DNA ejection from bacteriophage T5: analysis of the kinetics and energetics

Abstract

DNA ejection from bacteriophage T5 can be passively driven in vitro by the interaction with its specific host receptor. Light scattering was used to determine the physical parameters associated with this process. By studying the ejection kinetics at different temperatures, we demonstrate that an activation energy of the order of 70 k(B)T must be overcome to allow the complete DNA ejection. A complex shape of the kinetics was found whatever the temperature. This shape may be actually understood using a phenomenological model based on a multistep process. Passing from one stage to another requires the mentioned thermal activation of pressurized DNA inside the capsids. Both effects contribute to shorten or to lengthen the pause time between the different stages explaining why the T5 DNA ejection is so slow compared to other types of phage.

FIGURE 1

Static spectra of the light scattered by phage T5 before and after FhuA addition. The detected scattered intensity is plotted as a function of the angle θ. After addition of FhuA, the intensity (open circles) at the final state (i.e., once DNA ejection is achieved), becomes ∼15 times lower than the initial intensity measured without FhuA (solid circles).

FIGURE 2

Temperature effect on F(t) = (I(t) − I_final)/(I_init − I_final) plotted as a function of time. All experiments were done with a large excess of receptors. By simply decreasing the temperature from 41 to 5°C, the time required to achieve DNA ejection from phages shifts from a few tens of minutes to several days.

FIGURE 3

Ionic condition effect on the detected signal I(t) relative to its initial value I_init., plotted as a function of time. Spermidine 10mM and DNase were added in two incubating samples. These two samples differed in their NaCl content. When the ionic condition was not sufficient to condense DNA (open circles), the NaCl content being 100 mM, the ejection kinetics were similar to the curve measured without polyamines (solid line without symbols). In the opposite case, when the ionic condition was sufficient to condense DNA (solid symbol), the NaCl content being reduced to 10 mM, the relative signal remained at the final ratio value I_final/I_init = 0.61—measured the day after the kinetics experiment. In such an ionic condition, we observed a strong inhibition of the DNA release.

Figure s6

FIGURE 4

Interpretation of the continuous kinetics using a phenomenological model of successive events. Two intermediate lengths have been considered here corresponding to 50% and 10% of the genome length. The typical fraction of unejected DNA length for one phage is illustrated as a function of time in Fig. 4, right panel. Each transition from one stage to another is described by a first-order law. Between two stages, the DNA ejection itself is considered as instantaneous. In our experiments and at a given time, different fractions of phage having different unejected DNA lengths coexist and contribute to the detected signal. By fitting the data with this model, the fraction of each and its temporal dependence may be evaluated. An example is given in Fig. 4, left and middle panels, the temperature being 23°C. By adjusting the characteristic times describing the different decay rates, it is possible to reproduce accurately the experimental data by this model.

FIGURE 5

Interpretation of the temperature dependence of the DNA ejection kinetics. Since the curves seem parallel when plotted in log-lin scale (Fig. 2), a simple rescaling of the time t by an exponential allows us to obtain a good superimposition of the different data. Such a behavior is expected for an activation process, where the enthalpy ΔH required to activate the DNA ejection is compared to the thermal energy k_B T.