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 Skunavirus 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 Skunavirus genomes were analysed, revealing a strong correlation between phage diversity and the applied starter culture. These analysed Skunavirus 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 Skunavirus 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 Skunavirus sk1 infection was observed, indicative of their role in phage genome evolution and associated adaptation processes.