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

Abstract

The latent period of phage T4, normally ϳ25 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 T4⌬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. IMPORTANCELysis 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. The r genes of the T-even phages, first identified by laboratories of the Phage Group in the 1940s (1, 2), have a special place in the history of molecular biology. Detailed studies of the first three loci discovered-rI, rIIAB, and rIII-were foundational in working out the fundamentals of inheritance, genetic code, mutation, recombination, DNA repair, and gene structure (3-7). These mutable loci were originally discovered by their distinctive plaque morphology: large, clear, sharply defined plaques, easily distinguished from the small, fuzzy-edged, turbid plaques of the parental phages (1). The "r" designation meant "rapid lysis," which refers to the observation that the mutant phages isolated from the r-type plaques caused rapid, culture-wide lysis at ϳ25 min after infection, whereas cultures infected with the parental phages continued to increase in mass and accumulate progeny virions intracellularly for hours, in a state called "lysis inhibition"(LIN) (8). In the ensuing decades, more loci were classified as r genes based on mutant plaque phenotypes; at one point, r genes numbering u...

DominantVibrio choleraephage exhibits lysis inhibition sensitive to disruption by a defensive phage satellite

“…Next, we sought to identify an antiholin in ICP1. Antiholins can inhibit pore formation via multiple mechanisms: antiholin TMDs can directly interact with holins in the membrane altering quaternary structure (40,41), antiholin periplasmic domains can sense signals of superinfection while interacting with holins to block pore formation (42), and cytoplasmic antiholins can bind holins inside the cytoplasm (43). Because antiholins interacting directly with holins within the membrane will contain TMDs and previously characterized periplasmic antiholins contain TMDs related to their tethering to the membrane and subsequent release into the periplasm (44), we continued to focus on the ORFs in ICP1 containing at least one TMD.…”

“…After the endolysins digest the cell wall, spanins mediate the fusion between the inner and outer membranes to complete lysis of the cell. During LIN, antiholins inhibit holin oligomerization and triggering thereby stopping progression towards cell lysis (11)(12)(13).…”

“…In addition to changing plaque morphology, the T4 antiholin mutants demonstrate accelerated lysis kinetics in liquid culture (11,13), motivating us to test infection with ICP1 Δ arrA in liquid cultures. Attempts at making high titer stocks of Δ arrA ICP1 were unsuccessful (consistent with decreased phage yields due to a lack of LIN) hindering our ability to test infection at high MOI.…”

“…Consequently, LIN is considered an important evolutionary adaptation for phages in environments where host bacteria are scarce but free virions are plentiful (10). While such visually discernable phenotypes enabled early genetic inquiries, the molecular underpinnings of LIN remained unknown until recently and have only been identified and characterized in the T-even coliphages where LIN is mediated by holins and antiholins (11)(12)(13). Holins are the first step in canonical holin-endolysin-spanin lysis systems for Gram negative bacteria (14,15).…”

“…After the endolysins digest the cell wall, spanins mediate the fusion between the inner and outer membranes to complete lysis of the cell. During LIN, antiholins inhibit holin oligomerization and triggering thereby stopping progression towards cell lysis (11)(12)(13).Lytic phages are abundant in natural environments including marine ecosystems (16) and gut microbiomes (17), making LIN a relevant tool in the molecular arsenal of lytic phages infecting bacteria like Vibrio cholerae. Vibrio cholerae causes a substantial global health problem as the causative agent of the diarrheal disease cholera (18).…”