Oligomerization of the bacteriophage lambda S protein in the inner membrane of Escherichia coli

Abstract

Western blot (immunoblot) analysis of cell extracts from induced bacteriophage lambda lysogens probed with S-protein-specific antibody (raised against an S-,I-galactosidase fusion protein) demonstrated that the bacteriophage lambda S protein begins to appear 10 min after phage induction and is localized to the inner membrane at all times during the lytic cycle. Between 100 and 1,000 molecules of S protein per cell were present at the time of phage-induced lysis. Western blots of chemically cross-linked membranes from induced lysogens showed a ladder of bands at 18, 24, 32, and 42 kilodaltons (the S-protein monomer ran at 8 kilodaltons) that reacted with anti-S-protein antibody. Thus, the S protein appears to reside in the inner membrane as a multimer, and the molecular weights of the cross-linked species are consistent with those of S-protein homopolymers. Sodium dodecyl sulfate-resistant dimers were also detected when S protein was purified by immunoprecipitation.The lytic cycle of bacteriophage lambda concludes with the synchronous lysis of the host Escherichia coli cells and the efficient release of approximately 100 newly synthesized phage particles per cell. Two phage genes are sufficient for this synchronous lysis, as demonstrated by the cloning of the lambda lysis region into a plasmid under the transcriptional control of the lac operon (14,15). The lambda R gene encodes a soluble 17.5-kilodalton (kDa) transglycosylase that is synthesized in an active form in the cytoplasm and is capable of degrading the periplasmic peptidoglycan layer (4-7). The lambda S gene product increases the permeability of the inner membrane at 40 to 50 min postinfection and thereby initiates cell lysis (15,35,36,50). A third phage gene product, the Rz protein, is required for efficient lysis when the cells are grown in the presence of high concentrations of magnesium and is thought to encode an endopeptidase activity which cleaves peptide bonds that are present in the peptidoglycan layer (53). The simplest model for the mechanism of S gene-induced lysis is the formation or activation of a large nonspecific pore(s) through the inner membrane that allows the cytoplasmic R (and possibly Rz) gene product access to the periplasm at the time of lysis (50). This hypothesis accounts for the increased membrane permeability that was observed in the presence of the S protein and incorporates a reversible element (the opening and closing of the pore), which is compatible with experimental results that have been obtained by using the temperature-sensitive S allele (50).Initial reports identified both a 15-kDa acidic protein in the inner membrane fraction of lambda-infected cells and a 5.5-kDa protein found in phage particles as the products of the S gene (50,51 which suggests that there are two translational start sites controlled by an upstream stem-loop structure, resulting in the production of two proteins, S107 and S105, which differ by two N-terminal residues (8a). It has been proposed that the regulation of the timing of lysis might inv...

Micron-scale holes terminate the phage infection cycle

“…In addition, even at these enormous sizes, the perimeters of the holes are consistent with the numbers of S105 holins present at the time of lysis. Because each holin has three TMDs, which, as α-helices, are ∼1 nm in diameter, current estimates of ∼1,000 S105 molecules (35,36) could correspond to ∼3 μm of perimeter if all the proteins are in the perimeter and each TMD participates. To settle this issue, the ideal approach would be to tag S105 with a fluorescent moiety and use correlative microscopy to demonstrate the presence of the holin in the lesion.…”

S gene expression and the timing of lysis by bacteriophage lambda

“…On the order of 10 2 vesicles are derived from each cell, suggesting that membrane lesions may be widespread in the membranes of the killed cells and that no more than 10 to 40 S molecules are required to sustain the lesion. Cross-linking experiments have shown S oligomers up to 8-mers (9,47,48) and perhaps higher. Interestingly, the A52G mutant allele, which support extremely early lysis, triggers lysis at a much lower total S protein level, about 10-fold lower than that for the wild-type allele (19), which, according to the quantitation done here, indicates 300 to 600 molecules per cell.…”

“…A quantitative assessment of the accumulation of S proteins might help constrain possible models for the macromolecular structure of these membrane lesions. In phage and plasmid contexts, the levels of S protein which accumulate before the triggering of lysis have been estimated to be on the order of 300 to 1,000 molecules per cell on the basis of indirect results with S::lacZ fusion genes (25) and on yields of S protein purified by immunoaffinity chromatography (48). However, no direct measurement of S protein levels has been published.…”

Molecular Control of Bacterial Death and Lysis

“…Similar to bacteriophageencoded holins, Bax can cause membrane permeabilization in a process requiring protein oligomerization (7,77,286). Again, similar to holins, the molecular details of this process are obscure, but given the size of the proteins that are released (142) and the biophysical impact of Bax (and other proapoptotic proteins) on membranes (13), it is likely that "pore" formation involves lipid destabilization.…”