title: The Last r Locus Unveiled: T4 RIII Is a Cytoplasmic Antiholin.
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.
  
  
  
  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.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA Center for Phage Technology, Texas A&M University, College Station, Texas, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA Center for Phage Technology, Texas A&M University, College Station, Texas, USA ryland@tamu.edu.
authors.forename: Ry
authors.initials: R
authors.lastname: Young
mesh_terms: D017122:Bacteriophage T4 / Q000502:physiology*; D004926:Escherichia coli / Q000821:virology*; D017353:Gene Deletion; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D009154:Mutation; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D057074:Virus Release
chemical_list: D014764:Viral Proteins; C074649:rIII protein, Enterobacteria phage T4