The C-terminal sequence of the lambda holin constitutes a cytoplasmic regulatory domain

The C-terminal domains of holins are highly hydrophilic and contain cluster s of consecutive basic and acidic residues, with the overall net charge pre dicted to be positive. The C-terminal domain of lambda S was found to be cy toplasmic, as defined by protease accessibility in spheroplasts and inverte d membrane vesicles. C-terminal nonsense mutations were constructed in S an d found to be lysis proficient, as long as at least one basic residue is re tained at the C terminus. In general, the normal intrinsic scheduling of S function is deranged, resulting in early lysis. However, the capacity of ea ch truncated lytic allele for inhibition by the S107 inhibitor product of S is retained. The K97am allele, when incorporated into the phage context, c onfers a plaque-forming defect because its early lysis significantly reduce s the burst size. Finally, a C-terminal frameshift mutation was isolated as a suppressor of the even more severe early lysis, defect of the mutant SA5 2G, which causes lysis at or before the time when the first phage particle is assembled in the cell. This mutation scrambles the C-terminal sequence o f S, resulting in a predicted net charge increase of +4, and retards lysis by about 30 min, thus permitting a viable quantity of progeny to accumulate . Thus, the C-terminal domain is not involved in the formation of the letha l membrane lesion nor in the "dual-start" regulation conserved in lambdoid holins. Instead, the C-terminal sequence defines a cytoplasmic regulatory d omain which affects the timing of lysis. Comparison of the C-terminal seque nces of within holin families suggests that these domains have little or no structure but act as reservoirs of charged residues that interact with the membrane to effect proper lysis timing.