10.21203/rs.3.rs-8669920/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8669920/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBranched receptor-binding protein (RBP) systems enable bacteriophages to broaden their host range through the incorporation of two or more RBPs. Using the \u003cem\u003eKlebsiella\u003c/em\u003e podophages KP32, K11, and KP34 as model systems, we experimentally validated the interaction between the branching domain of the primary RBP (RBP1) and the conserved docking peptide of the secondary RBP (RBP2) as an essential architectural pair enabling dual-RBP incorporation into the virion.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSystematic engineering revealed that loss of either of these domains, the branching domain or the conserved peptide, abolishes RBP2 assembly, underscoring their structural role in organizing the branched configuration and demonstrating that the anchor domain is the sole element directly attaching the RBP complex to the virion. Exploiting this interaction, we engineered a chimeric phage based on the \u003cem\u003eKlebsiella\u003c/em\u003e KP32 scaffold that was capable of cross-genus infection and productive propagation on both \u003cem\u003eKlebsiella\u003c/em\u003e and \u003cem\u003eEscherichia\u003c/em\u003e hosts. In contrast to previous approaches that required replacement of entire tail modules, this strategy achieved host-range reprogramming through modular domain swapping and positional relocation of RBPs (i.e., exchanging RBP1 and RBP2 positions). Conversely, an Escherichia phage K1F scaffold was also successfully engineered to infect \u003cem\u003eKlebsiella\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur study confirms that the RBP branching domain and the conserved peptide function as specific interacting partners. Our findings establish the conserved peptide as a universal docking element and highlight the structural flexibility of podoviruses to accommodate RBPs from different positional and taxonomic contexts. Collectively, this work provides a mechanistic framework for rational phage engineering and defines a general design principle for generating customized therapeutic phages with an expanded host spectrum, including cross-genus infectivity.\u003c/p\u003e","manuscriptTitle":"Cross-genus phage design through branching domain and conserved peptide interactions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-04 08:32:30","doi":"10.21203/rs.3.rs-8669920/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-23T10:53:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-20T20:12:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-16T09:43:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41082678338500340701971884427210883601","date":"2026-02-02T20:40:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"66190383255000772524165772868339546136","date":"2026-02-02T12:03:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-02T11:22:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-23T16:23:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-23T16:20:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Biological Engineering","date":"2026-01-22T12:12:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-biological-engineering","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jbie","sideBox":"Learn more about [Journal of Biological Engineering](http://jbioleng.biomedcentral.com/)","snPcode":"13036","submissionUrl":"https://submission.nature.com/new-submission/13036/3","title":"Journal of Biological Engineering","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1cee210e-1075-4c58-a8b8-f3da9159ceed","owner":[],"postedDate":"February 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-16T13:39:48+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-04 08:32:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8669920","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8669920","identity":"rs-8669920","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}