The N-Terminal Domain of Tailspike Depolymerases Affects the Replication Efficiency of Synthetic <em>Klebsiella</em> Phages

Bacteriophage receptor-binding proteins are often attached to the tail via a conserved N-terminal adapter/anchor domain, presumed to function independently from the distal receptor-binding/catalytic domain. Using synthetic phage technology, we demonstrated that the N-terminal domain in Przondovirus phages KP192 and KP195 substantially modulates the receptor-binding and hydrolytic activities of their type A tailspikes. A bioinformatics analysis of related proteins revealed a high correlation between the N-terminal domain and the distal receptor-binding region. Furthermore, it was shown that an imperfect structural fit between the N-terminal domain and the adjacent tail proteins (gatekeeper and nozzle proteins) can reduce virion assembly efficiency, thereby impairing phage fitness. These results underscore the importance of selecting an appropriate N-terminal domain of receptor-binding proteins when engineering bacteriophages with altered host specificity.