JB Accepts, published online ahead of print on 6 November 2009

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J. Bacteriol. doi:10.1128/JB.01263-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

The N-terminal transmembrane domain of S is required for holin but not antiholin function

Rebecca White, Tram Anh T. Tran, Chelsey A. Dankenbring, John Deaton, and Ry Young

Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station TX 77843-2128; Department of Biology, Texas A&M University, College Station TX 77843-3528

The S gene encodes the holin, S105, and the antiholin, S107, which differs by its Met-Lys N-terminal extension. The model for the lysis-defective character of S107 stipulates that the additional N-terminal basic residue keeps S107 from assuming the topology of S105, which is N-out, C-in, with three TMDs. Here we show that the N-terminus of S105 retains its fMet residue, whereas the N-terminus of S107 is fully deformylated. This supports the model that in S105, TMD1 inserts into the membrane very rapidly but in S107, it is retained in the cytoplasm. Further, it reveals that, compared to S105, S107 has two extra positively charged moieties, Lys2 and the free N-terminal amino group, to hinder its penetration into an energized membrane. Moreover, an allele, S105_TMD1, deleted for TMD1, was found to be defective in lysis, insensitive to membrane depolarization and dominant to the wt allele, indicating the lysis-defective, antiholin character of S107 is due to the absence of TMD1from the bilayer rather than its ectopic localization at the inner face of the cytoplasmic membrane. Finally, the antiholin function of the deletion protein was compromised by the substitution of early lysis missense mutations in either the deletion protein or parental S105, but restored when both the S105_TMD1 and holin carried the substitution.