Decoding the molecular properties of mycobacteriophage D29 Holin provides insights into Holin engineering.

Holins are bacteriophage-encoded small transmembrane proteins that determine the phage infection cycle duration by forming non-specific holes in the host cell membrane at a specific time post-infection. Thus, Holins are also termed as "Protein clocks". Holins have one or more transmembrane domains, and a charged C-terminal region, which, although conserved among Holins, has not yet been examined in detail. Here, we characterize the molecular properties of mycobacteriophage D29 Holin C-terminal region, and investigate the significance of the charged residues and coiled coil (CC) domain present therein. We show that the CC domain is indispensable for Holin-mediated efficient bacterial cell lysis. We further demonstrate that out of the positively- and negatively-charged residues present in the C-terminal region, substituting the former, and not the latter, with serine, renders Holin non-toxic. Moreover, the basic residues present between the 59 and the 79 amino acids are the most crucial for Holin-mediated toxicity. We also constructed an engineered Holin, HolHC, by duplicating the C-terminal region. The HolHC protein shows higher toxicity in both and , and causes rapid killing of both bacteria upon expression, as compared to the wild-type. A similar oligomerization property of HolHC as the wild-type Holin allows us to propose that the C-terminal region of D29 Holin determines the timing, and not the extent, of oligomerization and, thereby, hole formation. Such knowledge-based engineering of mycobacteriophage Holin will help in developing novel phage-based therapeutics to kill pathogenic mycobacteria, including Holins are bacteriophage-encoded small membrane perforators that play an important role in determining the timing of host cell lysis towards the end of the phage infection cycle. Holin's ability to precisely time the hole formation in the cell membrane ensuing cell lysis is both interesting and intriguing. Here, we examined the molecular properties of the mycobacteriophage D29 Holin C-terminal region that harbours several polar charged residues and a coiled-coil domain. Our data allowed us to engineer Holin with an ability to rapidly kill bacteria and show higher toxicity than the wild-type protein. Due to their ability to kill host bacteria by membrane disruption, it becomes important to explore the molecular properties of Holins that allow them to function in a timely and efficient manner. Understanding these details can help us modulate Holin activity and engineer bacteriophages with superior lytic properties to kill pathogenic bacteria, curtail infections, and combat antimicrobial resistance.