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 lambda 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 lambda-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 microm. 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.

Fig. 1.

Induction of lambda lysis genes. (A) Plasmids used in this study. (B) Lysis curves of cultures carrying the following: no S105 plasmid (♢), p0 (▪), pS(•), pSR supplemented with 10 mM MgCl₂ (□), or pSRRzRz1 (○).The arrow shows the 60-min time point at which samples were taken for cryo-EM. IPTG, isopropyl-β-d-thiogalactopyranoside.

Fig. 2.

Cryo-EM of the S105 lesion. Cells were grown and imaged as described. (A) Cell expressing S105 is shown. (B) Area enclosed in the white dashed box is shown enlarged. The white solid lines indicate the location and extent of the lesion. The dashed white line indicates an area of semicontinuous membrane density. (C and D) Close-up views of the S105 lesions from two other cells. (Scale bar: 500 nm.) IM, inner membrane; OM, outer membrane.

Fig. 3.

Quantification of the number of S105 holes per cell (A), hole size variation (B), hole localization (C), and relation of hole size to location (D).

Fig. 4.

Cryoelectron tomography of an S105 lesion. (A, Inset) Cell shown expressing S105 was tilted ±55°, and a 3D reconstruction was calculated. A close-up of the lesion in projection is highlighted by the white arrow. (B) Segmentation of the envelope densities shows the outer membrane (blue) and inner membrane (orange). The 2.6-nm thick slices along z (C–H, spaced by 26 nm) show the appearance of a lesion and its subsequent disappearance over a z height of ∼125 nm. Arrows indicate the S105 lesion. (Scale bar: 250 nm.)