During λ infections, the holin S105 accumulates harmlessly in the membrane until, at an allele-specific time, suddenly triggering to form irregular holes of unprecedented size (>300 nm), releasing the endolysin from the cytoplasm, resulting in lysis within seconds. Here we used a functional S105-GFP chimera and real-time deconvolution fluorescence microscopy to show that the S105-GFP fusion accumulated in a uniformly distributed fashion, until suddenly, within 1 min, it formed aggregates, or rafts, at the time of lethal triggering. Moreover, the isogenic fusion to a nonlethal S105 mutant remained uniformly distributed, whereas a fusion to an earlylysing mutant showed early triggering and early raft formation. Protein accumulation rates of the WT, early, and nonlethal alleles were identical. Fluorescence recovery after photobleaching (FRAP) revealed that the nonlethal mutant and untriggered WT hybrids were highly mobile in the membrane, whereas the WT raft was essentially immobile. Finally, an antiholin allele, S105 ΔTMD1 -mcherryfp, in the product of which the S105 sequence deleted for the first transmembrane domain was fused to mCherryFP. This hybrid retained full antiholin activity, in that it blocked lethal hole formation by the S105-GFP fusion, accumulated uniformly throughout the host membrane and prevented the S105-GFP protein from forming rafts. These findings suggest that phage lysis occurs when the holin reaches a critical concentration and nucleates to form rafts, analogous to the initiation of purple membrane formation after the induction of bacteriorhodopsin in halobacteria. This model for holin function may be relevant for processes in mammalian cells, including the release of nonenveloped viruses and apoptosis.bacteriophage | latent period | peptide linker T he programmed formation of nonspecific, lethal membrane lesions, or "holes," is featured in many cytocidal phenotypes, including Bax-mediated apoptosis, virion release in infections of nonenveloped mammalian viruses, and the dissemination of important human toxins from bacteria (1-6). The most genetically tractable hole-formation process is host lysis in double-strand DNA bacteriophage infections (7,8). In the infection cycle of phage λ, lysis is a precisely timed event controlled by the holin, S105, a 105-aa product of the S gene (Fig. 1). S105 accumulates in the membrane throughout the morphogenesis period of the infection cycle. This accumulation has no effect on membrane integrity or the proton-motive force (PMF), as shown by noninvasive assays measuring the flagellar rotation speed (9), until suddenly triggering to form irregular holes of unprecedented size (>300 nm) (10). These holes allow release of the phage endolysin, R, from the cytoplasm, resulting in destruction of the cell wall within seconds (9). The holin triggering time is allele specific (11,12), in that it can be advanced or retarded by missense mutations throughout all three transmembrane domains (TMDs) of S105. Holins can be triggered prematurely by energy poisons; for S10...
Holins are small phage-encoded proteins that accumulate harmlessly in the cytoplasmic membrane during the infection cycle until suddenly, at an allele-specific time, triggering to form lethal lesions, or "holes." In the phages λ and T4, the holes have been shown to be large enough to allow release of prefolded active endolysin from the cytoplasm, which results in destruction of the cell wall, followed by lysis within seconds. Here, the holes caused by S105, the λ-holin, have been captured in vivo by cryo-EM. Surprisingly, the scale of the holes is at least an order of magnitude greater than any previously described membrane channel, with an average diameter of 340 nm and some exceeding 1 μm. Most cells exhibit only one hole, randomly positioned in the membrane, irrespective of its size. Moreover, on coexpression of holin and endolysin, the degradation of the cell wall leads to spherically shaped cells and a collapsed inner membrane sac. To obtain a 3D view of the hole by cryo-electron tomography, we needed to reduce the average size of the cells significantly. By taking advantage of the coupling of bacterial cell size and growth rate, we achieved an 80% reduction in cell mass by shifting to succinate minimal medium for inductions of the S105 gene. Cryotomographic analysis of the holes revealed that they were irregular in shape and showed no evidence of membrane invagination. The unexpected scale of these holes has implications for models of holin function.bacteriophage | cryoelectron tomography | Escherichia coli | holin | lambda B acteriophage lysis, the most frequent cytolethal event in the biosphere, is a precisely scheduled process controlled by proteins of the holin family (1). Holins are an extremely diverse class of small phage-encoded membrane proteins (2). The best studied holin is S105, a 105-residue polypeptide with three transmembrane domains (TMDs) encoded by the S gene of phage λ (3). Throughout the period of late gene expression and particle assembly, S105 accumulates in the cytoplasmic membrane of Escherichia coli without any effect on its integrity (4). Suddenly, at a programmed time, S105 triggers to form a lesion, or hole, in the membrane; this allows the λ-endolysin, R, to escape from the cytoplasm and attack the cell wall (2). In phages of Gram-negative hosts, there is a third step to complete the lysis pathway involving a protein or protein complex, the spanin, which connects the cytoplasmic and outer membranes (5, 6). In λ, the spanin complex consists of the cytoplasmic membrane protein, Rz, and the outer membrane lipoprotein, Rz1. This complex is essential for lysis in media containing millimolar concentrations of divalent cations, and thus is thought to act by disrupting the outer membrane, possibly by fusion with the inner membrane (6).Although the S105 holin has been extensively studied using genetic and biochemical approaches (3, 4, 7-9), nothing is known about the membrane holes except that they are nonspecific and large enough to allow escape of fully folded tetrameric R-β-galactosi...
Summary Holins control the length of the infection cycle of tailed phages (the Caudovirales) by oligomerizing to form lethal holes in the cytoplasmic membrane at a time dictated by their primary structure. Nothing is currently known about the physical basis of their oligomerization or the structure of the oligomers formed by any known holin. Here we use electron microscopy and single-particle analysis to characterize structures formed by the bacteriophage λ holin (S105) in vitro. In non-ionic or mild zwitterionic detergents, purified S105, but not the lysis-defective variant S105A52V, forms rings of at least two size classes, the most common having inner and outer diameters of 8.5 and 23 nm respectively, and containing approximately 72 S105 monomers. The height of these rings, 4 nm, closely matches the thickness of the lipid bilayer. The central channel is of unprecedented size for channels formed by integral membrane proteins, consistent with the non-specific nature of holin-mediated membrane permeabilization. S105 present in detergent-solubilized rings and in inverted membrane vesicles showed similar sensitivities to proteolysis and cysteine-specific modification, suggesting that the rings are representative of the lethal holes formed by S105 to terminate the infection cycle and initiate lysis.
Summary At a programmed time in phage infection cycles, canonical holins suddenly trigger to cause lethal damage to the cytoplasmic membrane, resulting in the cessation of respiration and the non-specific release of pre-folded, fully active endolysins to the periplasm. For the paradigm holin S105 of lambda, triggering is correlated with the formation of micron-scale membrane holes, visible as interruptions in the bilayer in cryo-electron microscopic images and tomographic reconstructions. Here we report that the size distribution of the holes is stable for long periods after triggering. Moreover, early triggering caused by an early lysis allele of S105 formed approximately the same number of holes, but the lesions were significantly smaller. In contrast, early triggering prematurely induced by energy poisons resulted in many fewer visible holes, consistent with previous sizing studies. Importantly, the unrelated canonical holins P2 Y and T4 T were found to cause the formation of holes of approximately the same size and number as for lambda. In contrast, no such lesions were visible after triggering of the pinholin S2168. These results generalize the hole formation phenomenon for canonical holins. A model is presented suggesting the unprecedentedly large size of these holes is related to the timing mechanism.
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