Real-time observations of single bacteriophage lambda DNA ejections in vitro

Abstract

The physical, chemical, and structural features of bacteriophage genome release have been the subject of much recent attention. Many theoretical and experimental studies have centered on the internal forces driving the ejection process. Recently, Mangenot et al. [Mangenot S, Hochrein M, Rädler J, Letellier L (2005) Curr Biol 15:430-435.] reported fluorescence microscopy of phage T5 ejections, which proceeded stepwise between DNA nicks, reaching a translocation speed of 75 kbp/s or higher. It is still unknown how high the speed actually is. This paper reports real-time measurements of ejection from phage lambda, revealing how the speed depends on key physical parameters such as genome length and ionic state of the buffer. Except for a pause before DNA is finally released, the entire 48.5-kbp genome is translocated in approximately 1.5 s without interruption, reaching a speed of 60 kbp/s. The process gives insights particularly into the effects of two parameters: a shorter genome length results in lower speed but a shorter total time, and the presence of divalent magnesium ions (replacing sodium) reduces the pressure, increasing ejection time to 8-11 s. Pressure caused by DNA-DNA interactions within the head affects the initiation of ejection, but the close packing is also the dominant source of friction: more tightly packed phages initiate ejection earlier, but with a lower initial speed. The details of ejection revealed in this study are probably generic features of DNA translocation in bacteriophages and have implications for the dynamics of DNA in other biological systems.

Fig. 1.

Images: time series of single genome ejections from λcI60 and λb221, taken at a frame rate of 4 s⁻¹. For each of the phages, the ejection in buffer with 10 mM NaCl (Upper) is significantly faster than ejection in 10 mM MgSO₄ (Lower). The 16-μm scale bar is approximately the contour length of a 48.5-kbp piece of DNA. Graphs show length of the DNA that has emerged from the capsid at each time point, as computed by using a computer image-processing algorithm together with DNA length standards as described in the text.

Fig. 2.

Graphs of ejection trajectories, comparing NaCl and MgSO₄ buffers and two genome lengths. Single-ejection events were analyzed as described in the text, resulting in trajectories giving the length of DNA out of the capsid as a function of time. These trajectories are aligned and plotted for visual comparison; the offset of the starting time of the ejection is not used in further analysis. The graphs show that ejection proceeds on a time scale of ≈1 s in NaCl buffer, or ≈10 s in MgSO₄ buffer. The ejection speeds of phages with different genome lengths appear similar in this view.

Fig. 3.

Averaged speeds of DNA ejection for λcI60 and λb221. The plot shows the DNA ejection speed as a function of the amount of DNA within the capsid, averaged in bins of width 2.5 kbp (shown as the horizontal error bars.) Vertical error bars are computed from the standard deviation of the calibration data; there are additional systematic deviations in all curves due to inaccuracies in calibration at the different ionic conditions. The curves for phages of different genome lengths lie close to each other, whereas most of the variation is caused by the difference in buffer conditions. A maximum of ≈60 kbp/s is reached in NaCl buffer, whereas the maximum in MgSO₄ buffer is ≈17 kbp/s. Vertical gray lines represent the genome lengths of λcI60 and λb221.

Fig. 4.

The number of ejections that have been triggered as a function of time. For each experiment, the total number of ejections that had been observed was plotted as a function of time; these are the same ejections that were used for the analysis above. Also plotted are exponential least-squares fits of the form a(1 − exp(−t/t₀)) + b, where t₀ is the time constant of triggering. To take into account the delay before LamB entered the flow chamber, we set t = 0 at the time of the first observed ejection.

Fig. 5.

The relationship between force and velocity. (Upper) Force on the DNA, as a function of the amount of DNA left within the capsid, according to theoretical calculations (M. Inamdar, personal communication). Calculations for both buffers were run according to the method of Purohit et al. (33), which requires two parameters, F₀ and c, that must be determined experimentally. We used F₀ = 12,000 pN/nm²; c = 0.30 nm for Mg buffer and F₀ = 660 pN/nm²; c = 0.52 nm for Na buffer, based on fitting to experimental data from Rau et al. (19). The calculations for Mg buffer were identical to those reported earlier (11). The force in Na buffer is significantly higher than that in Mg buffer. (Lower) Computed friction coefficient φ, showing the relationship between DNA packing within the capsid and its friction. The graph shows that φ generally increases with increasing DNA density. For low concentrations of DNA, φ is much lower for Na buffer than for Mg buffer. However, with more than ≈20 kbp in the capsid, φ becomes independent of the type of buffer. The value of φ appears to increase to a very high value when 100% of the DNA is packed. Error bars are computed as in Fig. 3.