Characterization of the repressor from the lambdoid phage HK022.

Abstract

The lambdoid coliphage HK022 can grow in either a lytic or lysogenic mode following infection. In λ phage the switch to either mode is controlled by the cI and Cro repressors which bind to operators in the λ chromosome and regulate gene expression of adjacent promoters. HK022 is organized similar to λ, but has its own unique repressors and operators which are different from λ. The major goal of this research was to characterize the properties of the CI repressor from HK022 to understand better how this protein regulates gene expression. Seven sites in HK022 with similar nucleotide sequences were identified by DNase I footprinting analyses as CI binding sites. Six were clustered in two groups of three, O(L)1-O(L)3 (operator left) and O(R)1-O(R)3 (operator right), while the remaining site O(FR) (operator far right), was alone. The positions of these sites on the HK022 chromosome from left to right was O(L)1-O(L)3, cI, O(R)3-O(R)1, cro, and O(FR). CI bound to adjacent operators, O(R)1 and O(R)2, with a high degree of cooperativity (cooperativity parameter, ω, of about 2000). When only a single site was present (as with O(FR)), the repressor also increased the affinity for adjacent non-specific DNA sites resulting in a "phased" pattern of binding. CI binding sites were defined as operators by analyses of virulent phage mutants, which were able to escape CI repression and grow on a lysogen. The vast majority of virulent mutants contained 2 mutations in O(R), with single mutations in O(R)1 and O(R)2, suggesting that mutations in both sites are required to overcome the high degree of cooperativity. However, one mutant contained only a single mutation in O(R) and a second mutation in the distant site, O(FR). When the single O(FR) mutation was recombined with a wild-type O(R), the resulting phage was not virulent, but virulent mutants arose from it at a high frequency by acquiring a single mutation in either O(R)1 or O(R)2. The mechanism of action by O(FR) is not understood.