Balint Kiss1, Hedvig Tordai1, Tamás Bozó1, Miklós Kellermayer1
1 Semmelweis University, Biophysics and Radiation Biology, Budapest
The outer leaflet of Gram-negative bacterial cell wall consists of lipopolysaccharide (LPS) and proteins. Among these molecules is the target that T7 bacteriophages recognize and adhere to during the initial step of infection. Adhesion induced structural changes initiating and performing DNA injection is not yet fully understood. Our main goal is to understand the details of viral infection via single molecule experiments.
We used total internal reflection fluorescence microscopy (TIRF) to study DNA ejection of surface-adhered phages. Furthermore, an optical tweezer system combined with confocal microscopy was used to conduct force measurements and image DNA ejection on single phage level. TIRF experiments showed that viral DNA release abruptly halts at apparently random points during ejection. Halting of the ejection process might not hinder translocation, because host cell RNA polymerases (RNAP) can pull the DNA through the membranes. For complete translocation RNAPs need to overcome the viral DNA clamping mechanism. To mimic the pulling effect of RNAPs phages were bound to the surface of microbeads and the end of the partially released DNA was grabbed by another microbead. Pulling the DNA with forces up to 60 pN (which is the required force to denaturate double stranded DNA) we couldn’t pull out the DNA any further.
TIRF microscopy and confocal microscopy combined with optical tweezers have proved to be an efficient tool to study the DNA release of T7 bacteriophages at single phage level. The observed release kinetics raise the question: What mechanisms lie behind the full release and translocation of T7 phage DNA? Our long-term goal is to create an optimal experimental setup wherein viral DNA translocation can be studied close to its natural environment.
Doctoral School: Basic and Translational Medicine
Program: Cellular and molecular biophysics
Supervisor: Miklós Kellermayer
E-mail address: firstname.lastname@example.org