1.E.8 The T4 Holin (T4 Holin) Family

T-even phage such as T4 use a holin-endolysin system for host cell lysis. Although the endolysin of phage T4 encoded by the e gene (Lysozyme E) was identified in 1961, the holin (product of gene t and called T-holin) was first characterized in 2001 (Ramanculov and Young, 2001a). It is unusually large (218 aas), twice as large as phage %u03BBholin S. The protein is highly hydrophilic with 49 acidic and basic residues distributed along its length and a single putative TMS near its N-terminus. Most of the protein is in the periplasm. The large periplasmic domain is a major determinant in the timing mechanism and is involved in lysis inhibition (LIN) (Ramanculov and Young, 2001b). LIN involves the antiholin rI protein of T4 (Ramanculov and Young, 2001c). Lysis inhibition is an effective strategy to coordinate lysis timing with phage particle maturation and to exclude other phage (Miller et al., 2003). The C-terminal periplasmic domain of T4 holin binds the periplasmic domain of T4 antiholin (RI; 97 aas) which like the holin, spans the membrane once (Tran et al., 2005).

  T-holin of T4 phage forms a 1:1 complex with the RI inhibitor which block aggregation and pore formation (Moussa et al. 2012).  The latent period of phage T4, normally ∼25 min, can be extended indefinitely if the infected cell is super-infected after 5 min. This phenomenon, designated as lysis inhibition (LIN), was first described in the 1940s and genetically defined by mutations in diverse T4 r genes. RI, the main effector of LIN, was shown to be secreted to the periplasm where, upon activation by super-infection 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, an 82 amino acid protein, is also required for LIN in both Escherichia coli B and K-12 strains. In T4ΔrIII infections, LIN was briefly established but was unstable (Chen and Young 2016). The overexpression of a cloned rIII gene alone impeded T-mediated lysis temporarily. However, co-expression of rIII and rI resulted in a stable LIN state. Bacterial two-hybrid assays and pull-down assays showed that RIII interacts with the cytoplasmic N-terminus of T, which is a critical domain for holin function. Chen and Young 2016 concluded that RIII is a T4 antiholin which blocks membrane hole-formation by directly interacting with the holin. Accordingly, they proposed 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.

Phage T4 T-holin (lysis protein) is identical to the holin from phage K3 and nearly identical to that from phage ARI. Residues 35-96 are 28% identical to residues 436-495 of a K uptake protein of Lactococcus lactis (gbAAK04721; TC family 2.A.72; Kup), suggesting an evolutionary relationship between a holin and a transporter. Unlike T4 holin T, holins usually have 1, 2 or 3 TMSs and a short C-terminal domain rich in basic residues.

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