Detection and characterization of protein methylation in bacteriophages and their host, <i>Cellulophaga baltica</i> , during infection

ABSTRACT Bacteria and their viruses (bacteriophages or phages) are in a dynamic “arms race” that balances predation and resistance, using various strategies to achieve dominance. To better understand these strategies, we infected the marine model bacterium Cellulophaga baltica with three previously characterized phages (phi18:1, phi18:4, and phi38:1), each with diverse genome types and infection efficiencies. Mass spectrometry-based proteomics measurements of the molecular signatures of the phage infection processes revealed that the percentage of peptides identified as methylated was approximately doubled in phage-infected cells (virocells) compared to uninfected cells, with a significantly increased frequency of host protein methylation observed in the early stage of the infections. Additionally, phage proteins displayed methylation in both virocells and free virions, neither of which has been previously reported. This suggests that protein post-translational methylation may be an important regulator in the phage-bacterial infection process. To explore this further, we assessed methylation patterns in virocells versus uninfected cells at a global level, as well as for a selected set of host proteins with known importance to phage infection, including elongation factor thermo unstable (EF-Tu), chaperone DnaK, and gliding motility proteins. Collectively, our results expand the molecular details of the phage-host arms race by highlighting dynamic protein methylation signatures in phage infection. IMPORTANCE The battle for survival between bacteria and their associated phages has broad ecological implications due to the evolutionary pressure each exerts on the other, as well as the unique phenotypes presented by phage-infected bacteria. The various mechanisms through which bacteria and their phages wage this battle is an active area of exploration. Here, we used the marine model bacterium Cellulophaga baltica and three of its phages to identify and examine a previously unreported method of regulation in phage-infected bacteria: protein methylation. We report the novel finding of methylation of phage proteins and demonstrate this methylation is not just restricted to viral proteins within the cell but is also found in free virions outside the cell. Furthermore, our study opens up future, protein-specific mechanistic investigation by highlighting virocell-unique methylation patterns on several host proteins with known importance to phage infection.