A comparison of the DNA binding and bending capacities and the oligomeric states of the immunity repressors of heteroimmune coliphages P2 and W 

Abstract

Bacteriophages P2 and WΦ are heteroimmune members of the P2-like family of temperate Escherichia coli phages. Temperate phages can grow lytically or form lysogeny after infection. A transcriptional switch that contains two con-vergent promoters, Pe and Pc, and two repressors regulate what life mode to enter. The immunity repressor C is the first gene of the lysogenic operon, and it blocks the early Pe promoter. In this work, some characteristics of the C proteins of P2 and WΦ are compared. An in vivo genetic analysis shows that WΦ C, like P2 C, has a strong dimerization activity in the absence of its DNA target. Both C proteins recognize two directly repeated sequences, termed half-sites and a strong bending is induced in the respective DNA target upon binding. P2 C is unable to bind to one half-site as opposed to WΦ, but both half-sites are required for repression of WΦ Pe. A reduction from three to two helical turns between the centers of the half-sites in WΦ has no significant effect on the capacity to repress Pe. However, the protein–DNA complexes formed differ, as determined by electrophoretic mobility shift experiments. A difference in spontaneous phage production is observed in isogenic lysogens.

Determination of the DNA-binding kinetics of three related but heteroimmune bacteriophage repressors using EMSA and SPR analysis

“…The number of C proteins in the respective complex is not known, but the SPR analysis does not indicate a higher number of C proteins per DNA molecule for P2 Hy dis C. A genetic analysis of the P2 C and Wϕ C proteins has shown that in the absence of DNA they form dimers, but not higher oligomeric structures (8). During gel-filtration all C proteins elute at the same positions, indicating also that P2 Hy dis C is a dimer in solution (data not shown).…”

“…A comparison of the complexes formed by P2 C and Wϕ C may indicate that they form similar complexes, but in the case of P2 there is no difference in the affinity to one half-site compared to the other. Using a circular permutation assay, the P2 and Wϕ C proteins have been shown to bend their respective DNA targets upon binding, and Wϕ C as opposed to P2 C is able to bind to only one half-site (8). This may explain why three retarded bands can be detected with Wϕ C but only two with P2 C.…”

“…Escherichia coli strain BL21(DE3) (5) containing plasmids pEE679 expressing P2 C (6), pEE922 expressing P2 Hy dis C (4), or pEE1020 expressing Wϕ C (8), were grown at 37°C in LB or M9 minimal medium with ampicillin (100 μg/ml). The proteins were overexpressed and purified as described previously (8).…”

“…The proteins were overexpressed and purified as described previously (8). The protein concentrations were determined by the Bradford method (10).…”

Assignment of 1H, 13C, and 15N chemical shift resonances of P2 C-repressor protein

“…The P2 C-repressor forms very stable dimers, but not higher oligomers in the absence of DNA (Ahlgren-Berg et al 2007). Earlier studies, however, have shown that the P2 C-repressor might bind as dimers to each half site and then form tetramers, which can be seen as four protected regions in a foot-print analysis.…”