Functional Evaluation of a Conserved <scp>HNH</scp> Endonuclease Gene in Lactococcal <i>Skunavirus</i> Genomes

Skunavirus is the most frequently encountered Lactococcus-infecting (bacterio)phage genus in dairy fermentations and may cause milk acidification failure or inefficiencies. During phage infection, DNA packaging into the phage prohead is a crucial step for the successful maturation of phage progeny. In the Skunavirus genus of lactococcal phages, a predicted HNH endonuclease-encoding gene adjacent to two putative DNA packaging genes is highly conserved. In the current study, we show that this gene, designated here as hrdP (HNH endonuclease related to DNA packaging), is essential for the production of infective Skunavirus sk1 virions. HrdP exhibits non-specific endonuclease activity, while deletion of hrdP from the sk1 genome results in loss of plaque-forming ability without hindering its DNA replication or protein production. Transmission electron microscopy analysis showed that the hrdP deletion mutant in sk1 is unable to produce intact virions. However, the proheads in this mutant displayed conformational changes associated with capsid expansion, suggesting that DNA packaging had initiated and progressed to a certain extent. This study provides valuable insights into the specific role of HrdP in Skunavirus members, highlighting it as a potential target for developing anti-phage strategies to enhance starter culture robustness.