PMID:27381920

Citation	Chen, Y and Young, R (2016) The Last r Locus Unveiled: T4 RIII Is a Cytoplasmic Antiholin. J. Bacteriol. 198:2448-57
Abstract	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.
Links	PubMed PMC4999926 Online version:10.1128/JB.00294-16
Keywords	Bacteriophage T4/physiology; Escherichia coli/virology; Gene Deletion; Gene Expression Regulation, Viral/physiology; Mutation; Viral Proteins/genetics; Viral Proteins/metabolism; Virus Release
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BPT4:LINR3		GO:1901218: negative regulation of holin activity	ECO:0000315:		P		E. coli bacteriophage T4 rIII According to Figure 3, lysis inhibition activity for RI and rIII are similar, and rIII is required in conjunction with rI for full antiholin activity.	complete CACAO 12937

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