GH15_072 (ORF72, named HolGH15 in the literature) is the holin of the virulent Staphylococcus aureus bacteriophage GH15, a 167-residue protein with two hydrophobic transmembrane helices and a long acidic, intrinsically disordered C-terminal tail. Its N-terminal domain organisation, two-helix topology and membership of the Phage_holin_1 (PF04531) superfamily identify it as a class II holin. Holins are the inner-membrane arm of the two-component phage lysis system: synthesised late in infection, they accumulate harmlessly in the cytoplasmic membrane of the infected cell until, at a time programmed into the protein's own sequence, they trigger, collapsing the proton motive force and tearing large non-specific lesions in the membrane. That event both destroys membrane integrity and releases the soluble endolysin into the periplasm to hydrolyse the peptidoglycan, so the cell bursts and the mature progeny virions escape. Heterologous expression of HolGH15 reproduces the canonical holin behaviour - growth and respiration arrest, premature triggering by the proton-motive-force uncoupler 2,4-dinitrophenol, and loss of cytoplasmic membrane and cell wall integrity with escape of cell contents - and both of its transmembrane domains are required for that activity. Purified HolGH15 is also bactericidal when applied from outside the cell, permeabilising the membranes of S. aureus, Listeria monocytogenes and several unrelated Gram-positive and Gram-negative species; this broad antibacterial activity is a property of the isolated protein rather than a role in the phage life cycle, and has been pursued as an antibacterial and food-safety application. GH15 also encodes the well-characterised endolysin LysGH15, the muralytic partner predicted by the holin-endolysin model.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0020002 host cell plasma membrane | IDA PMID:26873847 Identification and characterization of HolGH15: the holin of... | ACCEPT | Summary: CACAO row (assigned_by CACAO, dated 2025-03-31, evidence ECO:0006042). What the cited paper shows for this gene: HolGH15 is ORF72 of the Staphylococcus aureus phage GH15 genome, a protein with two hydrophobic transmembrane helices and the structural characteristics of a class II holin of the phage_holin_1 superfamily; on expression in Escherichia coli BL21 it arrests host growth, the arrest is triggered prematurely by the proton-motive-force uncoupler 2,4-dinitrophenol, transmission electron microscopy shows loss of cytoplasmic membrane and cell wall integrity with release of intracellular components, and both transmembrane domains are indispensable for that activity. The term matches: a holin's site of action and residence is the cytoplasmic (plasma) membrane of the infected host cell, and GO:0020002 is the interspecies cellular-component term for exactly that compartment. The UniProt entry independently carries two Phobius transmembrane helices (25-44, 56-73), Pfam PF04531 Phage_holin_1, NCBIfam TIGR01598 holin_phiLC3 and InterPro IPR006485 Phage-like_holin, and DNP-triggerable arrest only makes sense for a protein embedded in an energised membrane. Reason: Correct, appropriately specific, and the location where the core activity happens, so it is retained as a core function location. Two caveats were examined and neither changes the action. First, the cached publication is abstract-only (Europe PMC: isOpenAccess N, no PMC record; the publisher returns HTTP 403) and the abstract's membrane statements are predictions ("predicted to encode a membrane protein", "potentially consisted of two hydrophobic transmembrane helices"), so whatever direct localisation experiment backs the IDA is in the full text that the student curator read and this review cannot. Under the curator-deference rule that is not grounds to downgrade an experimental annotation whose claim is independently well supported. Second, the cells used were E. coli BL21, a heterologous expression host rather than GH15's host S. aureus, which brushes against the batch-1 CACAO shape "heterologous-system localization read as a host location" (Sindbis 6K in frog oocytes, phi29 p3 in a mammalian host cell nucleus). It does not instantiate it: the heterologous cell is itself a bacterium whose cytoplasmic membrane is the same structure as the real host's, GO:0020002 names no species, and the same paper shows the protein acting on S. aureus itself. The corpus precedent is sieA (BPP22), where GO:0020002 located_in is accepted for a phage protein in the bacterial inner membrane. Supporting Evidence: PMID:26873847 This protein exhibited the structural characteristics of class II holins and belonged to the phage_holin_1 superfamily PMID:26873847 Mutation analysis indicated that the two transmembrane domains of HolGH15 were indispensable for the activity of the full-length protein PMID:26873847 Expression of HolGH15 in Escherichia coli BL21 cells resulted in growth retardation of the host cells, which was triggered prematurely by the addition of 2,4-dinitrophenol PMID:24585055 the holin, accumulates harmlessly in the cytoplasmic membrane until triggering at an allele-specific time to form micron-scale holes file:BPG15/GH15_072/GH15_072-uniprot.txt Phage-like_holin |
| GO:0044659 viral release from host cell by cytolysis | IDA PMID:26873847 Identification and characterization of HolGH15: the holin of... | ACCEPT | Summary: CACAO row (assigned_by CACAO, dated 2025-03-03, evidence ECO:0001847). What the cited paper shows for this gene: HolGH15 is the holin of phage GH15 and behaves as a canonical one - heterologous expression halts host growth, 2,4-dinitrophenol triggers it prematurely, electron microscopy shows loss of cytoplasmic membrane and cell wall integrity with release of intracellular components, and both transmembrane domains are required. The term matches the biology: GO:0044659 is defined as "The dissemination of mature viral particles from a host cell by the rupture of cell membranes and the loss of cytoplasm", and the holin performs the membrane-rupture step of that process, after which wall hydrolysis by the endolysin and escape of the progeny follow at once. It is also the term the rest of GOA uses for lysis-system proteins, including lambda S (the archetypal canonical holin) and LysGH15, this same phage's endolysin. The defect in this row is the qualifier, and a second, weaker concern is the evidence code; both are recorded in source_defects. Reason: The claim is correct and the term is the right one, so the row is kept as a core function; what is wrong is the GAF qualifier. acts_upstream_of_or_within_positive_effect describes a gene product that influences a process it does not itself carry out, and this is the question the batch-3 screen raises for a holin, because a holin can be described either way: GO's own obsoleted holin term (GO:0015176) says the "Primary function of holins appears to be transport of murein hydrolases across the cytoplasmic membrane to the cell wall", and Young's review describes the holin as forming holes "allowing the endolysin to escape and attack the peptidoglycan". That upstream reading is about the endolysin's access to the wall, not about the process GO:0044659 names. The definition of GO:0044659 includes "the rupture of cell membranes and the loss of cytoplasm", and the holin executes that rupture itself: cryo-EM shows the micron-scale membrane lesions are "dependent solely on the function of the S105 protein", holin triggering with no endolysin present already collapses the proton motive force and bleeds the cytoplasm, and "Destruction of the murein and bursting of the cell are immediate sequelae". HolGH15 is a class II (two-TMD) canonical holin, not a pinholin, and the paper's own TEM shows it destroying membrane integrity and releasing cell contents. So the protein is within the process and the correct qualifier is involved_in. The comparator evidence is unusually clean: of the 50 experimental annotations to GO:0044659 in GOA, 44 use involved_in, among them lambda S (P03705, IDA, UniProt), PRD1 endolysin XV (P13559, IDA, CACAO) and LysGH15 (D6QY02, IDA, CACAO) - the endolysin of this same phage, from the same laboratory. All six experimental rows on this term carrying a non-standard positive-effect qualifier are CACAO rows, and two of those genes (Q9T1W9, Q9T1X2) have a paired UniProt row with the same term, same reference and same evidence code qualified involved_in, so a professional curator working from identical input chose involved_in. Separately, no experiment in the cited paper follows an infection or measures release of progeny virions: every assay is heterologous expression or exogenous application of purified protein, which is the situation in which this project's LysK review deliberately chose IC over an experimental code. That is recorded as a second, SUSPECTED defect rather than a reason to reject the claim, since the inaccessible full text could contain an infection experiment the abstract does not mention. Neither defect touches the term, the reference or the gene product. Supporting Evidence: PMID:26873847 The expression of HolGH15 caused morphological alterations in engineered E. coli cells, including loss of the cell wall and cytoplasmic membrane integrity and release of intracellular components, which were visualized by transmission electron microscopy PMID:26873847 Holins are phage-encoded hydrophobic membrane proteins that spontaneously and non-specifically accumulate and form lesions in the cytoplasmic membrane PMID:24585055 ranging in scale up to > 1ΞΌm, is dependent solely on the function of the S105 protein PMID:24585055 Without spanin function, lysis is blocked and progeny virions are trapped in dead spherical cells PMID:11018145 Destruction of the murein and bursting of the cell are immediate sequelae |
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Download this section (compressed HTML)Q: GO obsoleted both of its holin terms (GO:0015176 molecular function, GO:0015832 biological process) as classes of gene products, offering only GO:0019835 cytolysis in their place, and GO:0140911 pore-forming activity is defined for insertion into the membrane of another cell. Would GO consider an activity-level term for programmed permeabilisation of the membrane of the cell in which the protein is produced, so that holins can be annotated at the molecular function level and not only to process terms?
Q: When does HolGH15 trigger during a GH15 infection of Staphylococcus aureus, and does a holin-defective GH15 derivative fail to release progeny virions? This is the one experiment that would make the GO:0044659 annotation direct rather than inferred from holin class.
Q: Does HolGH15 form the micron-scale lesions of a canonical holin, or the small heptameric channels of a pinholin? The class II topology assignment rests on hydropathy alone, and the answer determines whether LysGH15 escapes through holes or is a depolarisation-activated enzyme.
Q: What does the long acidic, intrinsically disordered C-terminal region (residues 114-167, with an acidic stretch at 124-142) do? In lambda S and other holins the charged cytoplasmic tail influences triggering time, but it has not been tested in HolGH15, whose mutational analysis addressed only the two transmembrane domains.
Q: Why is purified HolGH15 bactericidal from outside the cell, and against species as distant as Pseudomonas aeruginosa and Klebsiella pneumoniae? A holin's natural substrate is the membrane of the cell that produced it, so exogenous activity on unrelated Gram-negative species implies a non-physiological route of insertion that is worth distinguishing from the life-cycle activity before the protein is developed as an antibacterial.
Experiment: Follow one-step growth of GH15 on Staphylococcus aureus, measuring optical density, proton motive force (with a membrane-potential dye) and extracellular versus intracellular plaque-forming units to separate the moment of membrane depolarisation from the moment of virion release. Repeat with an amber or deletion derivative of GH15_072 complemented in trans, and test whether 2,4-dinitrophenol advances lysis in the infected culture as it does on heterologous expression.
Hypothesis: HolGH15 is the lysis clock of phage GH15: it triggers at an allele-specific time during infection, and loss of its function blocks release of progeny virions.
Type: phage one-step growth / lysis timing
Experiment: Image triggered cells by cryo-electron tomography, as was done for lambda S105, P2 Y and T4 T, counting and sizing the membrane interruptions; in parallel reconstitute purified HolGH15 into planar bilayers or proteoliposomes and record conductance to distinguish discrete heptameric channels from large non-specific lesions.
Hypothesis: HolGH15 is a canonical holin that forms a small number of micron-scale lesions, rather than a pinholin forming many small channels.
Type: cryo-electron tomography and electrophysiology
Experiment: Co-express HolGH15 and LysGH15 in a Gram-positive surrogate and compare lysis with either protein alone, assaying peptidoglycan hydrolysis and release of a cytoplasmic reporter; test whether a holin-independent SAR-type signal peptide is present in LysGH15 by probing its membrane association before triggering.
Hypothesis: HolGH15 and LysGH15 are the functional holin-endolysin pair of GH15, and the holin is what gives LysGH15 access to the peptidoglycan.
Type: reconstitution of the lysis system
Experiment: Make progressive C-terminal truncations and charge-neutralising substitutions across residues 114-167, express each in E. coli and measure the time to growth arrest and the 2,4-dinitrophenol sensitivity, so that advance, delay and abolition of triggering can be distinguished as they are for lambda S alleles.
Hypothesis: The acidic disordered C-terminal tail of HolGH15 tunes its triggering time.
Type: structure-function analysis
Experiment: Compare the concentration dependence, lipid requirement and energy dependence of killing by externally applied HolGH15 with the properties of triggering on internal expression, using liposomes of defined composition, de-energised cells and the transmembrane-domain mutants already shown to abolish the internal activity. If the mutants retain exogenous killing, the two activities are mechanistically distinct.
Hypothesis: Exogenous bactericidal activity of purified HolGH15 proceeds by a different route of membrane insertion from the one used in the phage life cycle.
Type: comparative membrane permeabilization assay
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