Abstract
Western blot (immunoblot) analysis of cell extracts from induced bacteriophage lambda lysogens probed with S-protein-specific antibody (raised against an S--beta-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.
Highlights
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
In equivalent Western blots incubated with preimmune serum as the first antibody, neither the fusion proteins nor the S-protein fragment derived from collagenase cleavage of the SCZ5 protein was labeled
When a Triton X-100-extracted sample was mixed with an unextracted sample, the S-protein signal resembled that of the unextracted sample alone, as characterized by a decreased mobility and reduced labeling with antibody (Fig. 5, lane 5). These results indicate that a component in the unextracted sample interferes with the mobility of S protein in an sodium dodecyl sulfate (SDS)-polyacrylamide gel and either the efficiency with which S protein can transfer to the nitrocellulose or the reaction of S protein with anti-S-protein antibody during the Western blot procedure
Summary
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. 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]. Antibody raised against this S-p-galactosidase fusion protein was used to determine the time course of S-protein synthesis, its localization during the phage lytic cycle, and the state of oligomerization of S protein in the cell membrane
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