Holin/endolysin/spanin cell lysis

In most phages, active and timed lysis involves three steps corresponding to disruption of each of the three layers of the cell envelope: inner membrane, peptidoglycan and outer membrane .

Two proteins, an endolysin and a holin are usually required for programmed host cell lysis by dsDNA prokaryotic viruses . Additional proteins called spanins are involved in the lysis process in Gram-negative hosts. A multistep process allows a better control of lysis timing.

1. Cell membrane disruption (Gram-negative host, Gram-positive host)

Holins (from making "holes") are small membrane proteins that control the lysis timing. They accumulate in the host cell membrane until, at a programmed time (probably when reaching a critical concentration), they aggregate into oligomers and permeabilize the host cytoplasmic membrane inducing loss of polarization.

Canonical holins form large pores at one side of the bacteria, exposing locally the peptidoglycans to cytoplasmic canonical endolysin molecules . The outcome of this kind of lysis is a localized envelope catastrophe: "local blow-out".

Pinholins form small pores that result in membrane depolarization, triggering SAR endolysin activation and degradation of peptidoglycans in the whole cellular periplasmic space. The outcome of this kind of lysis is a general lysis of the outer membrane of the host cell.

Antiholins associate specifically with the holins or pinholins giving rise to inactive heteromultimers. This allows a larger accumulation of holin molecules without any risk of premature aggregation before the triggering time.

ssDNA and ssRNA prokaryotic viruses usually have a "single gene lysis" which is thought to inhibit the peptidoglycan layer polymerization.
The advantage of the two-component system is that holins allow a abrupt and time-determined lysis thus preventing slow host cell deterioration.

2. Peptidoglycan disruption (Gram-negative host, Gram-positive host)

Endolysins are muralytic enzymes that degrade the host cell wall. Depending on the absence or presence of a signal sequence, endolysins are classified as canonical endolysins (no signal sequence) and signal-anchor-release endolysins (SAR endolysins, N-terminal type II signal anchor). Canonical endolysins accumulate in the cytoplasm and are released at a precise timing through the large holes generated by the aggregation of holin molecules.

SAR endolysins are already bound on the periplasmic side of the cell membrane and thus do not need to transit through large holes in the cell membrane. Instead, their release occurs through membrane depolarization performed by pinholins, which make small holes in the host cell membrane (about 2 nm in diameter) .

3. Outer membrane disruption (Gram-negative host)

Spanins are essential for the lysis of bacteria with both internal (IM) and outer (OM) membranes (mostly Gram-negative). Lysis of these host bacteria additionally requires the disruption of the outer membrane. Spanins span the inner and outer membrane either as a unimolecular (u-spanin) or as a heterodimer molecule (IM-spanin or i-spanin + OM-spanin or o-spanin) .
After cell wall degradation by the endolysin, the spanin complexes are free to diffuse and aggregate. the lytic blowout occurs when sufficient spanin complexes are liberated within the degraded area of the PG. They mediate the fusion of the IM and OM, thereby disrupting the cell membranes .

Examples of lysis cassettes:

Virus	Family	Genus	Phage lysis protein	Reference
Phage T4	Myoviridae	T4likevirus	Endolysin	Programmed Escherichia coli cell lysis by expression of cloned T4 phage lysis genes M Morita, K Asami, Y Tanji, H Unno Biotechnol. Prog. June 2001; 17: 573-576
			Holin	Programmed Escherichia coli cell lysis by expression of cloned T4 phage lysis genes M Morita, K Asami, Y Tanji, H Unno Biotechnol. Prog. June 2001; 17: 573-576
			Spanin, outer membrane subunit	Rz/Rz1 lysis gene equivalents in phages of Gram-negative hosts Elizabeth J Summer, Joel Berry, Tram Anh T Tran, Lili Niu, Douglas K Struck, Ry Young J. Mol. Biol. November 9, 2007; 373: 1098-1112
			Spanin, inner membrane subunit	Rz/Rz1 lysis gene equivalents in phages of Gram-negative hosts Elizabeth J Summer, Joel Berry, Tram Anh T Tran, Lili Niu, Douglas K Struck, Ry Young J. Mol. Biol. November 9, 2007; 373: 1098-1112
			Antiholin	An ancient player unmasked: T4 rI encodes a t-specific antiholin E Ramanculov, R Young Mol. Microbiol. August 2001; 41: 575?583 Protein determinants of phage T4 lysis inhibition Samir H Moussa, Vladimir Kuznetsov, Tram Anh T Tran, James C Sacchettini, Ry Young Protein Sci. April 2012; 21: 571-582
Phage P2	Myoviridae	P2likevirus	Endolysin	Rz/Rz1 lysis gene equivalents in phages of Gram-negative hosts Elizabeth J Summer, Joel Berry, Tram Anh T Tran, Lili Niu, Douglas K Struck, Ry Young J. Mol. Biol. November 9, 2007; 373: 1098-1112
			Holin	Rz/Rz1 lysis gene equivalents in phages of Gram-negative hosts Elizabeth J Summer, Joel Berry, Tram Anh T Tran, Lili Niu, Douglas K Struck, Ry Young J. Mol. Biol. November 9, 2007; 373: 1098-1112 Functional analysis of a class I holin, P2 Y Kam H To, Jill Dewey, Jeremy Weaver, Taehyun Park, Ry Young J. Bacteriol. March 2013; 195: 1346-1355
			Antiholin	Functional analysis of a class I holin, P2 Y Kam H To, Jill Dewey, Jeremy Weaver, Taehyun Park, Ry Young J. Bacteriol. March 2013; 195: 1346-1355
			Spanin, inner membrane subunit	Rz/Rz1 lysis gene equivalents in phages of Gram-negative hosts Elizabeth J Summer, Joel Berry, Tram Anh T Tran, Lili Niu, Douglas K Struck, Ry Young J. Mol. Biol. November 9, 2007; 373: 1098-1112
			Spanin, outer membrane subunit	Rz/Rz1 lysis gene equivalents in phages of Gram-negative hosts Elizabeth J Summer, Joel Berry, Tram Anh T Tran, Lili Niu, Douglas K Struck, Ry Young J. Mol. Biol. November 9, 2007; 373: 1098-1112
Phage P22	Podoviridae	P22likevirus	Endolysin	Phage P22 lysis genes: nucleotide sequences and functional relationships with T4 and lambda genes D Rennell, A R Poteete Virology May 1985; 143: 280-289
			Holin	Phage P22 lysis genes: nucleotide sequences and functional relationships with T4 and lambda genes D Rennell, A R Poteete Virology May 1985; 143: 280-289
			Spanin, inner membrane subunit	Nucleotide sequence of the bacteriophage P22 gene 19 to 3 region: identification of a new gene required for lysis S Casjens, K Eppler, R Parr, A R Poteete Virology August 1989; 171: 588?598
			Spanin, outer membrane subunit	-
Phage φKMV	Podoviridae	Phikmvlikevirus	SAR endolysin	The lysis cassette of bacteriophage φKMV encodes a signal-arrest-release endolysin and a pinholin Yves Briers, Liesbet M Peeters, Guido Volckaert, Rob Lavigne Bacteriophage 2011; 1: 25-30
			Pinholin	The lysis cassette of bacteriophage φKMV encodes a signal-arrest-release endolysin and a pinholin Yves Briers, Liesbet M Peeters, Guido Volckaert, Rob Lavigne Bacteriophage 2011; 1: 25-30
			Spanin, inner membrane subunit	The lysis cassette of bacteriophage φKMV encodes a signal-arrest-release endolysin and a pinholin Yves Briers, Liesbet M Peeters, Guido Volckaert, Rob Lavigne Bacteriophage 2011; 1: 25-30
			Spanin, outer membrane subunit	The lysis cassette of bacteriophage φKMV encodes a signal-arrest-release endolysin and a pinholin Yves Briers, Liesbet M Peeters, Guido Volckaert, Rob Lavigne Bacteriophage 2011; 1: 25-30
Phage T7	Podoviridae	T7likevirus	Holin	Rz/Rz1 lysis gene equivalents in phages of Gram-negative hosts Elizabeth J Summer, Joel Berry, Tram Anh T Tran, Lili Niu, Douglas K Struck, Ry Young J. Mol. Biol. November 9, 2007; 373: 1098-1112
			Spanin, inner membrane subunit	Rz/Rz1 lysis gene equivalents in phages of Gram-negative hosts Elizabeth J Summer, Joel Berry, Tram Anh T Tran, Lili Niu, Douglas K Struck, Ry Young J. Mol. Biol. November 9, 2007; 373: 1098-1112
			Spanin, outer membrane subunit	Rz/Rz1 lysis gene equivalents in phages of Gram-negative hosts Elizabeth J Summer, Joel Berry, Tram Anh T Tran, Lili Niu, Douglas K Struck, Ry Young J. Mol. Biol. November 9, 2007; 373: 1098-1112
Phage lambda	Siphoviridae	Lambdalikevirus	Endolysin	Cell lysis by induction of cloned lambda lysis genes J Garrett, R Fusselman, J Hise, L Chiou, D Smith-Grillo, J Schulz, R Young Mol. Gen. Genet. 1981; 182: 326-331
			Holin	Holin triggering in real time Rebecca White, Shinobu Chiba, Ting Pang, Jill S Dewey, Christos G Savva, Andreas Holzenburg, Kit Pogliano, Ry Young Proc. Natl. Acad. Sci. U.S.A. January 11, 2011; 108: 798-803
			Spanin, inner membrane subunit	Spanin function requires subunit homodimerization through intermolecular disulfide bonds Joel D Berry, Manoj Rajaure, Ry Young Mol. Microbiol. April 2013; 88: 35-47
			Spanin, outer membrane subunit	Spanin function requires subunit homodimerization through intermolecular disulfide bonds Joel D Berry, Manoj Rajaure, Ry Young Mol. Microbiol. April 2013; 88: 35-47
Phage T1	Siphoviridae	Tunalikevirus	Endolysin	Rz/Rz1 lysis gene equivalents in phages of Gram-negative hosts Elizabeth J Summer, Joel Berry, Tram Anh T Tran, Lili Niu, Douglas K Struck, Ry Young J. Mol. Biol. November 9, 2007; 373: 1098-1112
			Holin	Rz/Rz1 lysis gene equivalents in phages of Gram-negative hosts Elizabeth J Summer, Joel Berry, Tram Anh T Tran, Lili Niu, Douglas K Struck, Ry Young J. Mol. Biol. November 9, 2007; 373: 1098-1112
			u-Spanin	Rz/Rz1 lysis gene equivalents in phages of Gram-negative hosts Elizabeth J Summer, Joel Berry, Tram Anh T Tran, Lili Niu, Douglas K Struck, Ry Young J. Mol. Biol. November 9, 2007; 373: 1098-1112