| Description |
This work analyzes the DNA translocating mechanisms by the “P22-like†bacteriophages Salmonella enterica phage P22 and Shigella flexneri phage Sf6. DNA packaging into virions during assembly and ejection of the DNA into target cells is examined. To explore DNA packaging, interactions of the terminase proteins (TerS and TerL) and their role in recognizing the correct DNA to be packaged are explored experimentally in vivo. Evidence was obtained showing that the N-terminal domain of TerS is involved in the recognition of bacteriophage DNA, and the C-terminal domain of TerS likely interacts with TerL. The P22 pac site was shown to be necessary and sufficient to induce DNA packaging. Additionally, the Sf6 pac site was identified and shown to be similar to the P22 pac site. The phenotypes of a pac-null P22 mutant and mutations that alter TerS were analyzed. For the first time, the pac site was shown to be essential for P22 lytic growth. Mutations altering the TerS protein that affect pac site recognition were shown to be located in various locations on the protein. Additionally, a mutation in terL was also identified that allows normal progeny phage production in the absence of a functional pac site. This is the first evidence that TerL can work in conjunction with TerS to enable DNA recognition. In our studies of DNA ejection from the virion, a number of insights were obtained. The tail needle (gp26 protein) is the plug that keeps the DNA inside the complete virion, while the ejection proteins (gp7, gp20, and gp16) are required to deliver the DNA into the cytoplasm after gp26 release. We found that alterations of the C-terminal protein domain at the tip of the tail needle can have a strong affect on the rate at which DNA is released into the host cytoplasm, indicating that it may serve as a secondary switch controlling DNA delivery. We also examined the numbers and locations of the three different ejection proteins present in the complete P22 virion and found that they occupy space that is also available to the DNA inside the viral head. |
