The latent period of phage T4， normally ∼25 min， can be extended indefinitely if the infected cell is superinfected after 5 min. This phenomenon， designated lysis inhibition (LIN)， was first described in the 1940s and is genetically defined by mutations in diverse T4 r genes. RI， the main effector of LIN， has been shown to be secreted to the periplasm， where， upon activation by superinfection with a T-even virion， it binds to the C-terminal periplasmic domain of the T4 holin T and blocks its lethal permeabilization of the cytoplasmic membrane. Another r locus， rIII， has been the subject of conflicting reports. In this study， we show that RIII， an 82-amino-acid protein， is also requ... More
The latent period of phage T4， normally ∼25 min， can be extended indefinitely if the infected cell is superinfected after 5 min. This phenomenon， designated lysis inhibition (LIN)， was first described in the 1940s and is genetically defined by mutations in diverse T4 r genes. RI， the main effector of LIN， has been shown to be secreted to the periplasm， where， upon activation by superinfection with a T-even virion， it binds to the C-terminal periplasmic domain of the T4 holin T and blocks its lethal permeabilization of the cytoplasmic membrane. Another r locus， rIII， has been the subject of conflicting reports. In this study， we show that RIII， an 82-amino-acid protein， is also required for LIN in both Escherichia coli B strains and E. coli K-12 strains. In T4ΔrIII infections， LIN was briefly established but was unstable. The overexpression of a cloned rIII gene alone impeded T-mediated lysis temporarily. However， coexpression of rIII and rI resulted in a stable LIN state. Bacterial two-hybrid assays and pulldown assays showed that RIII interacts with the cytoplasmic N terminus of T， which is a critical domain for holin function. We conclude that RIII is a T4 antiholin that blocks membrane hole formation by interacting directly with the holin. Accordingly， we propose an augmented model for T4 LIN that involves the stabilization of a complex of three proteins in two compartments of the cell: RI interacting with the C terminus of T in the periplasm and RIII interacting with the N terminus of T in the cytoplasm.
