The Last r Locus Unveiled: T4 RIII Is a Cytoplasmic Antiholin

• Published in: Journal of bacteriology Vol. 198; no. 18; pp. 2448 - 2457
• Main Authors: Chen, Yi, Young, Ry
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 15.09.2016
• Subjects: Amino acids; Bacterial proteins; Bacteriology; Bacteriophage T4 - physiology; Binding sites; Biochemistry; Cytoplasm; E coli; Escherichia coli; Escherichia coli - virology; Gene Deletion; Gene expression; Gene Expression Regulation, Viral - physiology; Mutation; Viral Proteins - genetics; Viral Proteins - metabolism; Virus Release
• ISSN: 0021-9193; 1098-5530
• DOI: 10.1128/JB.00294-16

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 T4Δ rIII 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. IMPORTANCE Lysis inhibition is a unique feature of phage T4 in response to environmental conditions, effected by the antiholin RI, which binds to the periplasmic domain of the T holin and blocks its hole-forming function. Here we report that the T4 gene rIII encodes a cytoplasmic antiholin that, together with the main antiholin, RI, inhibits holin T by forming a complex of three proteins spanning two cell compartments.