Structure of the lethal phage pinhole

Pang, Ting; Savva, Christos G.; Fleming, Karen G.; Struck, Douglas K.; Young, Ry

doi:10.1073/pnas.0907941106

Cited by 78 publications

(200 citation statements)

References 36 publications

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“…Previous studies have shown that the holin from lambda bacteriophage 21 has two TMDs, and that TMD 1 is dispensable for pore formation and lysis (Pang et al, 2009). However, our results showed that both TMD 1 and TMD 2 of HolGH15 are indispensable for the function of the full-length protein.…”

Section: Discussioncontrasting

confidence: 65%

“…The typical holin forms very large pores (large enough for 500 kDa proteins) that allow the non-specific release of endolysins and other proteins into the cytoplasm (Bläsi & Young, 1996;Wang et al, 2003). However, the holin protein of lambda phage 21 forms pinholes that only depolarize the membrane (Pang et al, 2009(Pang et al, , 2010a(Pang et al, , b, 2013. Holins are also important for many activities, such as classification, gene transfer (Lang & Beatty, 2001), biofilm formation (Ranjit et al, 2011) and biotechnology material production (Gao et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Identification and characterization of HolGH15: the holin of Staphylococcus aureus bacteriophage GH15

Song

Xia

Jiang

et al. 2016

Journal of General Virology

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Holins are phage-encoded hydrophobic membrane proteins that spontaneously and nonspecifically accumulate and form lesions in the cytoplasmic membrane. The ORF72 gene (also designated HolGH15) derived from the genome of the Staphylococcus aureus phage GH15 was predicted to encode a membrane protein. An analysis indicated that the protein encoded by HolGH15 potentially consisted of two hydrophobic transmembrane helices. This protein exhibited the structural characteristics of class II holins and belonged to the phage_holin_1 superfamily. 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. 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. HolGH15 exerted efficient antibacterial activity at 37 8C and pH 5.2. Mutation analysis indicated that the two transmembrane domains of HolGH15 were indispensable for the activity of the full-length protein. HolGH15 showed a broad antibacterial range: it not only inhibited Staphylococcus aureus, but also demonstrated antibacterial activity against other species, including Listeria monocytogenes, Bacillus subtilis, Pseudomonas aeruginosa, Klebsiella pneumoniae and E. coli. At the minimal inhibitory concentration, HolGH15 evoked the release of cellular contents and resulted in the shrinkage and death of Staphylococcus aureus and Listeria monocytogenes cells. To the best of our knowledge, this study is the first report of a Staphylococcus aureus phage holin that exerts antibacterial activity against heterogeneous pathogens.

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Section: Discussioncontrasting

confidence: 65%

Section: Introductionmentioning

confidence: 99%

Identification and characterization of HolGH15: the holin of Staphylococcus aureus bacteriophage GH15

Song

Xia

Jiang

et al. 2016

Journal of General Virology

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“…In addition, the behavior of purified S 21 68 and S105 in the nonionic detergent dodecylmaltoside supports the notion that the canonical holin has much more scope for protein-protein interactions (20). S105 was found to form highly oligomeric ring structures estimated to contain >70 molecules, whereas S 21 68 forms a heptameric homooligomer in dodecylmaltoside (4).…”

Section: Discussionsupporting

confidence: 59%

“…The results of a combined biochemical, genetic, ultrastructural, and computational approach indicate that the prototype pinholin, S 21 68 ( Fig. 1 A and B), of lambdoid phage 21 of Escherichia coli forms heptamers with a central lumen <2 nm in diameter (4), too small to allow the passage of endolysin (5). Phages using pinholins, therefore, require a distinct class of muralytic enzymes called the signal anchor release (SAR) endolysins, which are exported in a membrane-tethered enzymatically inactive form during the latent period.…”

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confidence: 99%

Visualization of pinholin lesions in vivo

Pang

Fleming

Pogliano

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Lambdoid phage 21 uses a pinholin-signal anchor release endolysin strategy to effect temporally regulated host lysis. In this strategy, the pinholin S 21 68 accumulates harmlessly in the bilayer until suddenly triggering to form lethal membrane lesions, consisting of S 21 68 heptamers with central pores <2 nm in diameter. The membrane depolarization caused by these pores activates the muralytic endolysin, R 21, leading immediately to peptidoglycan degradation. The lethal S 21 68 complexes have been designated as pinholes to distinguish from the micrometer-scale holes formed by canonical holins. Here, we used GFP fusions of WT and mutant forms of S 21 68 to show that the holin accumulates uniformly throughout the membrane until the time of triggering, when it suddenly redistributes into numerous small foci (rafts). Raft formation correlates with the depletion of the proton motive force, which is indicated by the potential-sensitive dye bis-(1,3-dibutylbarbituric acid)pentamethine oxonol. By contrast, GFP fusions of either antiholin variant irsS 21 68, which only forms inactive dimers, or nonlethal mutant S 21 68 S44C , which is blocked at an activated dimer stage of the pinhole formation pathway, were both blocked in a state of uniform distribution. In addition, fluorescence recovery after photobleaching revealed that, although the antiholin irsS 21 68-GFP fusion was highly mobile in the membrane (even when the proton motive force was depleted), more than one-half of the S 21 68-GFP molecules were immobile, and the rest were in mobile states with a much lower diffusion rate than the rate of irsS 21 68-GFP. These results suggest a model in which, after transiting into an oligomeric state, S 21 68 migrates into rafts with heterogeneous sizes, within which the final pinholes form.bacteriophage | membrane protein | membrane potential | structured illumination microscopy I n general, the phage infection cycle is terminated by the function of the holin, a small viral membrane protein (1). Throughout the morphogenesis period, holins accumulate harmlessly in the cytoplasmic membrane until suddenly forming lethal membrane lesions (holes). This event, called triggering, occurs at an allele-specific time. The lesions formed by canonical holins are very large, with diameters of micrometer-scale, allowing the escape of prefolded phage endolysins from the cytoplasm (2, 3). However, another important class of holins, the pinholins, form holes too small for the passage of protein. The results of a combined biochemical, genetic, ultrastructural, and computational approach indicate that the prototype pinholin, S 21 68 ( Fig. 1 A and B), of lambdoid phage 21 of Escherichia coli forms heptamers with a central lumen <2 nm in diameter (4), too small to allow the passage of endolysin (5). Phages using pinholins, therefore, require a distinct class of muralytic enzymes called the signal anchor release (SAR) endolysins, which are exported in a membrane-tethered enzymatically inactive form during the latent period. When the pinholins trig...

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“…This would undoubtedly kill the cell. Indeed, b-barrel toxins, such as alpha-haemolysin, as well as many a-helical antimicrobial peptides use this 'hole poking' mechanism to kill foreign cells [6][7][8].…”

Section: Introduction: the Challenges Of Unfolded Outer Membrane Protmentioning

confidence: 99%

A combined kinetic push and thermodynamic pull as driving forces for outer membrane protein sorting and folding in bacteria

Fleming

2015

Phil. Trans. R. Soc. B

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One contribution of 12 to a theme issue 'The bacterial cell envelope'. In vitro folding studies of outer membrane beta-barrels have been invaluable in revealing the lipid effects on folding rates and efficiencies as well as folding free energies. Here, the biophysical results are summarized, and these kinetic and thermodynamic findings are considered in terms of the requirements for folding in the context of the cellular environment. Because the periplasm lacks an external energy source the only driving forces for sorting and folding available within this compartment are binding or folding free energies and their associated rates. These values define functions for periplasmic chaperones and suggest a biophysical mechanism for the BAM complex.

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Structure of the lethal phage pinhole

Cited by 78 publications

References 36 publications

Identification and characterization of HolGH15: the holin of Staphylococcus aureus bacteriophage GH15

Identification and characterization of HolGH15: the holin of Staphylococcus aureus bacteriophage GH15

Visualization of pinholin lesions in vivo

A combined kinetic push and thermodynamic pull as driving forces for outer membrane protein sorting and folding in bacteria

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