Lysis

Lysis

One problem the phage must solve is lysing the cells at the right moment. Most likely this time is a compromise, worked out over the eons, between maximizing the number of completed phage particles released and minimizing the interval after infection or induction until some completed phage are released. Although infection can begin by slipping lambda’s DNA molecule into the cell through a small hole, the release of newly assembled phage particles requires something more drastic. At the least, holes large enough for the phage head must be punched in the rigid peptidoglycan layer, and the inner and outer membranes must be ruptured as well.

Three lambda proteins are known to participate in the lysis process. They are all late proteins synthesized under control of the Q gene product. The first to be identified is the product of the lambda R gene. Originally this was called the lysozyme for its ability to lyse cells, then for a while it was mistakenly called an endopeptidase or endolysin for the specific bonds in the peptidoglycan the enzyme was thought to cleave, and now it is correctly known to be a transglycosylase (Fig. 14.10) that cleaves between N-acetylglucosamine and N-acetylmuramic acid. Another lambda-encoded protein also degrades the peptidoglycan layer of the cell. It is the product of the R_Z gene, and it is an endopeptidase. The third protein required for lysis is the product of the S gene. Experiments indicate that this protein forms a pore through the inner membrane so that the R and R_Z products, which are cytoplasmic proteins, are provided access to their peptidoglycan substrate.

Figure 14.10 The structure of thepeptidoglycan layer and the bonds cleaved by the R and Rz proteins.

The behavior of cells infected with S^- phage is consistent with the idea that the S gene codes for a pore. As expected, the R and R_Z products accumulate in the cytoplasm of such cells, and lysis does not occur unless the inner membrane is damaged. Chloroform treatment or freez-ing and thawing are two methods for disrupting the integrity of the inner membrane in S^- mutants. Protein synthesis, DNA synthesis, and respi-ration do not shut off in cells infected with S^- phage as they normally do 40 minutes after infection with S⁺ phage. The shutoff of macro-molecular synthesis that normally occurs 40 minutes after infection results from leakage of crucial intracellular components, as at this time the cell loses its ability to concentrate small molecules intracellularly.

S^- mutants facilitate work with lambda. First, since macromolecular synthesis does not shut off at 40 minutes, phage continue to be made and the phage yield per infected or induced cell is raised from about 100 to about 500. Second, phage may easily be harvested from the cell growth medium by centrifuging cells full of phage into pellets, resus-pending in small volumes, and lysing by the addition of chloroform. As a result, large quantities of highly concentrated phage are easily obtained.