ORF72 of the Staphylococcus aureus phage GH15 genome, designated HolGH15, encodes a predicted membrane protein with two hydrophobic transmembrane helices that has the structural characteristics of a class II holin and belongs to the phage_holin_1 superfamily.

HolGH15 has the structural hallmarks of a class II holin (two transmembrane domains) and belongs to the phage_holin_1 family, the family UniProt also assigns from Pfam PF04531 and NCBIfam TIGR01598.

Expression of HolGH15 in E. coli arrests host growth, and the arrest is triggered prematurely by the proton-motive-force uncoupler 2,4-dinitrophenol - the diagnostic behaviour of a genuine holin rather than of a generically toxic membrane protein.

Transmission electron microscopy of cells expressing HolGH15 shows loss of cytoplasmic membrane and cell wall integrity with release of intracellular contents, i.e. cytolysis.

Both transmembrane domains are required for HolGH15 activity, tying the membrane lesion to the membrane-embedded part of the protein.

Applied from outside the cell, purified HolGH15 kills S. aureus and Listeria monocytogenes by evoking release of cellular contents, and inhibits a range of unrelated species. This is the isolated protein acting as an antibacterial agent, not a step of the GH15 life cycle.

The paper's stated novelty is the heterologous antibacterial activity of a staphylococcal phage holin; no phage infection, lysis-timing or progeny-release experiment is reported in the abstract.

Externally applied HolGH15 permeabilises the Listeria monocytogenes cytoplasmic membrane, releasing cytoplasmic beta-galactosidase, and kills the cells by destroying membrane integrity.

Electron microscopy attributes the bactericidal effect of HolGH15 specifically to membrane permeabilisation and loss of membrane integrity, confirming that the protein's activity is on the membrane itself.

In canonical phage lysis the holin itself ruptures the cytoplasmic membrane: it accumulates harmlessly there and then triggers to form micron-scale holes.

Cryo-electron microscopy and tomography show that holin triggering alone produces massive interruptions in the cytoplasmic membrane, and that the lesions depend solely on the holin protein - the holin performs the membrane rupture, it does not merely permit it.

Holin triggering in the absence of any endolysin halts growth and respiration, bleeds cytoplasmic ions and small molecules to the medium and collapses the proton motive force - the phenotype Song et al. observed on heterologous expression of HolGH15.

Premature triggering by the uncoupler 2,4-dinitrophenol is the single most diagnostic property of a holin, which is what makes the DNP result in PMID:26873847 identify HolGH15 as a genuine holin.

The alternative reading of holin function - that it acts upstream by letting the endolysin out - is about the endolysin's access to the peptidoglycan, not about whether the holin participates in lysis.

Lysis is what releases the progeny virions: when the final step is blocked the virions stay trapped inside the cell.

Holins are small membrane proteins that accumulate in the membrane and, at a genetically programmed time, permeabilise it to the folded endolysin; destruction of the murein and bursting of the cell follow immediately.