Genomic and functional divergence analysis of closely related Teseptimavirus phages reveals dual HNH endonuclease positioning and its role in plaque formation.

Antibiotic resistance has renewed interest in bacteriophages as antimicrobials, yet the complexity of phage-host interactions often leads to inconsistent infection outcomes, underscoring the need to unravel the molecular basis of phenotypic diversity among closely related phages. Here, we compared two closely related T7-like phages, polyvalent phages C5 and N30, both belonging to Teseptimavirus, which share 89.67% genomic identity and an identical host range. While C5 exhibited a higher adsorption rate, plaque size, and efficiency of plating, N30 showed stronger bacteriostatic activity and larger burst sizes, highlighting the importance of a comprehensive evaluation of phage infection traits for therapeutic or biocontrol applications. Comparative genomics revealed high overall similarity between phages C5 and N30 but notable divergence in a hypothetical protein and a histidine-asparagine-histidine (HNH) endonuclease. Broader analysis across Teseptimavirus further uncovered two distinct genomic positions of the HNH endonuclease: either adjacent to virion assembly proteins in the genome middle or next to the terminase large subunit at the genome end, suggesting functional specialization in capsid assembly and DNA packaging. Tertiary structure prediction and comparison of the HNH endonucleases revealed moderate structural similarity despite moderate sequence divergence. Furthermore, a single nucleotide substitution in the HNH gene of N30 markedly increased its efficiency of plating in one tested host strain, demonstrating that terminase-associated HNH endonuclease plays a pivotal role in plaque formation. Together, these findings provide the first evidence of dual HNH endonuclease positioning within Teseptimavirus and identify the HNH endonuclease as a key genetic determinant of plaque formation, offering new insights into the molecular basis of phage infection variability and host adaptation.IMPORTANCEClosely related bacteriophages often display strikingly different infection phenotypes, yet the genetic basis of this variation remains poorly understood. Here, we uncover a previously unrecognized feature within the genus Teseptimavirus: the HNH endonuclease can occupy two distinct genomic positions, either associated with virion assembly or DNA packaging modules, suggesting functional specialization. We further demonstrate that this enzyme is closely linked to infection phenotypes, as even a single nucleotide substitution in the HNH gene significantly alters the efficiency of plating. These findings identify the HNH endonuclease as a key determinant of phage infectivity and reveal that its genomic context may shape functional outcomes. This work provides new insight into how subtle genetic differences drive phenotypic divergence among closely related phages and highlights the HNH endonuclease as a promising target for rational phage optimization in therapeutic and biocontrol applications.