Molecular Mechanism of Holin Transmembrane Domain I in Pore Formation and Bacterial Cell Death

Bacterial cell lysis during bacteriophage infection is timed by perfect orchestration between components of the holin-endolysin cassette. In bacteria, progressively accumulating holin in the inner membrane, retained in its inactive form by antiholin, is triggered into active hole formation, resulting in the canonical host cell lysis. However, the molecular mechanism of regulation and physical basis of pore formation in the mycobacterial cell membrane by D29 mycobacteriophage holin, particularly in the nonexistence of a known antiholin, is poorly understood. In this study, we report, for the first time, the use of fluorescence resonance transfer measurements to demonstrate that the first transmembrane domain (TMI) of D29 holin undergoes a helix <-> beta-hairpin conformational interconversion. We validate that this structural malleability is mediated by a centrally positioned proline and is responsible for controlled TMI self-association in membrana, in the presence of a proton gradient across the lipid membrane. We demonstrate that TMI is sufficient for bacterial growth inhibition. The biological effect of D29 holin structural alteration is presented as a holin self-regulatory mechanism, and its implications are discussed in the context of holin function.