Micron-scale holes terminate the phage infection cycle

Abstract

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...

The lambda spanin components Rz and Rz1 undergo tertiary and quaternary rearrangements upon complex formation

“…3 Briefly, the host RY16504 carrying the plasmid p0 was induced for 60 min with 1 mM IPTG at A550 ¼ 0.4 and 5 lL of culture was immediately applied to holey carbon films (C-Flat, Protochips). Grids were plunge frozen in liquid ethane using an FEI Vitrobot.…”

“…2 First, after accumulating harmlessly in the IM throughout late gene expression, the holin S105, encoded by S, suddenly forms extremely large, irregular holes. 3 Besides terminating macromolecular synthesis and virion assembly, this event allows the lambda endolysin R, a soluble cytoplasmic transglycosylase, to escape to the periplasm, thus allowing the second step, degradation of the PG, to occur. The destruction of the PG is followed by the third step, which requires the products of the last two genes, Rz and Rz1.…”

Active Bax and Bak are functional holins

“…In addition, recent cryo-electron microscopy and tomography studies revealed that the lesions caused by holin S105 of phage l could be visualized as interruptions in the bilayer spanning very large areas of the cytoplasmic membrane, averaging >350 nm in diameter (Dewey et al 2010). Assuming these lesions correspond to the holes, this latter observation fully accounted for the ability of holins to allow the rapid, nonspecific escape of proteins not only of endolysins, of which the l R endolysin, a soluble, 15-kDa monomeric protein is typical, but also of much larger proteins, including the ;500-kDa R-LacZ chimeras (Dewey et al 2010). However, the lesions that are formed by the Bax proteins in the bacterial cytoplasmic membrane do not appear to be of the near micron-scale holes like those formed by the S105 holin, since they could not be observed using cryo-electron microscopy at a 50-nm resolution (data not shown).…”

Visualization of pinholin lesions in vivo

“…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.…”