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

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.