Isolation of Excision-Deficient Mutants

Isolation of Excision-Deficient Mutants

As expected, the int mutants can be helped to lysogenize by complementation. Not surprisingly, these mutants are also found to excise poorly without assistance. Hence Int protein is also required for excision. Although it would seem that the process of integration should be readily reversible with the same components that are used for integration, surprisingly, a phage-encoded protein is required for excision but not integration.

A phenomenon known as heteroimmune curing forms the basis of a simple demonstration that excision requires a protein in addition to Int protein. If lambda lysogens are infected with a heteroimmune phage like λimm⁴³⁴, most cells are lysed, but many of the survivors are found

λ i m m^{4 3 4} + Lysogen   - >    Some nonlysogens

to have been cured of the lambda. This is called superinfection curing. Apparently the superinfecting heteroimmune phage provides diffusible products that facilitate excision of the prophage. Some Int⁺ deletion phage, but not others, can promote curing when they superinfect lysogens of different immunity. Thus something in addition to Int protein must be involved in excision.

The isolation of a nonsense mutation in the gene required for excision proved that it coded for a protein. This isolation used heat-pulse curing. If a lysogen of λCI₈₅₇ growing at 32° is heated to 42° for five minutes and then grown at 32°, the heat-sensitive CI₈₅₇ repressor is first denatured, and phage growth begins. Then, after the cells are cooled to 32°, the repressor renatures and further phage development ceases before suf-ficient phage products have accumulated to kill the cells. In the five minutes of derepression however, sufficient phage proteins are synthe-sized that lambda can excise from the chromosome. After further cell growth, the excised lambda genome is diluted away, and daughter cells that are cured of lambda appear at high frequency.

Heat-pulse curing was used to isolate excision-defective mutants in the following way (Fig. 18.5). A mutagenized stock of λCI₈₅₇ was used to lysogenize cells. This step selected for phage retaining the ability to lysogenize. The lysogenic cells resulting from this step were grown at 32° and then replicated onto three petri plates. One was incubated at 32°, one was incubated at 42°, and the third was incubated at 42° for five minutes, at 32° for two hours, and then at 42° overnight.

Figure 18.5 Temperature protocols for the identification ofxis⁺andxis^-by theinability of the latter to excise following a short heat induction. Each of the three temperature time lines represents the conditions the three petri plates were exposed to.

The first plate kept viable copies of all the colonies. The second plate showed which colonies were infected with lambda, as growth at 42° would induce the phage and kill lysogens. Growth on the third plate indicated which colonies could heat-pulse cure. On this plate, the lambda lysogens would heat-pulse cure, and the cured cells in such a colony would be capable of growing into colonies during the subsequent growth at 42°. Any colony whose cells possessed excision-defective phage could not heat-pulse cure and would therefore be killed by the attempted growth of the unexcised prophage during the subsequent extended exposure to 42°.

Later, a simpler method for detecting excision-defective mutants was devised. This scheme uses cells in which lambda has mistakenly inte-grated into a site within the gal genes that resembles the authentic attB site. The cells are Gal^- as a result. Infection of these cells by heteroim-mune phage able to provide Int and Xis proteins in trans catalyzes excision of the phage from the gal genes, and the cells become Gal⁺. If such infected cells are plated on galactose plates, plaques with Gal⁺ revertants are red on medium containing galactose indicator dye and Gal^- plaques are white. With this convenient assay, the excision abilities of many different phage can be assayed on a single galactose indicator plate.

Using the plate assay for excision, Enquist and Weisberg performed a thorough genetic analysis of the att-int-xis region of the lambda chromosome. They isolated and characterized hundreds of int and xis point mutations. The mutations fell into two complementation groups indicating that two genes were involved. One codes for the Int protein and one for the Xis protein. The mutations were mapped with a set of deletions ending in the region. The large number of mutants studied permitted a reasonable estimation of the sizes of the genes. The int gene appeared to be about 1,240 base pairs long, and the xis gene was very small, only 110 base pairs long. No additional phage genes acting in trans and directly involved in the integration or excision process werediscovered.