Isolation of Integration-Defective Mutants

Isolation of Integration-Defective Mutants

A regulated BOB’-POP’-specific recombination system would solve lambda’s problem of restricting integration and excision reactions to the right time and right sites on the phage and host genomes. That lambda possesses such a recombination system could be proven by the isolation of lambda mutants unable to lysogenize due to the absence of an integration enzyme. The main difficulty in such a proof is a common problem in genetics—that of identifying the desired mutant.

At the time that integration mutants were being sought, a lambda mutant was already known that permitted good guesses to be made about the properties of the desired integration-defective mutants. The lambda mutation deletes the phage b2 region. It extends into the POP’ region and leaves the phage deficient in its integration abilities. Most of the λb2 phage that infect cells without lysing them establish repression but do not integrate into the chromosome. They therefore form turbid plaques similar to those of wild-type lambda. If cells from the turbid center of such a plaque are streaked on solidified medium and allowed to proceed through additional generations of growth in the absence of superinfecting phage, the λb2 are diluted away since they do not repli-cate by their own ori, and they are not integrated into the chromosome to be replicated by the host. Therefore the resultant cells are nonlysogenic.

When λb2 phage and wild-type λ coinfect cells, the wild-type λ cannot complement the defect in the λb2. This shows that the defect in λb2 is in a site required for the integration, and not in a protein that can act in trans. Even so, the λb2 provide a good indication of the behavior to be expected of mutants defective in any phage-encoded proteins required for integration. Mutants defective in the ability to integrate should form turbid plaques but not stably integrate. They could have mutations in phage proteins required for the process of integration or mutations in the DNA sites directly used in integration. Because the number of nucleotides in the DNA sites ought to be much smaller than the coding region for the integration proteins, the majority of integra-tion-defective mutants isolated ought to be in the integration proteins.

The two steps of growing cells to dilute away the nonintegrated lambda and testing for lambda immunity can be combined. The candi-date cells are streaked on plates containing the pH indicators eosine yellow and methylene blue as well as having about 10⁷λCI^- mutants spread on the surface. The cells spread on the plate grow, divide, and occasionally encounter a λCI^-. If a cell infected by a λCI^- particle does not possess lambda immunity, the infecting phage is not repressed, and it grows and lyses the cell. This releases some acid and changes the color of the pH indicators. Adjacent cells may also be infected and lysed, but because the colony stops growing before all cells can be lysed, some cells remain in the colony. A colony of nonlysogenic cells spotted on these plates therefore grows into a ragged, purple colony, whereas colonies of immune cells yield smooth, pink colonies.

A brute-force pick-and-spot technique based on the knowledge of the behavior of λb2 mutants on the indicating plates permitted the isolation of lambda mutants unable to lysogenize. These are called int mutants. Some of them were nonsense mutants, proving that the phage encodes a protein that is required for integration.