The Mechanisms of Genome Entry in Bacteriophage HK97

The process of entry of the bacteriophage genome into host cells is poorly understood. In this thesis, I used bacteriophage HK97 as a model to investigate the mechanisms of entry. Phage genome entry begins with adsorption of HK97 to the outer membrane receptor, LamB. Next, the phage DNA passes from the phage capsid through the barrier of the cell envelope to reach the cytoplasm where it can be replicated. The mechanism behind the transfer of the DNA is unknown, though it involves interactions between host and phage components. Using host gene knockout mutants, I searched for proteins involved in entry of bacteriophage HK97 and identified required proteins found within the host cell envelope, FkpA and PtsG. These proteins are not required by all phages including some with a high degree of sequence identity compared to phage HK97. I also found a phage HK97 protein, gp15, whose expression was able to block entry of the same phages that required both PtsG and FkpA. Changes in the requirement of a phage for either FkpA and PtsG for entry and the sensitivity of a phage to gp15 correlated with sequence divergence in one particular phage protein, the tape measure protein (TMP). I illustrated that TMP sequence is responsible for dictating the requirements for FkpA and PtsG as well as the sensitivity to gp15 by exchanging TMPs between phages. The requirement for PtsG and the sensitivity to gp15 were mapped to residues between 452-600 within the HK97 TMP. PtsG mediates entry of the HK97 genome through a direct interaction with these residues within the TMP, and gp15 blocks entry by trapping the PtsG TMP complexes to prevent the formation of the structure that mediates entry.Ph.D