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.



Catalao MJ., Gil F., Moniz-Pereira J., Sao-Jose C. and Pimentel M. (2013). Diversity in bacterial lysis systems: bacteriophages show the way. FEMS Microbiol Rev. 37(4):554-71.

Chen, Y. and R. Young. (2016). The Last r Locus Unveiled: T4 RIII Is a Cytoplasmic Antiholin. J. Bacteriol. 198: 2448-2457.

Maciejewska, B., B. Roszniowski, A. Espaillat, A. Kęsik-Szeloch, G. Majkowska-Skrobek, A.M. Kropinski, Y. Briers, F. Cava, R. Lavigne, and Z. Drulis-Kawa. (2016). Klebsiella phages representing a novel clade of viruses with an unknown DNA modification and biotechnologically interesting enzymes. Appl. Microbiol. Biotechnol. [Epub: Ahead of Print]

Miller, E.S., E. Kutter, G. Mosig, F. Arisaka, T. Kunisawa, and W. Rüger. (2003). Bacteriophage T4 genome. Microbiol. Mol. Biol. Rev. 67: 86-156.

Moussa, S.H., J.L. Lawler, and R. Young. (2014). Genetic Dissection of T4 Lysis. J. Bacteriol. 196: 2201-2209.

Moussa, S.H., V. Kuznetsov, T.A. Tran, J.C. Sacchettini, and R. Young. (2012). Protein determinants of phage T4 lysis inhibition. Protein. Sci. 21: 571-582.

Ramanculov, E. and R. Young. (2001c). An ancient player unmasked: T4 rI encodes a t-specific antiholin. Mol. Microbiol. 41: 575-583.

Ramanculov, E. and R. Young. (2001a). Functional analysis of the phage T4 holin in a λ context. Mol. Genet. Genomics 265: 345-353.

Ramanculov, E. and R. Young. (2001b). Genetic analysis of the T4 holin: timing and topology. Gene 265: 25-36.

Tran, T.A., D.K. Struck, and R. Young. (2007). The T4 RI antiholin has an N-terminal signal anchor release domain that targets it for degradation by DegP. J. Bacteriol.189: 7618-7625.

Tran, T.A.T., D.K. Struck, and R. Young. (2005). Periplasmic domains define holin-antiholin interactions in T4 lysis inhibition. J. Bacteriol. 187: 6631-6640.


TC#NameOrganismal TypeExample

Lysis protein, T, of 218 aas and 1 TMS. T holin activity is regulated by the largely periplasmic anti-holin RI (P13304) and the cytoplasmic antiholin RIII which act synergistically (Chen and Young 2016). RI is degraded by DegP (Tran et al., 2007).  Mutations in all 3 topological domains if RI (the N-terminal cytoplasmic domain, the single TMS, and the C-terminal periplasmic domain) can abrogate holin function (Moussa et al. 2014).

Phage T4

Lysis protein or holin (P06808) of Phage T4


T-holin (196 aas) of phage RB43.


T-holin of phage RB43


Putative holin of 208 aas and 1 TMS.

Proteobacterial viruses

Holin of pectobacterial phage My1


Putative holin (lysis protein) of enterobacterial phage T5 (218 aas; 1 TMS) (Catalão et al. 2012).

Enterobacterial phage

Putative holin of phage T5


T holin lysis mediator of 215 aas and 1 TMS.  Phage KP27 protects against multidrug resistant strains of Klebsiella (Maciejewska et al. 2016).

Holin of Klebsiella phage KP27


Holin of 240 aas and 2 N-terminal TMSs. It interacts (via its C-terminus) with antiholin (via its C-terminus); this interaction blocks the holin homomultimerization and delays host cell lysis. It also interacts (via its N-terminus) with the lysis inhibition accessory protein rIII; this interaction stabilizes the holin-antiholin complex thereby resulting in a robust block of the hole formation.

Holin of Acinetobacter phage AbTZA1