(A) Amino acid sequence of T and its derivatives. The predicted TMD of T is boxed and shaded. The rV mutants r2(I39V) and r3(T75I) are indicated above the sequence. In T^his, a hexahistidine tag (HHHHHHGG) was inserted between the residues G132 and Y133 (underlined). For the ^ssPhoAΦT_CTD construct, the signal sequence of PhoA, shown below the T sequence, was substituted for residues 1 to 55 of T. For the FtsI_TMDΦT construct, the transmembrane domain of FtsI, shown in a shaded box below the T sequence, was substituted for residues 2 to 69 of T. For the T_TMDφPhoA construct, residues 1 to 70 of T were substituted for residues 1 to 26 in the full-length primary gene product of phoA; the PhoA sequence begins with PVLE (from PhoA residue 27 on, as indicated). (B) Amino acid sequence of RI and its derivatives. The predicted signal sequence of RI is boxed, with the leader peptidase I cleavage site predicted by the SignalP program (http://www.cbs.dtu.dk/services/SignalP/) (2) indicated by a carat. In the c-myc-tagged version of RI, the c-myc tag (QKLISEEDL) was inserted after the terminal glutamate at position 97, while in the GFPΦRI^cmyc constructs, the entire GFP sequence was inserted after the initial Met residue. For the ^ssPhoAφRI_CTD construct, the signal sequence of PhoA, shown below the RI sequence, was substituted for residues 1 to 24 of RI. For the RI_NTDφPhoA construct, the predicted signal sequence of RI replaced residues 1 to 26 in the uncleaved precursor to mature PhoA. The sole known rI missense mutation, R78P, is shown by a down arrow.

The C-terminal domains of T and RI are the determinants of LIN. (A) ^ssPhoAΦRI_CTD is necessary and sufficient for LIN. CQ21(λkanΔ(SR)) cells carrying the indicated plasmids were induced at time zero, and culture turbidity was monitored as a function of time: ⊠, pT4T; ▪, □, pT4T and pZA-RI; ○, •, pT4T and pZA-^ssPhoAΦRI_CTD; ▴, pT4T and pZA-RI_NTDΦPhoA; ⧫, pS105; ⋄, pS105 and pZA-RI; ▿, pS105 and pZA-^ssPhoAΦRI_CTD. To demonstrate premature triggering, KCN was added to two cultures (pT4T and pZA-RI, □; and pT4T and pZA-^ssPhoAΦRI_CTD, ○) at the time indicated by the solid arrow. (B) Phage accumulation during ^ssPhoAΦRI_CTD-mediated LIN. CQ21(λ-t^his) carrying the indicated plasmids was induced at time zero, and culture turbidity (solid symbols) and phage accumulation (open symbols) were monitored as a function of time. ○, •, no plasmid; ⋄, ⧫, pZA-RI; □, ▪, pZA-^ssPhoAΦRI_CTD. (C) Periplasmic T_CTD interferes with LIN. CQ21(λkanΔ(SR)) cells carrying the indicated plasmids were induced at time zero, and culture turbidity was monitored as a function of time. •, pT4T; ○, pT4T and pZA-^ssPhoAΦT_CTD; □, pZA-^ssPhoAΦT_CTD; ▵, pT4TRI; ▿, pT4TRI and pZA-^ssPhoAΦT_CTD; ⋄, pT4TRI and pZA-T_CTD; ▴, pT4TRI and pZAT_TCD; ▾, pT4TRI and pZA- FtsI_TMDΦT_70-218; ▪, pT4TRI and pZA-T_TMDΦPhoA. (Inset) Detail for growth of cells carrying pT4T alone (•) and pT4T and pZA-^ssPhoAΦT_CTD (○). (D) Periplasmic T_CTD blocks LIN during T4 phage infections. CQ21 cells carrying either pZA32-luc (solid symbols) or pZA-^ssPhoAΦT_CTD (open symbols) were induced at time zero and were grown without infection (○, •) or infected at a multiplicity of infection of 10 with either T4D (□, ▪) or T4rI (▵,▴). In panels A, C, and D, CHCl₃ was added at the time indicated by the arrow.

T and RI form a complex. (A) Immunoprecipitations were performed with samples containing either ^ssPhoAΦT_CTD (lanes 2 to 4), both ^ssPhoAΦT_CTD and GFPΦRI^cmyc (lanes 5 to 7), or GFPΦRI^cmyc only (lanes 8 to 10), prepared from induced cells carrying either of the plasmids pZA-^ssPhoAΦT_CTD or pZE-GFPΦRI^cmyc or both. Primary antibodies for immunoprecipitations: lanes 2, 5, and 8, rabbit preimmune serum; lanes 3, 6, and 9, anti-T rabbit antibody; lanes 4, 7, and 10, anti-GFP monoclonal antibody. Samples were analyzed by SDS-PAGE and immunoblotted with either anti-T or polyclonal anti-c-myc, as indicated to the right. (B) Same as panel A, except that the rV variant ^ssPhoAΦT_CTD^T75I, produced from the plasmid pZA-^ssPhoAΦT_CTD^T75I, was used. The molecular mass appears in lane 1 for each blot.

(A) Topology of class I, II, and III holins (35, 38). (B) Features of plasmid and phage constructs. All plasmids used for inductions are based on the structure of pS105 (14) (upper structure), with the λ late promoter and the promoter-proximal genes of the late transcriptional unit (lower structure), except that in the plasmid pT4T, the S gene in the λ SRRzRz1 lysis cassette of pER157 (30) is replaced by the phage T4 t gene. In plasmid pT4TRI, the rI gene is inserted 13 nucleotides downstream of the Rz gene of pT4T. “X” designates areas where homologous recombination can occur for generation of λt recombinants.

Localization of T and RI chimeras. Subcellular fractions were prepared and analyzed by SDS-PAGE and Western blotting as described in Materials and Methods. (A) Cells carrying pZA-^ssPhoAΦRI_CTD^cmyc were grown in the absence (lanes 2 and 3) or the presence (lanes 4 and 5) of 1 mM azide for 10 min in advance of induction. Cells from these cultures were collected by TCA precipitation and centrifugation, resuspended in SDS-PAGE sample buffer, and subjected to SDS-PAGE and Western blotting using anti-RI antisera as the primary antibody. Lane 1, molecular mass standards; lanes 2 and 4, samples from uninduced cultures; lanes 3 and 5, samples from induced cultures. (B) Cells carrying the pZA-RI_NTDΦPhoA were induced, fractionated, and analyzed by SDS-PAGE and Western blotting using anti-PhoA as the primary antibody. Lane 1, molecular mass standards; lane 2, mature form of PhoA; lane 3, blank; lane 4, cells from an uninduced culture; lane 5, cells from an induced culture; lane 6, total cell lysate; lane 7, 1,000 × g pellet; lane 8, 1,000 × g supernatant; lane 9, 100,000 × g supernatant (soluble fraction); lane 10, 100,000 × g pellet (membrane fraction); lane 11, detergent-extractable (1% NP40) membrane fraction; lane 12, detergent-insoluble fraction. (C) Cells carrying pZA-^ssPhoAΦT_CTD were grown in the absence (lanes 2 to 6) or the presence (lanes 7 to 11) of 1 mM azide for 10 min in advance of induction, harvested, fractionated, and analyzed by SDS-PAGE and Western blotting using anti-T antisera as the primary antibody. Lane 1, molecular mass standards; lanes 2 and 7, uninduced cells; lanes 3 and 8, induced cells; lanes 4 and 9, cells after spheroplasting; lanes 5 and 10, spheroplasts; lanes 6 and 11, periplasm.

Periplasmic T_CTD causes T4 to form r-type plaques. Lawns of cells of MDS12 tonA::Tn10 lacI^q1 (A), MDS12 tonA::Tn10 lacI^q1 harboring uninduced pZA-^ssphoAΦT_CTD (B), or MDS12 tonA::Tn10 lacI^q1 harboring induced ^ssPhoAΦT_CTD (C) were infected with either T4D or T4rI phage.