The Pinholin of Lambdoid Phage 21: Control of Lysis by Membrane Depolarization

Abstract

The phage 21 holin, S 21 , forms small membrane holes that depolarize the membrane and is designated as a pinholin, as opposed to large-hole-forming holins, like S . Pinholins require secreted SAR endolysins, a pairing that may represent an intermediate in the evolution of canonical holin-endolysin systems.For most phages, the termination of each infection cycle is the strictly programmed and regulated lysis of the host, brought about by two phage-encoded proteins (28). One of these, the endolysin, is capable of degrading the cell wall, while the second, the holin, is a small membrane protein which controls endolysin function. During the assembly of progeny virions, holin molecules accumulate in the cytoplasmic membrane without damaging the host. Then, at a time dictated by their primary structure, holins trigger to disrupt the cytoplasmic membrane. For many phages, like and T4, this event releases to the periplasm an endolysin that has accumulated fully folded and enzymatically active in the cytosol. By contrast, phages P1 and 21 encode endolysins that are exported by the host sec system and accumulate in the periplasm as enzymatically inactive proteins tethered to the membrane by an Nterminal SAR (signal anchor-release) domain (25, 26). These SAR endolysins become enzymatically active when their SAR domains exit the membrane to generate the mature, soluble form in the periplasm. This process occurs spontaneously at a low rate but is greatly accelerated when the cytoplasmic membrane is deenergized. Thus, for phages encoding SAR endolysins, holins need only to depolarize the membrane in order to fulfill their role in controlling the timing of lysis. The formation of large membrane lesions like those resulting from S triggering (22) were small and showed a considerable size variation; this heterogeneity persisted when phage from large and small plaques were replated (Fig. 1B to D). Thus, like phage P1 but unlike and T4, the S 21 holin gene is nonessential for plaque formation (7,8,10,27 (Fig. 2A). S 21 and S are not functionally equivalent. We next designed experiments to determine if S 21 68 and R 21 could complement the lysis defect of phages S am R ϩ and S ϩ R am , respectively. Previously, we had reported that, when expressed from the pUC18 derivative pTZ18R, the S 21 gene appeared to be the functional equivalent of S (2). However, the lysis of the culture was not complete even an hour after its onset, despite the fact that the S 21 protein was produced at supraphysiological concentrations from the very-high-copy-number plasmid. For this reason, we repeated these experiments with various alleles of S 21 68 and R 21 transactivated from the late promoter on a medium-copy-number plasmid, in trans to lysisdefective prophages. This system was shown in other studies to support lysis with approximately normal timing (1, 6). As can be seen in Fig. 2B, expression of R 21 from the plasmid complemented the lysis defect of an induced S ϩ R am lysogen, with lysis beginning 55 min after induction and completed within 10 m...

Structure of the lethal phage pinhole

“…Thus, for SAR endolysins, the holin protein need only produce lesions large enough to allow the passage of ions through and depolarization of the cytoplasmic membrane. Indeed, unlike lesions formed by the holin, lesions formed by the phage 21 holin did not allow the passage of either endolysin or a periplasmic marker-tagged GFP protein (8). The term ''pinholin'' has been proposed to differentiate the small-hole (pinhole) forming character of the phage 21 holin from the canonical holins that form large, nonspecific holes.…”

“…Recently, an alternative and remarkably different class of holin-endolysin systems has emerged (6)(7)(8). This class, represented by the lambdoid bacteriophage 21, utilizes endolysins having novel N-terminal secretory signals called signal-anchor release (SAR) domains.…”

Visualization of pinholin lesions in vivo

“…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.…”

Insights into the regulation of bacteriophage endolysin: multiple means to the same end

“…Pinholins, like holins, form holes in the cytoplasmic membrane of the host. However, unlike holins, the holes formed by pinholins are not of sufficient size to allow for the diffusion of endolysin, and yet are large enough to cause leakage of protons leading to the disruption of proton motive force, thus resulting in the depolarization of the membrane (Park et al, 2007;Pang et al, 2010Pang et al, , 2013. As mentioned earlier, the O. oeni phage endolysin Lys44 is transported by a cleavable SAR signal sequence (São-Jose et al, 2000).…”