Functional assembly of the lambda S holin requires periplasmic localization of its N-terminus

Bacteriophage-lambda-induced host-cell lysis requires two phage-encoded pro teins, the S holin and the R transglycosylase. At a specific time during in fection, the holin forms a lesion in the cytoplasmic membrane that permits access of the R protein to its substrate, the peptidoglycan. The lambda S g ene represents the prototype of holin genes with a dual-start motif; they e ncode two proteins, a lysis effector and a lysis inhibitor. Although the tw o S proteins differ only by two amino acids (Met-l and Lys-2) at the N-term inus, the longer product (S107) acts as an inhibitor of the lysis effector (S105). The functional difference between the proteins has been previously ascribed to the Lys-2 residue in S107. It was therefore of interest to dete rmine the subcellular localization of the N-terminus of either S protein. T o study the membrane topology of the S proteins, we used the topology probe TEM beta-lactamase and an N-terminal tag derived from the Pseudomonns aeru ginosa phage Pf3 coat protein. We show that both S proteins have a type III (N-out/C-in) topology. The results provide insight into the regulatory mec hanism imposed by the dual-start motif and will be discussed in terms of a model for temporal regulation of the S-dependent "hole" in the membrane.