Functional interactions of Gp22 and Gp23 with Gp21 DNA helicase define the replication module of dairy-relevant Ceduovirus phage 94p4

Lactococcus lactis and Lactococcus cremoris strains play a vital role in dairy fermentations, yet their industrial use is threatened by bacteriophage infections that can lead to production failures. Replicative helicases are motor phage proteins driving viral genome replication, and their activity is often supported by accessory proteins involved in helicase recruitment, loading onto DNA, and regulation. However, in most phages of Gram-positive bacteria, such factors remain largely uncharacterized. Here, we identify and functionally characterize 2 proteins, Gp22 and Gp23, encoded by genes located immediately downstream of the helicase gene gp21 in the lytic Ceduovirus phage 94p4 infecting L. lactis and L. cremoris strains. Our findings demonstrate that Gp22 is a DNA-binding protein lacking inherent helicase activity which attenuates the DNA unwinding function of Gp21. In contrast, Gp23 lacks both DNA binding and intrinsic unwinding activity, but significantly enhances DNA unwinding by Gp21. Protein interaction assays confirmed that both Gp22 and Gp23 bind directly to Gp21, suggesting they function as helicase-associated accessory proteins. This study provides the first functional insight into 2 phage-encoded proteins that modulate the Gp21 helicase and may regulate DNA processing during phage replication. These findings advance our understanding of the molecular mechanisms behind phage propagation in Lactococcus spp., bacteria that are integral to starter cultures used in the dairy industry worldwide. By characterizing replication-related phage proteins, this work contributes to expanding the knowledge on phage biology with potential relevance for the development of phage control strategies in the dairy industry.