Visualization of pinholin lesions in vivo.

Lambdoid phage 21 uses a pinholin-signal anchor release endolysin strategy to effect temporally regulated host lysis. In this strategy, the pinholin S(21)68 accumulates harmlessly in the bilayer until suddenly triggering to form lethal membrane lesions, consisting of S(21)68 heptamers with central pores <2 nm in diameter. The membrane depolarization caused by these pores activates the muralytic endolysin, R(21), leading immediately to peptidoglycan degradation. The lethal S(21)68 complexes have been designated as pinholes to distinguish from the micrometer-scale holes formed by canonical holins. Here, we used GFP fusions of WT and mutant forms of S(21)68 to show that the holin accumulates uniformly throughout the membrane until the time of triggering, when it suddenly redistributes into numerous small foci (rafts). Raft formation correlates with the depletion of the proton motive force, which is indicated by the potential-sensitive dye bis-(1,3-dibutylbarbituric acid)pentamethine oxonol. By contrast, GFP fusions of either antiholin variant irsS(21)68, which only forms inactive dimers, or nonlethal mutant S(21)68(S44C), which is blocked at an activated dimer stage of the pinhole formation pathway, were both blocked in a state of uniform distribution. In addition, fluorescence recovery after photobleaching revealed that, although the antiholin irsS(21)68-GFP fusion was highly mobile in the membrane (even when the proton motive force was depleted), more than one-half of the S(21)68-GFP molecules were immobile, and the rest were in mobile states with a much lower diffusion rate than the rate of irsS(21)68-GFP. These results suggest a model in which, after transiting into an oligomeric state, S(21)68 migrates into rafts with heterogeneous sizes, within which the final pinholes form.