Genetic and biochemical analysis of dimer and oligomer interactions of the lambda S holin

Abstract

Bacteriophage lambda uses a holin-endolysin system for host cell lysis. R, the endolysin, has muralytic activity. S, the holin, is a small membrane protein that permeabilizes the inner membrane at a precisely scheduled time after infection and allows the endolysin access to its substrate, resulting in host cell lysis. lambda S has a single cysteine at position 51 that can be replaced by a serine without loss of the holin function. A collection of 27 single-cysteine products of alleles created from lambda S(C51S) were tested for holin function. Most of the single-cysteine variants retained the ability to support lysis. Mutations with the most defective phenotype clustered in the first two hydrophobic transmembrane domains. Several lines of evidence indicate that S forms an oligomeric structure in the inner membrane. Here we show that oligomerization does not depend on disulfide bridge formation, since the cysteineless S(C51S) (i) is functional as a holin and (ii) shows the same oligomerization pattern as the parental S protein. In contrast, the lysis-defective S(A52V) mutant dimerizes but does not form cross-linkable oligomers. Again, dimerization does not depend on the natural cysteine, since the cysteineless lysis-defective S(A52V/C51S) is found in dimers after treatment of the membrane with a cross-linking agent. Furthermore, under oxidative conditions, dimerization via the natural cysteine is very efficient for S(A52V). Both S(A52V) (dominant negative) and S(A48V) (antidominant) interact with the parental S protein, as judged by oxidative disulfide bridge formation. Thus, productive and unproductive heterodimer formation between the parental protein and the mutants S(A52V) and S(A48V), respectively, may account for the dominant and antidominant lysis phenotypes. Examination of oxidative dimer formation between S variants with single cysteines in the hydrophobic core of the second membrane-spanning domain revealed that positions 48 and 51 are on a dimer interface. These results are discussed in terms of a three-step model leading to S-dependent hole formation in the inner membrane.

FIG. 1

Primary structure, membrane topology, translational control region, and transactivation of λ S. (A) Primary structures of λ S. ===, transmembrane domains as predicted by the TMHMM program (http://www.cbs.dtu.dk/services/TMHMM-1.0/) (25); XXX, the highly charged, dispensable C-terminal region (5); #, the two start codons of S. The positions of single-cysteine substitutions are indicated by asterisks above the sequence. (B) Membrane topology of λ S. The three α-helical transmembrane domains are numbered from 1 to 3. (C) Dual-start motif of λ S. The boxed sequences indicate the Shine-Dalgarno sequences for the dual translational start sites of S. The lengths of both protein products are given in amino acid (aa) residues. (D) The lambda lysis genes lie in an overlapping cluster in the lysis cassette downstream of the single late gene promoter pR′. Expression of the lysis genes from a prophage and/or transactivation plasmid is dependent on the protein Q that is required for antitermination of the terminator tR′. For dominance and antidominance tests, the holin S105 was expressed from a prophage and a second S105 variant (S105*) was expressed from a medium-copy-number transactivation plasmid (pS105*) (Table 2) (23, 24).

FIG. 2

Lysis phenotypes of single-cysteine-containing S mutants. (A) MC4100(λCmΔSR) cells carrying the plasmids pKB1 (Sam7; ●), pS105 (wild type; C51; ▾), pS105_C51S (○), pS105_C51S/D47C (■), pS105_C51S/A48C (□), pS105_C51S/T49C (▴), pS105_C51S/M50C (▵), pS105_C51S/A52C (⊙), pS105_C51S/I53C (⧫), pS105_C51S/I54C (◊), and pS105_C51S/A55C (X) were induced and monitored for turbidity until cell lysis was completed or for 110 to 120 min after induction. (B) Expression of single-cysteine-containing S mutants in trans to S105. MC4100(λCmS105) cells carrying the same plasmids as in panel A were induced and monitored for turbidity. All single-cysteine S mutants showed an antidominant lysis phenotype with lysis occurring as fast as or faster than with two copies of the parental S105 holin (▾).

FIG. 3

Lysis phenotype and DSP cross-linking pattern of different S variants. (A) MC4100(λKnΔSR) cells carrying the plasmids pS105 (●), pS105_C51S (○), pS105_A52V (■), and pS105_C51S/A52V (□) were induced and monitored for turbidity. (B) 60 min after induction, membrane samples of MC4100(λKnΔSR) plus pKB1 (Sam7) and the same strains as in panel A were prepared and treated with 16 mM DSP cross-linker. The cross-linking experiment was performed as described in Materials and Methods and analyzed by Western blotting. Lane 1, molecular mass (m) marker; lane 2, pKB1(Sam7); lane 3, pS105; lane 4, pS105_C51S; lane 5, pS105_A52V; lane 6, pS105_C51S/A52V. The masses of prestained molecular standards are given in kDa on the left. S-specific bands are marked by arrows. (C) Left gel, Triton X-100-soluble inner membrane samples of MC4100 cells carrying λRG1-derived prophages were prepared and analyzed by Western blotting as described in Materials and Methods with the alteration that the samples were mixed with 2× sample buffer containing 2.8 M β-mercaptoethanol. Lane 1, molecular mass standards (m); lane 2, S_G80S; lane 3, Sam7. Right gel, membrane samples of induced MC4100 cells carrying λRG1- or λRG1-derived prophages bearing defective S alleles were prepared. DSP cross-linking and Western blot analysis were performed as described in Materials and Methods. The lanes labeled with + and − indicate the presence and absence, respectively, of the cross-linking agent during sample preparation. The amino acid change for each S variant is given below the panel. An X below the panel indicates S proteins which form higher oligomers upon DSP treatment. The masses of prestained molecular standards are given in kDa on the left. S monomer and oligomer bands are marked by arrows.

FIG. 4

Expression of S105_A48V and S105_A52V in trans to S105 and dimerization with S105τ94. (A) MC4100(λCmΔSR) cells carrying the plasmid pS105_A48V (▵) or pS105_A52V (◊) and MC4100(λCmS105) cells carrying the plasmid pKB1 (Sam7; X), pS105 (●), pS105_A48V (▴), or pS105_A42V (⧫) were induced and monitored for turbidity. (B) Oxidation and sample preparation for Western blot analysis were performed as described in Materials and Methods. Lane 1, molecular mass (m) marker; lane 2, mixed cultures MC4100(λCmΔSR) plus pS105τ94 and MC4100(λCmΔSR) plus pS105_A52V; lane 3, MC4100(λCmS105τ94) plus pS105_A52V; lane 4, MC4100(λCmΔSR) plus pS105_A52V; lane 5, mixed cultures MC4100(λCmΔSR) plus pS105τ94 and MC4100(λCmΔSR) plus pS105_A48V; lane 6, MC4100(λCmS105τ94) plus pS105_A48V; lane 7, MC4100(λCmΔSR) plus pS105_A48V. Labeling of the panel is as in Fig. 3.

FIG. 5

Dimer formation between S molecules with single cysteines in TM2. S proteins were tested for heterodimer formation under oxidative conditions and analyzed by Western blotting as described in Materials and Methods. Samples were prepared from MC4100(λCmS104τ94) (A) and from MC4100(λCmS104τ94_C51S/I53C) (B) cells harboring a transactivation plasmid. The lanes are labeled with the S allele on the transactivation plasmid. S-specific monomer and dimer bands, as well as the masses of prestained molecular mass (m) standards in kDa, are indicated. These Western blots show the heterodimer formation between different pairs of S molecules; the results are summarized in Table 3. (C) α-Helical wheel projection of the second transmembrane domain of λ S, with 3.6 amino acids per turn. The positions and original amino acids individually replaced by cysteine are indicated in single-letter code in this scheme.

FIG. 6

Model for hole formation. At least three steps, described in the text, are required for hole formation: first, dimerization of λ S; second, oligomerization; and third, a concerted conformational change which is equivalent to the triggering of hole formation. The S alleles which are blocked in different steps during the process of hole formation are indicated above the individual steps. S₁, monomer; S₂ dimer; S_n oligomer; deg, degradation of S molecules; dim_cι and oligo_cι dimers and oligomers detected by DSP cross-linking; dim_ox, dimers detected by cysteine-specific disulfide bond formation.