Topological dynamics of holins in programmed bacterial lysis

Abstract

The fate of phage-infected bacteria is determined by the holin, a small membrane protein that triggers to disrupt the membrane at a programmed time, allowing a lysozyme to attack the cell wall. S(21)68, the holin of phage 21, has two transmembrane domains (TMDs) with a predicted N-in, C-in topology. Surprisingly, TMD1 of S(21)68 was found to be dispensable for function, to behave as a SAR ("signal-anchor-release") domain in exiting the membrane to the periplasm, and to engage in homotypic interactions in the soluble phase. The departure of TMD1 from the bilayer coincides with the lethal triggering of the holin and is accelerated by membrane depolarization. Basic residues added at the N terminus of S(21)68 prevent the escape of TMD1 to the periplasm and block hole formation by TMD2. Lysis thus depends on dynamic topology, in that removal of the inhibitory TMD1 from the bilayer frees TMD2 for programmed formation of lethal membrane lesions.

Fig. 1.

Properties of holins and holin genes. (A) Primary structure of S²¹68 and the N-terminal domains of Lyz, R²¹, and PhoA. The codon numbering for S²¹68 follows that of the full-length gene product, S²¹71 (see C for the dual-start structure at the beginning of S²¹). The SAR sequences of Lyz and R²¹ (7, 8) and TMD1 of S²¹68 (16) are shown in the yellow boxes, with the residues that are polar or neutral in terms of hydrophobicity in blue and the hydrophobic residues in red. TMD2 of S²¹68 is shown in an uncolored box. The red stars separated by three Xs above the TMDs of S²¹ indicate GxxxG-like motifs that may mediate interhelical interactions (19). The vertical arrow above the PhoA sequence indicates the normal signal sequence cleavage site (27). In ^ssPhoAΦS²¹68, the indicated sequence from PhoA is fused to the Met₄ codon of S²¹. The position of the S16C missense change is indicated by a vertical arrow below the TMD1 sequence. The N terminus of S²¹68 was given two additional positive charges by inserting the sequence RYIRS between positions 4 and 5. To generate S²¹68_ΔTMD1, the indicated sequence was deleted from S²¹68. The vertical arrow between residues 66 and 67 indicates the position where the sequence G₂H₆G₂ was inserted in the allele used for purification. (B) Holin topologies. The topologies of the λ antiholin, S107 (a), and holin, S105 (b), are shown (6, 13, 23, 28). (c) and (d) show S²¹71 and S²¹68 with two TMDs, respectively. In (e), the lethal form of S²¹ is shown with its TMD1 in the periplasm. (C) Translational control region of the λ and 21 holin genes (11, 15, 16). Filled and empty stars show starts of the long (antiholin) and short (holin) gene products. Shine-Dalgarno sequences for the first and second translational starts of S^λ are indicated by striped and empty boxes, respectively. The single Shine–Dalgarno sequence of S²¹ is indicated by a striped box. The horizontal inverted pairs of arrows show the RNA stem loops controlling the dual starts. For S21, the vertical arrow shows the Met-4 → Leu mutation, which eliminates the production of the holin from the allele referred to as S²¹71. The Lys residues conferring antiholin character to the longer translational product in both the S and S²¹ genes are shown in red.

Fig. 2.

TMD1 exits the membrane. Aliquots from cultures expressing the indicated genes were collected by TCA precipitation and analyzed by SDS/PAGE under reducing or nonreducing conditions, as indicated. Except for A, where anti-Lyz antisera were used, separated proteins were detected by Western blotting using antisera raised against the C-terminal peptide of S²¹. Lane 1 in all panels, lanes 6 and 9 in D and lane 4 in G contain molecular mass standards. In B–G, a star and a double star indicate the positions of the monomer and dimer forms of S²¹68_S16C, respectively. (A) TMD1 of S²¹68 can substitute for the SAR domain of P1 Lyz. Lanes 2 and 3, S²¹68_TMD1ΦLyz_ΔSAR; lanes 4 and 5, lyz; m, membrane fraction; s, soluble fraction. FtsI and R^λ were used as controls for the membrane and soluble fractions, respectively (see SI Fig. 6). (B) Dimerization of S²¹68_S16C via its TMD1. Lanes 2 and 3, S²¹68_S16C; lanes 4 and 5, RYIRSΦS²¹68_S16C. Samples taken at 40 min after induction were prepared with or without the reducing agents DTT and β-mercaptoethanol, as indicated. (C) Disulfide formation reflects specific TMD1–TMD1 interactions. Lane 2, vector control; lane 3, S²¹68; lane 4, S²¹68_S16C; lane 5, S²¹68_G14C. (D) Protease sensitivity of S²¹68_S16C dimers. In this panel, a large format Tris–Tricine gel system was used to allow resolution of the dimer- and monomer-related degradation products. Spheroplasts were prepared, induced for the expression of the indicated S²¹68 allele, and subsequently digested with proteinase K as described in Materials and Methods. Lanes 2–5, spheroplasts expressing S²¹68_S16C and treated with protease for 0 (lanes 2 and 4) or 5 min (lanes 3 and 5); in lanes 2 and 3, the sample loading buffer contained reducing agents. Lanes 7–8, spheroplasts expressing RYIRSΦS²¹68_S16C and treated with protease for 0 (lane 7) or 5 min (lane 8). Lanes 10–11, induced spheroplasts carrying the plasmid vector treated with protease for 0 (lane 10) or 5 min (lane 11). Symbols θθ and θ to the right of lane 5 indicate the position of the major degradation product of the dimer and monomer forms, respectively. A filled circle to the right of lane 8 indicates the position of the RYIRSΦS²¹68_S16C monomer form. (E) Dimerization of S²¹ proteins increases as a function of time after induction. Lanes 2–6, S²¹68_S16C; lanes 7–11, S²¹71_S16C. Samples were taken at the times (minutes) indicated above the lanes and subjected to SDS/PAGE without reduction. (F) Direct export of TMD1 accelerates dimerization of the S²¹ holin. Lanes 2–5, ^ssphoAΦS²¹68_S16C. Samples were taken at indicated times (minutes) and subjected to SDS/PAGE without reduction. (G) Collapse of the membrane potential accelerates dimerization of S²¹ proteins. Twenty minutes after induction, DNP was added to one of duplicate cultures expressing either S²¹68_S16C or S²¹71_S16C. Five minutes later, samples were taken from all four cultures and subjected to SDS/PAGE without reduction. Lanes 2 and 3, S²¹68_S16C; lanes 5 and 6, S²¹71_S16C.

Fig. 3.

The TMDs of S²¹68 have opposing functions. In each experiment, cultures of MG1655 lacI^q1 tonA::Tn10 bearing a plasmid carrying the indicated S²¹ and R²¹ alleles under the control of the λ late promoter, pR′, and a compatible transactivating plasmid, pQ, carrying the λ late-activator gene Q under the control of a hybrid lac-ara promoter (see SI Table 1). The cultures were induced at time 0, and turbidity was followed as a function of time. Constructs used in A, B, E, and F contained the wild-type R²¹ gene, whereas those used in C carried, instead, R²¹_E35Q, encoding an enzymatically inactive form of R²¹. (A) Expression of S²¹68 in combination with R²¹ results in abrupt host lysis. (●, ○), S²¹68 R²¹; ♦, S²¹68 amR²¹. DNP was added (arrow) to one culture (●) at 20 min after induction. (B) Expression of S²¹68_ΔTMD1 in combination with R²¹ results in abrupt host lysis. (●, ○), vector control; (■, □), S²¹68_ΔTMD1 R²¹; DNP was added (arrows) to two of the cultures (■, ○) at 25 min after induction. (C and D) Induction of different S²¹68 alleles in the absence of endolysin causes lethality. C shows growth curves after induction: (○, curve a), vector control; (■, curve b), S²¹68; (■, curve c), S²¹68_ΔTMD1; (♦, curve d), ^ssphoAΦS²¹68. D shows cell survival at 60 min after induction, assessed as colony forming units (CFU) and expressed as a percentage of the control. (E) TMD1 of S²¹68 antagonizes the holin activity of its TMD2. (●), S²¹68 R²¹; (■), S²¹68_S16C R²¹; (♦), RYIRSΦS²¹68_S16C R²¹. (F) The SAR domain of P1 Lyz cannot inhibit hole formation by TMD2 of S²¹68. (○), SAR_lyzΦS²¹68_ΔTMD1, uninduced; (●), SAR_lyzΦS²¹68_ΔTMD1, induced; (■), RYIRS-SAR_lyzΦS²¹68_ΔTMD1, induced.

Fig. 4.

Potential homotypic and heterotypic TMD interactions in the function of S²¹. (A) Helical projections of TMDs of S²¹. Stars indicate GxxxGxxxG-like motifs of each TMD (G10, G14, and G18 in TMD1; G40, S44, and G48 in TMD2). Hydrophilic or neutral residues in each TMD are circled and shaded. (Left) the SAR domain (TMD1) of S²¹ (residues 7–27), with the position of the S16C mutation highlighted by an asterisk. (Right) TMD2, the hole-forming domain of S²¹ (residues 36–60), with a potential hydrophilic surface that may line the lethal membrane lesion indicated by the arc. (B) Pathway to hole formation involves inhibited (i) and active (ii) topological isomers of S²¹. See Discussion for details.