Phage N4 uses a SAR endolysin-holin system for host cell lysis

Bacteriophages (phages) cause active host cell lysis to terminate their infection and release progeny into the environment. Some phages delay their lysis, and thereby increase progeny yield, in a phenomenon called lysis inhibition (LIN). There are two dissimilar phages of Escherichia coli known to exhibit LIN: T4 and N4. Studies of phage T4 have demonstrated a multi-protein mechanism that stalls lysis and maintains the LIN state in response to superinfection in a high phage population density. However, the lysis proteins responsible for T4 lysis and LIN are not conserved with phage N4. In this study, we characterize the phage N4 proteins involved in lysis by molecular and genetic means. We define the functions of the minimal gene set required for lysis through heterologous expression and complementation. We also demonstrate that this complete lysis cassette is sufficient to induce LIN under high-density phage infection conditions. Furthermore, by sequence comparison with a selected mutant library that does not induce LIN, we have identified genomic regions both within and outside the lysis cassette involved in N4 LIN. We propose a model where N4 lysis proteins that are responsible for executing rapid N4 lysis can be regulated to induce LIN. Despite the lack of conservation with T4 components, our study suggests that direct modulation of lysis initiation may be common and provides a springboard for identifying other phages that regulate their phage production numbers in this way. ImportanceBacteriophages (phages) are viruses that only kill bacteria. To do so, they use lysis proteins targeted at the structural components of a bacterial cell. There are commonalities between the phages that infect bacteria with similar structures, but there are also interesting differences we can exploit to learn more about specific phages or their bacterial hosts. In this study, we characterize the lysis proteins a phage called N4 uses to kill E. coli bacteria. We also provide the basis for understanding how regulation of those proteins is linked to pathways that control phage yield by a mechanism that seems to differ from the one previously reported example in phage T4. Future studies on the relationship between lysis proteins and phage yield could provide handles to use this information for optimal phage production.