Genome and Internal Protein Ejection from DNA Viruses
Because of the unique simplicity of their life cycles, compared to all other evolving organisms, double-stranded (ds) DNA bacteriophages have served as an extremely valuable model for elucidating the basic physics and molecular biology of gene replication and expression. In this dissertation, we present experimental work on two phages, lambda and P22, to study the general genome delivery (ejection) mechanisms of dsDNA bacteriophage.
Using lambda phage, we systematically investigated the effects on DNA genome ejection of external osmotic pressure controlled by the concentration of osmolyte (PEG 8000) and the presence of polyvalent cations (tetravalent polyamine spermine, Sp4+). We found that the internal pressure of the capsid decreases from 38 to 17 atm as the [Sp4+] is increased from 0 to 1.5 mM. The existence of Sp4+ can also induce incomplete ejection under zero osmotic pressure when its concentration reaches 0.15 mM or higher; for [Sp4+] below this threshold, the ejection is complete. Further, we observed that the self-attraction induced by Sp4+ affects the configurational dynamics of the encapsidated genome, causing it to get stuck in a broad range of non-equilibrated structures.
In order to further study the DNA ejection mechanism from phage capsids, we have systematically determined how DNA transcription in vitro is affected by the presence of different osmolyte and viscogen molecules, so that we can test the transcription-pulling hypothesis of genome delivery in the presence of crowded environments mimicking the cytoplasm of the bacterial cell hosts of phages. We found that at high concentrations of DNA templates, macromolecules can increase the RNA yield due to crowding effects on the initiation step of transcription, while small molecules decrease the yield because of viscosity effects on the elongation step. Experiments carried out at low concentrations show a decrease in yield for large and small molecules, confirming the dominant effect of viscosity effects in the elongation step.
Having established and quantified the nature of the spontaneous driving force for DNA delivery, we then studied the ejection behavior of internal proteins from another phage, P22, and their functions in an in vitro osmotic suppression system controlled by PEG 8000. We found that the Outer Membrane Protein A (OmpA) from Salmonella, the natural bacterial host of P22, can significantly enhance the rate of DNA ejection in the presence of the primary receptor, LPS. While the DNA is ejected in the presence of LPS, no ejection of the internal proteins occurs unless OmpA is also present. We also find that their ejection is largely complete before any of the genome is ejected. This finding helps us understand the possible roles that the internal proteins play during infection.