Stable micron-scale holes are a general feature of canonical holins

Abstract

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 S(21) 68. 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.

Figure 1

Topology diagram of holin classes. Examples of each holin class are represented by the holins used in this study. Class I (P2Y and S105), class II (S²¹68), class III (T4T).

Figure 2

Hole formation by S105 in the context of an induced prophage. A culture of E. coli MC4100 ΔtonA λCamΔSR pS105R_amRz_amRz1_am was thermally induced and grown until holin triggering, as monitored by the optical density of the culture. Cells were immediately plunge435 frozen in ethane and observed by cryo-TEM as described in Materials and Methods. (A) Cryo-micrograph of an E. coli cell bearing an S105 lesion. Inset: close-up view of the area in which a lesion is observed in the inner membrane. OM: Outer membrane. IM: Inner membrane. The white line indicates the location and extent of the lesion. Scale bar corresponds to 500 nm. (B) Number of holes/cell observed. (C) Hole size distribution. (D) Hole distance from nearest pole vs. hole size.

Figure 3

Stability of the S105-derived lesions. A culture of E. coli MG1655 ΔtonA lacI^q ΔlacY pQ pS105R_amRz_amRz1_am was induced with IPTG. Samples were removed for plunge freezing at the indicated times after holin triggering and analyzed as described for Fig. 2.

Figure 4

Early triggering by S105_A52F or by S105 wt with the addition of KCN. E. coli MC4100 ΔtonA λCamΔSR carrying either pS105R_amRz_amRz1_am or an isogenic plasmid with the S105_A52F allele were thermally induced and KCN was added to the former culture 15 minutes later. Cells were plunge frozen and imaged as described for Fig. 2. (A) Number of holes/cell observed and (B) hole size distribution for S105_A52F. (C) Number of holes/cell and (D) hole size distribution for wt S105 prematurely triggered with KCN.

Figure 5

Hole formation by different holins. (A) E. coli MC4100 ΔtonA λCamΔSR cells carrying a plasmid isogenic to pS105R_amRz_amRz1_am but with the indicated heterologous holin gene substituted for S105 were thermally induced, sampled and analyzed as described for Fig. 2. (A) P2 Y; (B) T4 t. Insets: close-up views of the areas in which inner membrane lesions are observed. The white lines indicate the location and extent of the lesions Scale bars correspond to 500 nm. (C) Number of holes/cell for P2 Y and T4 T. (D) Hole size distribution for P2 Y and T4 T. (E) Hole distance from nearest pole for P2 Y and T4 T.