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

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. 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 phage 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 the entire RBP, the adaptor and nozzle proteins, 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. 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 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.