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Home page > Kinetics and energetics analysis of DNA ejection from bacteriophages

Contributors: M. de Frutos, E. Raspaud Collaborators: L. Letellier (IBBMC, CNRS UMR 8619), S. Brasiles, P. Tavares (VMS, CNRS UMR 2472)

Bacteriophages (bacterial virus) are complex molecular assemblies which multiplication relies on bacteria infection. Our aim is to understand the physical mechanisms and forces that drive the DNA release from the phage capsid. For the first time, light scattering was used to determine the kinetics of this process, measured in vitro conditions. For a solution sufficiently diluted, the intensity scattered by the particles is sensitive to their mass (phagic proteins plus encapsidated DNA). The DNA ejection induces a loss of phage mass and consequently, a decrease of scattering intensity as a function of time (see figure). For bacteriophage T51, a complex shape was observed for the ejection kinetics. A detail analysis of this shape shows the existence of pauses during the ejection at specific positions of the phagic genome. This particular behaviour of phage T51 has to be related to the two steps in vivo transfer. A study of the ejection kinetics as a function of temperature allowed us to investigate the thermodynamical properties of these systems. The characteristic times describing the transfer pauses appear to be consistent with an Arrhenius law, allowing us to determine the activation barrier which governs the mechanism. The nature of the proteins regulating the DNA transfer is still unknown but comparison with other phages should be instructive to elucidate the exact mechanism.

DNA is propelled outside the phage by the high pressure resulting from its confinement inside the capsid3. As a consequence, the amount of ejected DNA at the equilibrium is a function of the difference between the inner and outer pressures. As expected, we observed that a reduction of the capsid pressure 2 by the addition of a DNA condensing agent (spermine) results in a decrease of the amount of ejected DNA. More interestingly, our results demonstrate the existence of an additional force related to the presence of a DNA condensate outside the capsid. We have observed this effect in vitro but this mechanism likely plays a role in vivo, when the pressure inside the bacterial cytoplasm is opposed to DNA transfer. Condensing agents (polycations and proteins) are present inside the bacteria cytoplasm and can then act as an additional force pulling on the DNA.

Schematic representation of the DNA release from a single bacteriophage and curve showing the kinetics observed by light scattering for the DNA ejection from a phage population.

References :

1) DNA ejection from bacteriophage T5: analysis of the kinetics and energetics, M. de Frutos, L. Letellier, E. Raspaud ,Biophys. J. 88: 1364-1370 (2005).

2) Effect of spermine and DNase on DNA release from bacteriophage T5, M. de Frutos, S. Brasiles, P. Tavares, E. Raspaud, EPJ E 17, 429 – 434 (2005).

3) Osmotic pressure inhibition of DNA ejection from phage, A. Evilevitch, L. Lavelle, C. M. Knobler, E. Raspaud, W. M. Gelbart, PNAS 100: 9292-9295 (2003).