A Single-Molecule Hershey-Chase Experiment

Abstract

Summary Ever since Hershey and Chase used phages to establish DNA as the carrier of genetic information in 1952, the precise mechanisms of phage DNA translocation have been a mystery [1]. Although bulk measurements have set a time-scale for in vivo DNA translocation during bacteriophage infection, measurements of DNA ejection by single bacteriophages have only been made in vitro. Here, we present direct visualization of single bacteriophages infecting individual Escherichia coli cells. For bacteriophage λ, we establish a mean ejection time of roughly 5 min with significant cell-to-cell variability, including pausing events. In contrast, corresponding in vitro single-molecule ejections are more uniform and finish within 10 s. Our data reveal that when plotted against the amount of DNA ejected, the velocity of ejection for two different genome lengths collapses onto a single curve. This suggests that in vivo ejections are controlled by the amount of DNA ejected. In contrast, in vitro DNA ejections are governed by the amount of DNA left inside the capsid. This analysis provides evidence against a purely intrastrand repulsion-based mechanism and suggests that cell-internal processes dominate. This provides a picture of the early stages of phage infection and sheds light on the problem of polymer translocation.

Langevin dynamics simulation of DNA ejection from a phage

“…The assembled structures of DNA under the motor forces of experimental relevance are not in equilibrium and as a result the stochasticity in the ejection kinetics is a reflection of the extent of the nonequilibrium conformations of the packaged DNA inside the capsid. Our simulation results are entirely consistent with the in vitro results of Chiaruttini et al [21] and the in vivo results of Van Valen et al [7].…”

“…One of the major puzzles is the occurrence of pauses during DNA ejection. In the recent in vivo single-molecule Hershey-Chase experiment, Van Valen et al [7] have shown that there are significant variations in the ejection time, although it is the same molecule that is being ejected. The ejection trajectories exhibit abundant, but sporadic, pauses.…”

“…By monitoring DNA ejection from a phage against a solution with controllable osmotic pressure, the ejection can be stopped midway by a threshold value of the external osmotic pressure [8,10,13]. The ejection rate of DNA in the in vitro experiments is an order of magnitude faster than that in the in vivo experiments [7]. The roles of electrostatic interactions and multivalent ion binding to DNA have also been investigated [17,19].…”

Phage DNA Dynamics in Cells with Different Fates

“…There are different ways to label phage particles fluorescently (8,9,14,20,(30)(31)(32), and the key issue is to ensure that the labeled phages are functional and well-behaved for the examination of viral life cycle. Recently, we constructed two fluorescent phages, l LZ1 and l LZ2 , based on leyfp (8,20).…”

“…Cell recognition in the phase-contrast channel was performed using the Schnitzcell routine (gift of Michael Elowitz, California Institute of Technology), cell lineage tracking was performed by a homemade script, and spot recognition was similar to Spatzcells (29). We performed short movies with 30 …”