Diversity and functional characterization of HNH endonucleases encoded by lactococcal Skunavirus phages

Homing, a biological phenomenon involving enzyme-mediated genetic exchange by homologous recombination, has been highlighted as a potential driver of phage genome evolution. In the current study, 18 lactococcal phages, belonging to the <i>Skunavirus</i> genus, were isolated from Dutch dairy facilities, and their genomes were sequenced. Together with 71 phages from previous studies involving Dutch dairy fermentation facilities, a total of 89 <i>Skunavirus</i> genomes were analysed, revealing a strong correlation between phage diversity and the applied starter culture. These analysed <i>Skunavirus</i> genomes were predicted to encode a total of 212 intact HNH endonucleases (HNHEs), which were classified into families based on structural homology and their insertion locations on the genome. Members of the I-HmuI-like HNHE family were observed to be present among most analysed genomes, though they varied in individual <i>Skunavirus</i> phages in both their number and genomic locations. Phylogenetic analysis revealed that these I-HmuI-like HNHEs cluster together according to their insertion locations and the corresponding starter cultures. Furthermore, the so-called genetic marker exclusion activity of particular expressed HNHEs against <i>Skunavirus</i> sk1 infection was observed, indicative of their role in phage genome evolution and associated adaptation processes.