Using multiple methods including cryoTEM observations of phages in suspended vitrified films or cryosections of infected bacteria, we address the question of bacteriophage T5 DNA organization in the full capsid and during encapsidation and release of the genome.
- The organization of DNA in the full capsid has been debated over the years. We propose that the hexagonally packed DNA is organized into multiple domains separated by defect walls [2]. Ongoing tomography observations will be presented.
- DNA release form the bacteriophage capsid involves physical and biological processes. It is possible to follow the ejection of DNA from the bacteriophage in vitro after interaction of the phage with its purified receptor. CryoTEM experiments let us visualize the portion of DNA kept inside the capsid at different steps of the ejection and provide information on its structure [1-3]. From these experiments, we discuss how the structure of confined DNA changes with the ionic conditions (isotropic, versus fully or partially toroidal). CryoTEM and fluorescence approaches reveal the presence of pauses during T5 ejection (moments where the ejection speed slows down to zero) allowing partially filled capsids to be imaged. We discuss the importance of ionic conditions and presence/absence of a support film in the location of these pauses [4]. We also discuss the reasons why pauses have not been detected in other phages like Lambda. Our work highlights the role of DNA organization inside the bacteriophage capsid on the stochastic and out of equilibrium nature of the ejection process.
- DNA organization during encapsidation inside the bacteria is analysed on cryosections of infected bacteria (work in progress) and comparisons are driven with capsids containing various amounts of DNA under multiple ionic conditions.
[1] A Leforestier, F Livolant, Proc. Natl. Acad. Sci. 2009, 106, 9157–9162.
[2] A Leforestier, F Livolant, J. Mol. Biol, 2010, 396, 384–395.
[3] A. Leforestier, A. Siber, F. Livolant , R. Podgornik, Biophys. J. 2011, 201, 100, 2209–2216
[4] M. De Frutos, A. Leforestier, and F. Livolant 2014, Biophys. Rev. Lett. 09, DOI: 10.1142/S1793048013500069